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Factors influencing the aerobic respiration of Escherichia coli Rainnie, Donald James

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FACTORS INFLUENCING THE AEROBIC RESPIRATION OF ESCHERICHIA COLI by DONALD JAMES RAINNIE B.Sc., University of Manitoba, 1964 M.Sc. , University of Saskatchewan, A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department o f Biochemistry We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1973 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of z/^c^??/s7/ey The University of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT Facultative anaerobic bacteria such as Escherichia coli are capable of obtaining energy from glycolysis, aerobic respiration, and anaerobic respiration. Although a considerable amount of information is available on the production of ATP in E. coli by glycolysis, very l i t t l e is known about the production of energy by aerobic or anaerobic respiration. Since the properties of aerobic respiration were experimentally more readily attacked, an investigation of the factors influencing the aerobic respiration and respiratory chain-linked energy production of E. coli was undertaken. The principal technique utilized in these investigations was the polarographic determination of oxygen tension. Investigation of the pro-perties of a commercially available, vibrating-reed, oxygen electrode revealed that silver ions were released from the silver anode of the uncoated oxygen electrode into the buffer solution surrounding the electrode. Loss of silver from the electrode was dependent on the buffer concentration and the type of buffer, and was relatively independent of the presence or absence of the polarizing voltage. It is postulated that the release of silver from the anode of the oxygen electrode involved chelation by the buffer ions. This problem was avoided subsequently by using a Clark oxygen electrode. The pH, buffer ion and the buffer concentration of the assay medium were observed to influence the rate of respiration of E. coli. In addition the buffer ion and the pH influenced the linearity of oxygen consumption with time. Glycylglycine-KOH buffer, pH 7.0, at a concentration of 300 mM was determined as "optimal" according to the criteria of: (i) supporting a high respiratory rate; (ii) supporting a constant rate of oxygen utilization; and ( i i i ) maintenance of these characteristics of the E. coli cell suspension i i i for a greater period of time than required to complete the experiment. The applicability of these c r i t e r i a of classical enzyme kinetics to the determination of "optimal" conditions for the investigation of systems involved in energy conservation i s questioned. During the simultaneous measurement of acid production and oxygen consumption, a 15 to 30 second lag in acid production was observed to occur during the transition from aerobic to anaerobic glucose u t i l i z a t i o n . This, observation is discussed with respect to the information currently a v a i l -able on the regulation of the amphibolic pathways of E. c o l i . Silver ions inhibited the oxidation of endogenous substrates, glucose, glycerol, D- and L-lactate, acetate, succinate and fumarate by intact-cell suspensions of E. c o l i . The oxidation of formate was only sl i g h t l y i n h i -bited under the conditions which resulted in complete inhibition of respir-ation on the previously indicated substrates. The oxidation of glucose and glycerol was more sensitive to s i l v e r ions than that of D- or L-lactate, fumarate or succinate. This was attributed to inhibition of glyceraldehyde -3-phosphate dehydrogenase. Before the onset of the inhibition by s i l v e r ions there was a period when respiration was stimulated. This effect was similar to that given by 2,4-dibromophenol. With both compounds the degree of stim-ulation was larger in iron-sufficient than in iron-deficient c e l l s . It i s postulated that s i l v e r ions uncouple respiratory chain-linked energy pro-duction as well as inhibiting the respiratory chain and glyceraldehyde -3-phosphate dehydrogenase in E. c o l i . Growth and c e l l respiration were affected when iron became limiting in batch cultures of E. c o l i growing on succinate. A decrease occurred in the efficiency with which succinate was converted to c e l l mass, in the iv respiratory control ratio, and in the levels of nonheme iron, cytochrome , and NADH and succinate oxidase a c t i v i t i e s . On addition of f e r r i c citrate to the iron-limited cultures the above components returned at d i f -ferent rates to the levels found in iron-sufficient c e l l s . The concentration of nonheme iron, the respiratory control ratio and the efficiency of con-version of succinate to c e l l mass recovered more rapidly than.the level of cytochrome b^ and the oxidase a c t i v i t i e s . Succinate oxidase activity re-covered more rapidly than either succinate dehydrogenase or cytochrome b^ levels. It i s postulated that nonheme iron i s involved in respiratory chain-linked energy production in E. c o l i . v TABLE OF CONTENTS Page ABSTRACT i i i TABLE OF CONTENTS vi LIST OF TABLES x LIST OF FIGURES « . . . x i ABBREVIATIONS xv ACKNOWLEDGEMENTS xix Chapter 1. INTRODUCTION " 1 1.1 Transport 2 1.2 Amphibolic pathways 15 1.3 Respiratory chain 28 1.4 Oxidative phosphorylation 45 1.5 The influence of s i l v e r ions on growth and enzyme act i v i t y 58 1.6 The influence of iron limitation on respiration and energy conservation 63 1.7 The objectives of the research reported in this thesis » . . . . . . . 67 2. MATERIALS AND METHODS 70 2.1 Materials 70 2.1.1 Bacteria 70 2.1.2 Chemicals 71 2.1.3 Equipment 73 2.1.4 Media 75 2.2 Methods 76 2.2.1 Culturing proced\ires for bacteria 76 2.2.1.1 Maintenance of stock cultures 77 2.2.1.2 Growth of cultures for inoculation . . . 77 2.2.1.3 Culture of the bacteria . 77 2.2.2 Demonstration of s i l v e r release from the Aminco oxygen electrode 77 2.2.2.1 Growth and harvesting 78 2.2.2.2 Measurement of oxygen consumption . . . 79 2.2.2.3 Determination of c e l l v i a b i l i t y . . . . 80 v i Page 2.2.3 Measurement of s i l v e r release from Aminco oxygen electrode 80 2.2.3.1 Sample preparation 80 2.2.3.2 Atomic absorption spectro-photometry 81 2.2.4 The influence of p'H, buffer ion and buffer concentration on the respir-ation of E. c o l i 81 2.2.4.1 Growth and harvesting 81 2.2.4.2 Measurement of oxygen consumption . 82 2.2.4.3 Protein determinations 83 2.2.5 The influence of s i l v e r on the respir-ation of E. c o l i 83 2.2.5.1 Growth and harvesting . 84 2.2.5.2 Measurement of oxygen consumption . . . . . . . . . 85 2.2.5.3 Concurrent measurement of oxygen consumption and proton production . . . . 85 2.2.5.4 Assay for glyceraldehyde -3-phosphate dehydrogenase 86 2.2.6 The influence of iron limitation on respiration of E. c o l i 87 2.2.6.1 Growth and harvesting 87 2.2.6.2 Estimation of the efficiency of conversion of succinate to c e l l mass 89 2.2.6.3 Measurement of the effect of dibromophenol and s i l v e r nitrate on oxygen consumption 89 2.2.6.4 Respiratory control ratio 96 2.2.6.5 Determination of total iron and nonheme iron 90 2.2.6.6 Preparation of c e l l extracts 91 2.2.6.7 Determination of cytochromes a_2 and b^ „ 91 2.2.6.8 Assay for succinate dehydrogenase , . 92 2.2.6.9 Assay for succinate oxidase 94 2.2.6.10 Assay for NAJJH oxidase 94 2.2.6.11 Glucose determinations 94 3. PART I: SILVER ION AND THE RESPIRATION AND ENERGY-COUPLING OP E. COLI ' 95 3.1 Results . 95 3.1.1 Release of s i l v e r from the Aminco oxygen electrode . . . . . . . . . 95 3.1.2 Factors influencing the release of s i l v e r from the Aminco oxygen electrode . . . 105 v i i Page 3.1.3 The influence of pH, buffer ion and ' buffer concentration on the respir-ation of E. c o l i 108 3.1.4 Inhibition of the respiration of E. c o l i by added s i l v e r nitrate 117 3.1.5 Selection of a carbon source for the growth of E. c o l i to be used for the investigation of the uncoupling of respiration by s i l v e r nitrate . . 129 3.1.6 Uncoupling of the respiration of E. c o l i by added s i l v e r nitrate .142 3.2 Discussion 142 3.2.1 The measurement of oxygen tension with an oxygen electrode 142 3.2.2 A proposed mechanism for the release of s i l v e r from the anode of the Aminco oxygen electrode 150 3.2.3 The influence of pH, buffer ion and buffer concentration on the respiration of B. c o l i 152 3.2.4 The inhibition of the respiration of E. c o l i by s i l v e r ions 154 3.2.5 Selection of a carbon source for the growth of E. c o l i to be used for the investigation of the uncoupling of respiration by s i l v e r nitrate 167 3.2.6 The uncoupling of the respiration of E. c o l i by added s i l v e r nitrate 172 3.2.7 Redox potential as an indicator of the oxygen level of batch cultures of E. c o l i • • « 174 4. PART II: IRON LIMITATION AND THE RESPIRATION AND ENERGY-COUPLING OF E. COLI o 177 4.1 Results o 177 4.1.1 The influence of iron limitation on the respiration and energy-coupling of E. c o l i 177 4.2 Discussion 205 4.2.1 Batch culture versus continuous culture 205 4.2.2 The influence of iron limitation on the respiration and energy-coupling of E, c o l i . , 209 5. PART III: THE TRANSITION FROM AEROBIC TO ANAEROBIC GLUCOSE UTILIZATION 222 5.1 Results and discussion 222 v i i i Page 5.1.1 The lag in acid production hy E. c o l i associated with the transition from aerobic to anaerobic glucose u t i l i z a t i o n . . . . 222 BIBLIOGRAPHY 2^2 APPENDICES A 2 5 0 B 252 C . 255 ix LIST OF TABLES Table Page 1.1 Enzymes of the amphibolic pathways of E. c o l i ^ 3.1 The v i a b i l i t y of E. c o l i before and after the cessation of oxygen consumption . . . . . 97 3.2 Time course for the release of s i l v e r from the Aminco oxygen electrode, with the presence or absence of polarizing voltage 3.3 Effect of concentration of buffer on the release of s i l v e r from the Aminco oxygen electrode . . . . . . . . 109 3.4 The influence of the concentration of glycylglycine-KOH buffer, pH 7.0, on the respiration rate of E. c o l i 115 3.5 The influence of the duration of suspension in 300 mM glycylglycine-KOH buffer, pH 7.0, at 0°C, on the res-piration of E. c o l i 116 3.6 The influence of s i l v e r nitrate and reduced glutathione on glyceraldehyde-3-phosphate dehydrogenase activity . . . . 128 3.7 The influence of the addition of s i l v e r nitrate on the i n i t i a l rate of oxygen consumption . . . . . . . . 130 3.8 The stimulation of the respiration of E. c o l i by 2,4-di-bromophenol and s i l v e r nitrate . . . . . 147 4.1 The level of iron-containing respiratory chain components in c e l l extracts of iron-limited, succinate-grown, E. c o l i prior to, and following the addition of f e r r i c citrate (final cone. 6 uM) 185 4.2 Enzyme ac t i v i t i e s in c e l l extracts of iron-limited, suc-cinate-grown, E. c o l i following the addition of f e r r i c citrate (final cone. 6 pill) . 189 x LIST OF FIGURES Figure Page 1.1 Pathways for the catabolism by E. c o l i of common nutrients . . . 3 1 . 2 The phosphoenolpyruvate phosphotransferase system 5 1.3 The phosphoenolpyruvate phosphotransferase system 5 1.4 Glycerol and L- a -glycerophosphate dissimilation in E. c o l i 8 1.5 Amphibolic pathways of E. c o l i 17 1.6 Models of the electron transport systems of E. c o l i . . . . 44 2.1 Culture apparatus 8 8 2 . 2 Dithionite reduced-minus-oxidized difference spectrum . . . 93 3.1 Effect of c e l l concentration on oxygen consumption by E. c o l i suspensions as measured with the Aminco oxygen electrode 96 3 . 2 Prevention of cessation of respiration of E. c o l i by the addition of reduced glutathione 99 3.3 Respiration of E. c o l i as measured with Clark-type oxygen electrode 100 3.4 Stimulation of respiration via the addition of reduced glutathione 101 3.5 Retention of the cessation of respiration following the replacement of Aminco electrode by Clark-type electrode 103 3.6 Inhibition of the oxygen consumption of E. c o l i by a substance released by the Aminco electrode . . . . . . . 104 3.7 Inhibition by s i l v e r of the respiration of E. c o l i as measured with Clark-type oxygen electrode 106 3.8 Buffer dependence of the release of s i l v e r from the Aminco oxygen electrode . . . . . 107 3.9 Buffer ion and pH dependence of the respiration of E. c o l i 111 x i Figure Page 3.10 The influence of buffer ions on the oxygen consumption traces of E. c o l i . . 113 3.11 The influence of the concentration of glycy l -glycine-KOH buffer, pH 7*0, on the respiration rate of E . c o l i 114 3.12 Inhibition of the endogenous respiration of E. c o l i by s i l v e r . . . . . 118 3.13 Inhibition of the glucose-dependent (A) and formate-dependent (B) respiration of E. c o l i by s i l v e r 119 3.14 Inhibition of the acetate-dependent respiration of E. c o l i by s i l v e r 120 3.15 Inhibition of the glycerol-dependent (A) and glucose-dependent (B) respiration of E. c o l i by s i l v e r 121 3.16 Inhibition of the D-lactate-dependent (A) and glucose-dependent (B) respiration of E. c o l i by s i l v e r . . . . . . 122 3.17 Inhibition of the L-lactate-dependent (A) and succinate-dependent (B) respiration of E. c o l i by s i l v e r . . . . . . 123 3.18 Inhibition of the fumarate-dependent (A) and glucose-dependent (B) respiration of E. c o l i by s i l v e r 124 3.19 Inhibition of the respiration and acid production of E. c o l i by s i l v e r 127 3.20 Growth of E. c o l i on 0.4$ glucose in a medium containing 6 uM f e r r i c citrate . . . . . . < > . . ° <> « « 132 3.21 Growth of E. c o l i on 0.4$ glucose . ....<, 134 3.22 Growth of E. c o l i on 0.4$ glycerol 136 3.23 Growth of E. c o l i on 0.8$ DL-lactate .138 3.24 Growth of E. c o l i on 0.8$ acetate 139 3.25 Growth of E. c o l i on 0.6$ succinate . . . . . . . . . . . . 141 3.26 Stimulation of the respiration of E. c o l i by 2,4-dibromophenol . . . . . . . . . . . . . . 143 x i i Figure Page 3.27 Stimulation of the respiration of E. c o l i "by-si l v e r nitrate 144 .3.28 The influence of potassium nitrate on the respir-ation of E. c o l i 145 3.29 The dependence of the respiratory control ratio (RG.R) on the uncoupler concentration 146 4.1 Plateau in the oxygen level of a culture of E. c o l i growing on 0 . 6 $ succinate 178 4 . 2 Growth and oxygen level ( A ) , efficiency (B ) and cytochrome b-j levels (c) of a culture of E. c o l i growing on 0 . 6 $ succinate 179 4.3 The response of growth and oxygen level (A), and nonheme iron and cytochrome b^ levels (B) to the addition of f e r r i c citrate (Fe) to a culture of E. c o l i growing on 0 . 6 $ succinate 1 8 2 4.4 A semi-log plot of the growth data from Fig. 4.3 • • • • 184 4.5 The response of growth and oxygen level (A) and, enzyme levels (B) to the addition of f e r r i c citrate to a culture of E. c o l i growing on 0 . 6 $ succinate . . . . 1 8 7 4.6 The response of growth and oxygen level (A) efficiency (B) and cytochrome b^ levels (c) to the addition of sodium citrate (SC) to a culture of E. c o l i growing on 0 . 6 $ succinate . . . . . . . . 190 '4.7 The response of growth and oxygen level to the addition of f e r r i c citrate (Fe) and ammonium sulfate (AS) to an iron-deficient, nitrogen-limited culture of E. c o l i growing on 0 . 6 $ succinate 193 4.8 The response of growth and oxygen level to the addition of f e r r i c citrate (Fe) to a culture of E. c o l i growing on 0 . 6 $ succinate in a medium containing the trace metals Ca 2 +, Zn 2 +, Co 2 +, Mn 2 + and Cu 2 + 1 9 4 4 . 9 The growth and oxygen level (A) and cytochrome a^, cytochrome b^ and nonheme iron levels (B) of an iron-sufficient culture of E. c o l i growing on 0 . 6 $ succinate 1 9 5 x i i i Figure Page 4.10 The growth and oxygen level (A) and efficiency (B) of an iron-sufficient culture of E. c o l i growing on 0.6$ succinate' 197 4.11 The growth oxygen level and redox potential of an iron-sufficient culture of E. c o l i growing on 0.6$ succinate 199 4.12 A semi-log plot of the growth data from Fig. 4.9 200 4.13 The growth and oxygen level (A) and cytochrome h-| and nonheme iron content (B) of an iron-limited culture of E. c o l i growing on 0.4$ glucose 202 4.14 A semi-log plot of the growth data from Fig. 4.13 204 4.15 The response of growth and oxygen level (A) e f f i c i -ency (B) and the respiratory control ratio, RCR(c) to the addition of f e r r i c citrate (Fe) to a culture of E. c o l i growing on 0.6$ succinate 206 5.1 Oxygen consumption and acid production during aerobic and anaerobic u t i l i z a t i o n of glucose by intact E. c o l i . . 223 5.2 Oxygen consumption and acid production during aerobic and anaerobic u t i l i z a t i o n of glucose by Tris-EDTA permeabilized E. c o l i 225 5.3 The release of ultraviolet absorbing material from E. c o l i . . . . . . . . . . . . 231 xiv ABBREVIATIONS A.A. (or a.a.) - amino acid Acetyl-CoA (or acetyl-S-CoA) - acetyl coenzyme A Acetyl - acetyl phosphate ADP - adenosine-5 1-diphosphate ALA - S -aminolevulinic acid AMP - adenosine-5'-monophosphate ;AMP - 3 1 ,5 1-adenosine monophosphate atm - atmosphere ATP - adenosine-5 1-triphosphate ATPase - adenosine triphosphatase C 2 - two carbon compound c 4 - four carbon compound C^-dicarboxylio acids - four carbon dicarboxylic acids CCCP - carbonyl cyanide m-chlorophenylhydrozone CDP - cytidine-5'-diphosphate CoA - coenzyme A cyt. - cytochrome DBP - 2 ,'4-dibrorao phenol DCCD - dicyclohesylcarbodiimide DCIP - 2,6-dichlorophenolindophenol DHAP - dihydroxyacetonephosphate DNA - deoxyribonucleic acid DNP - 2,4-dinitrophenol DTNB - dithionitrobenzene EDTA — ethylenediamine tetraacetic acid XV EDTH EPR FAD FDP Fe FMN F-6-P fp (or Fp) 3-GAP (or GAP) GDP Glu L- «-glycerol-P G-1-P G-6-P GSH GTP HEPES HQNO hexose-1-P hexose-6-P IA IAA ITP CC-KG Mg 2 +(Ca 2 +)-ATPa3e energy-dependent transhydrogenase electron paramagnetic resonance flavi n adenine dinucleotide fructose-1,6-diphosphate nonheme iron f l a v i n mononucleotide fructose-6-phosphate flavoprotein glyceraldehyde-3-phosphate guanosine-5 1-diphosphate glucose L- «-glycerophosphate glucose-1-phosphate glucose-6-phosphate glutathione, reduced guanosine-5'-triphosphate N-2-hyd roxyethylpipe raz ine-N'-2-ethane sulfonic acid 2-heptyl-4-hydroxyquinoline-N-oxide hexose-1-phosphate hexose-6-phosphate iodoacetic acid iodoacetamide inosine -5 1-triphosphate oc-oxoglutarate (or oc-ketoglutarate) /Ig 2 +- or Ca 2 +-£ triphosphatase 2+ 2  - Mg   -stimulated adenosine xvi MOPS NAD+ NADH NADP+ NADPH NAD(P)H NEM OAA PBP PCMB PEP 2- PGA 3- PGA 6-P-gluconate P-HPr P i PIPES PMPS PMS Pyr RCR mRNA S1 S 1 " p - morpholinopropane sulfonic acid - nicotinamide adenine dinucleotide - nicotinamide adenine dinucleotide, reduced - nicotinamde adenine dinucleotide phosphate - nicotinamide adenine dinucleotide phosphate, reduced - pyridine nucleotides, reduced - N-ethylmaleimide - oxaloacetic acid - pentabromophenol - p_-chloromercuribenzoate - phosphoenolpyruvate - 2-phosphoglyceric acid - 3-phosphoglyceric acid - 6-phosphogluconate - phospho-HPr - inorganic phosphate - piperazine-N,N'-"bis(2-ethane sulfonic acid) - p_-mercuriphenylsulfonate - phenazine methosulfate - pyruvate - respiratory control ratio - messenger ribonucleic acid - sugar-) - sugari phosphate x v i i SDS - sodium dodecylsulfate succinyl-CoA - succinyl coenzyme A TGA cycle - tricarboxylic acid cycle TES - ?I-tris(hydroxymethyl )methyl-2-amino-ethane sulfonic acid TPP - thiamine pyrophosphate Tris - tris(hydroxymethyl)aminomethane Triose-P - triose phosphate TTFA - thenoyltrifluoroacetone TTFB - 4,5,6,7-tetrachloro-2-trifluoromethyl benzimadozle uv - ultraviolet.' UQ-8 - ubiquinone-8 UQ*-8 - ubisemiquinone-8 vit«K2(40) - vitamin K 2(40) x v i i i ACKNOWLEDG EM3NTS The author wishes to thank his supervisor, Dr. P. D. Bragg, for his suggestions, advice, constructive criticism and patience during the experimental work and the preparation of the manuscript. The author also wishes to thank Drs. G. T. Beer, D. G. Kilburn and J. F. Richards, of his Ph. D. committee,for their advice on problems encoun-tered in the research, and their comments on the i n i t i a l draft of the thesis. To Ms. C. Hou, Drs. I.C. Kim and P. L. Davies, the author expresses his appreciation of their assistance and cooperation. A special thanks to Ms. Elaine Yoshizawa, for her untiring efforts in typing the thesis, and to Ms. Carol Tsuyuki for her assistance in proof reading the f i n a l copy. The financial support of a Medical Research Council Studentship and a University of British Columbia Graduate Fellowship are acknowledged with thanks fThis work was financed by a Medical Research Council grant. xix "... For certain limited purposes a l l (these) things can be discussed in isolation and specific hypotheses made about their functioning. But i t is only when they are assembled into a machine that they make complete sense, and when the assembly and i t s laws are ignored, unnecessary ad hoc hypotheses may appear needed to explain in terms of an isolated part of the machine facts which simply reflect the essential relation of this part to others." - Dean & Hinshelwood, 1966. xx 1 1. INTRODUCTION Although the regulation of mitochondrial respiration, and energy production linked to the respiratory chain, by adenine nucleotides and inorganic phosphate has been known for some time, the characteristics of these processes in bacterial systems have remained largely undefined. Con-sequently, efforts in Dr. Bragg's laboratory have been directed towards ob-taining an understanding of the functioning of the respiratory chain of Escherichia c o l i (E. c o l i ) , and the regulation of respiratory chain-linked energy production in this organism. Within the framework of this ultimate goal, the objectives of the research presented in this thesis were (i) to investigate the site(s) of si l v e r ion inhibition of the aerobic respiration of E. c o l i with a view to the possible u t i l i z a t i o n of s i l v e r ions as a tool for the further in v e s t i -gation of respiratory chain function and regulation, and ( i i ) to investigate the involvement of iron in the function of the E. c o l i respiratory chain, with respect to the rate of electron transport, and to the coupling of energy conservation to the respiratory chain. The principal analytical procedure that was u t i l i z e d in the investi-gation of the factors influencing the aerobic respiration of E. c o l i was the measurement of the oxygen tension of the assay or culture medium. Since the aerobic respiration of an intact organism as measured by the disappearance of oxygen from the environment reflects the function and kinetic character-i s t i c s of a l l pathways and systems which provide reducing equivalents and oxygen to the terminal oxidase(s) of the electron transport chain, the intro-duction w i l l include a discussion of: (i) the sequence and regulation of the 2 major systems in E. c o l i which are involved in providing substrates to the respiratory chain, and ( i i ) the characteristics of the respiratory chain of this organism. 1.1 Transport Compounds with potential for providing reducing equivalents to the electron transport chain of E. c o l i are numerous (Figure 1 . 1 ) . The prime requisite for metabolism of any of these nutrients by the c e l l i s the penetration of the compound into the c e l l . This may occur by one or more of the following mechanisms: (i) group translocation; ( i i ) active transport; ( i i i ) f a c i l i t a t e d diffusion; or (iv) free diffusion. The distinguishing properties of these mechanisms have been outlined by Hayashi and Lin ( 1 9 6 5 ) , Kaback (1972) and Harold ( 1 9 7 2 ) . To date, research into the penetration of potential carbon and/or energy sources into E. c o l i has primarily been con-cerned with the following compounds or systems: (i) the phosphoenolpyruvate (PEP) phosphotransferase system; ( i i ) glycerol; ( i i i ) L-a-glycerolphosphate; (iv) acetate; (v) formate; (vi) C^-dicarboxylic acids; and ( v i i ) the D-lactate oxidase coupled active transport system. The phosphoenolpyruvate phosphotransferase system was f i r s t reported by Kundig et a l . , in 1964 and has become the classic example of uptake by group translocation, involving, in this instance, vectorial phosphorylation. The system in E. c o l i consists of three components; HPr, a heat stable, low molecular weight protein produced constitutively which functions as a phos-phate carrier; Enzyme I which is a soluble, sugar nonspecific enzyme, also produced constitutively which catalyzes the phosphorylation of HPr by PEP; and Enzyme II, a group of membrane-bound enzymes of varying degrees of sugar sp e c i f i c i t y , some produced constitutively , some inducible, which catalyze Di- and Oligosaccharides 3 Pentoses Gluconate Hexoses Glycerol Triose-P Fats Pyruvate ( 2H, c o 2 y Aspartate Lactate Propionate Pyrimidines Serine Glycine Cysteine Alanine Valine Isoleucine(Part) Leucine Isoleucine(Part) Aromatic A.A.(Part) Isocitrate ot-Oxoglut arat e Glutamate Proline Histidine Aromatic A.A.(Part) 'Succinate' Fig. 1.1 Pathways for the catabolism by E. c o l i of common nutrients, (Kornberg, 1970) 4 the transfer of the phosphoryl group from phosphorylated HPr to the sugar. This two-step process i s summarized in Figure 1.2. Subsequently, Kundig and Roseman (l972a,b) have described the further resolution of Enzyme II into a sugar specific component, IIA, and a nonspecific component, IIB. In addition, the system has an absolute requirement for a divalent cation, and for phosphatidyl glycerol for optimal ac t i v i t y . There may be a fourth protein component of unknown function, and required for induced systems only. The sugars phosphorylated are a l l of the D-configuration and include glucose, mannose, fructose, their corresponding hexosamines and N-acetylhexosamines, CC-methyl glucoside, galactose and thiomethylgalactoside. A l l , with the exception of fructose are phosphorylated at C-6. Fructose i s phosphorylated at G-1. The requirement for PEP as phosphate donor i s specific and cannot be replaced by any of the nucleoside 5-mono-, d i - , or triphosphates or any of a number of other potential phosphate donors (Kundig et a l . , 1964). The PEP phosphotransferase system i s present and functional i n cytoplasmic membrane vesicles of E. c o l i . Kaback and co-workers have ex-ploited the reduced complexity of the membrane vesicles to investigate the mechanism and regulation of the PEP translocation system (Kaback, 1970a,b). The data are consistent with the hypothesis that the PEP phosphotransferase system, in isolated membrane preparations, i s subject to "product" inhibition by glucose-6-phosphate (G-6-P) and to "feedback" inhibition by glucose-1-phosphate (G-1-P). Inhibition by these sugar phosphates is noncompetitive, and the inhibitor sites are distin c t , accessible from both sides of the membrane, possibly close physically and are under independent control. In addition to inhibition by sugar phosphates, growth conditions appear to influence the properties of the PEP phosphotransferase system. Enzyme I: « 2 + Phosphoenolpyruvate + HPr v ^ N Phospho~HPr + Pyruvate Enzyme II: Phospho HPr + Sugar Mg' 2+ Sugar-phosphate + HPr Net: (Enz I + Enz II): Mg' 2+ Phosphoenolpyruvate + Sugar Sugar-phosphate + Pyruvate HPr Fig. 1.2 The phosphoenolpyruvate phosphotransferase system, Extracellular Membrane Intracellular II P<~ HPr • s r P HPr + PEP •Pyruvate Fig. 1.3 The phosphoenolpyruvate phosphotransferase system. (Roseman, 1969) 6 Kaback (1970b) reported that the carbon source on which the c e l l s were grown — glucose, glycerol or succinate — affected ( i ) the rate and extent to which the sugar phosphate was accumulated, but not the amount of sugar phosphorylated, and ( i i ) the sensitivity of the i n i t i a l rate of uptake to glucose-6-phosphate and to glucose-1-phosphate inhibition. Membrane vesicles prepared from ce l l s in different phases of growth also showed differences in sugar phosphate transport and sensitivity to glucose-6-phosphate i n h i b i -tion. Similarly, Kundig and Roseman (,1971a) indicated that the amount of HPr decreased substantially when the c e l l s reached the stationary phase of growth. Present knowledge of the phosphoenolpyruvate phosphotransferase system may be presented schematically as in Figure 1.3. Elucidation of the systems involved in glycerol uptake and cc-glycerolphosphate transport in E. c o l i has been carried out entirely by Lin and associates (Hayashi et a l . t 1964; Hayashi and Lin, 1965; Zwaig and Lin, 1966; Cozzarelli et a l . , 1968; Sanno et a l . , 1968; Berman and Lin, 1971)• The results of their investigations may be summarized as follows. The d i s -similation of glycerol and L-a-glycerolphosphate by E. c o l i requires the induction of several gene products: (i) a specific protein postulated to med-iate the f a c i l i t a t e d diffusion of glycerol into the c e l l ; ( i i ) glycerol kinase; ( i i i ) L- a-glyerolphosphate dehydrogenase; and, (iv) the L - a -glycerolphosphate transport system. Synthesis of these gene products, coded by genes widely separated on the chromosome, is sensitive to catabolite re-pression and i s negatively controlled by a single regulatory locus glp R, whose product i s neutralized by the inducer L-a-glycerolphosphate. L- a-glycerolphosphate i s accumulated by the cells through the 7 mediation of active transport whereas glycerol uptake occurs via f a c i l i t a t e d -diffusion with the glycerol subsequently trapped through conversion to.L- ot-glycerolphosphate by the ATP-dependent glycerol kinase (Figure 1.4). The act i v i t y of the glycerol kinase is regulated by feedback inhibition by fructose-1,6-diphosphate. This provides an additional means of excluding the u t i l i z a t i o n of glycerol during glucose metabolism. The following properties of acetate uptake by E. c o l i have been re-ported by Wagner et a l . , (1972): (i) acetate uptake by c e l l s grown on acetate was more rapid than by cells grown on glucose; ( i i ) was competitively i n h i b i t -ed by propionate but not by butyrate; and ( i i i ) demonstrated saturation kin-etics. Based on these preliminary results they concluded that a specific system exists for the uptake of acetate by E. c o l i but that i t does not involve active transport as there was no detectable free acetate within the c e l l . By analogy with the mechanism proposed by Klein et a l . , (1971) for fatty acid transport, Wagner et a l . , suggested that the uptake of acetate may occur by vectorial acylation. The failure of formate to sustain osmotic pressure across the c e l l membrane of E. c o l i was interpreted by Bovell et_ a l . , (l963a,b) to indicate penetration of this compound into the c e l l by free diffusion. Measurement of the permeability of E. c o l i to sodium formate by a volume distribution tech-nique supported this interpretation. E. c o l i are capable of growing aerobically using C^-dicarboxylic acids as the sole source of carbon. The penetration of these acids into the intact bacterial c e l l and membrane vesicles has been studied by Kay and Kornberg (1971) and Lo et a l . , (l972a,b), and Rayman et a l . , (l972a,b), respectively. The transport system was induced by C4-dicarboxylic acids and was subject to 8 Extracellular Glycerol L-ot-Glycerol-P Membrane Intracellular f a c i l i t a t e d diffusion active transport Glycerol (-) aerobic d ehyd rogenase --FDP •L-a-Glycerol-P DHAP. :GAP anaerobic dehydrogenase Pig. 1.4 Glycerol and L-a-glycerolphosphate dissimilation i n E. c o l i . (Berman and Lin, 1 9 7 1 ) 9 catabolite repression. Kay and Kornberg failed to observe even a transient-l y raised intracellular concentration of C^-dicarboxylic acid concomitant with their uptake and suggested that the energy required for the trans-location was supplied by the rapid removal of the C^-dicarboxylic acid through oxidative metabolism subsequent to entering the c e l l . However, Lo et a l . , (1972a,b) and Rayman et a l . , (l972a,b) reported succinate accumu-lation against a concentration gradient by intact E. c o l i and by membrane vesicles. The accumulation of succinate by active transport was suggested by: (i) recovery of succinate from the c e l l and membrane vesicles i n an un-altered form, ( i i ) the high temperature coefficient and nonlinearity of the temperature dependence of the transport system, and ( i i i ) the inhibition of succinate transport by inhibitors of respiration and uncouplers of energy conservation (Lo et a l . , 1972a,b; Rayman et a l . , I972a,b). The Kj,, and v m a x values for fumarate and for succinate, competitive uptake studies and studies u t i l i z i n g dicarboxylic acid transport negative mutants, demonstrated that a single transport system functioned in the transport of the C^-dicarboxylic acids. The stereospecificity of the system was not absolute, but there was absolute s p e c i f i c i t y with respect to chain length and the requirement for the presence of two free carboxyl groups. (Kay and Romberg, 1971). Active accumulation of a compound requires energy expenditure. Lo et a l . , (1972b) clearly showed that the PEP phosphotransferase system was not involved in any way i n the succinate uptake process and suggested that the inhibition of succinate transport by inhibitors of respiration and uncoupling agents indicated that the respiratory chain and possibly oxidative phosphor-ylation were required for succinate transport. Additional information on 10 this speculation has been provided by Rayman e_t a l . , (l972a,b) using membrane vesicles derived from a strain of E. c o l i lacking succinate de-hydrogenase and fumarate reductase. They reported that succinate was ac-cumulated in the presence of D-lactate, or ascorbate with phenazine metho-sulfate (ascorbate-PMS), but was not stimulated by the addition of adeno-sine triphosphate (ATP) or adenosine diphosphate (ADP). The characteristics of the transport system in the membrane vesicles were essentially identical with that of the intact c e l l . A notable difference, however, was the ob-servation that compounds known to interfere with phosphorolytic and trans-phosphorylation reactions were without significant effect on succinate trans-port by the membrane vesicles. This result indicated that phosphorylated high energy intermediates were not involved i n energizing succinate trans-port. In general, the characteristics of the D-lactate-linked, or ascorbate-PMS-linked succinate transport system as reported by Rayman et a l . , (1972b) were very similar to those of other D-lactate oxidase coupled transport systems previously described (Kaback, 1972). By analogy to the mechanism proposed by Kaback and Barnes (1971) for ^-galactoside transport via E. c o l i membrane vesicles, Rayman et a l , , (1972b) proposed that succinate uptake was mediated by alternate reduction and oxidation of a succinate carrier i n membranes. The characteristics of D-lactate oxidase coupled transport systems w i l l be discussed i n more detail below. The D-lactate oxidase coupled transport systems of E. c o l i cyto-plasmic membrane vesicles have been reported by Kaback and his colleagues to be responsible for the accumulation of amino acids, y#-galactosides, galac-tose, glucuronic acid, arabinose, glucose-6-phosphate, manganous ions, and potassium ions in the presence of valinomycin, at rates comparable to those 11 of the intact c e l l . Oxidation of DL-ct-hydroxybutyrate and succinate also stimulate the accumulation of these compounds but at a lower rate than ob-served with D-lactate. Although the rate of accumulation i s slower, the transport efficiency (Konings and Freese, 1972) calculated for y3-galacto-sides from the data of Barnes and Kaback (.1971) indicated that the trans-port of y<3-galactosides linked to oxidation of DL- cc-hydroxybutyrate was 2 to 3 times more efficient than that coupled to D-lactate oxidation although the authors suggest that DL-a-hydroxybutyrate i s a substrate for the D-lactate oxidase system. D-lactate and succinate were converted nearly stoichiometrically to pyruvate and fumarate respectively. There appeared to be no requirement for the generation of high energy phosphate compounds but there was an absolute requirement for electron transport. There was no evidence for any chemical transformation of the substrate during concentrative uptake driven by res-piration (Kaback, 1972; Barnes and Kaback, 1970). Recently, Simoni and 2+ Shallenberger (1972) have presented evidence which indicated that the Mg (Ca 2 +)-stimulated adenosine triphosphatase (Mg 2 +(Ca 2 +)-ATPase) was required for the transport of amino acids linked to D-lactate oxidation. The results of reduced-minus-oxidized difference spectra and the level of aerobic steady-state reduction of respiratory chain components of the membrane vesicles with D-lactate, succinate or reduced nicotinamide adenine dinucleotide (NADH) as electron donor was interpreted as indicating that the D-lactate, succinate and NADH oxidases u t i l i z e d the same cytochrome system, and consequently, that the coupling between transport and D-lactate oxidase could not be related either to rates of electron flow to oxygen or to a unique cytochrome system coupled to D-lactate dehydrogenase. The 12 addition of increasing concentrations of NADH or succinate to the trans-port assay system in the presence of a fixed concentration of D-lactate failed to further stimulate transport. On the contrary, i f the succinate dehydrogenase was more active than the D-lactate dehydrogenase the addition of succinate inhibited transport. Since succinate did not inhibit the par-t i a l l y purified D-lactate dehydrogenase nor the D-lactate-dichlorophenol-indophenol (DCIP) reductase, these results, and those obtained from the difference spectra suggested that the site of coupling of transport to D-lactate oxidation must occur prior to the entry of the electrons into the cytochrome system. A comparison of the sensitivity of D-lactate-dependent respiration and D-lactate oxidation-coupled transport to inhibitors of electron trans-port and uncouplers of energy conservation demonstrated ( i ) the same degree of sensitivity to the respiratory chain inhibitors amytal, 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), and sodium cyanide, ( i i ) nearly complete inhibition of D-lactate coupled transport by azide or antimycin with no effect on D-lactate oxidation, and ( i i i ) a marked inhibition of D-lactate oxidase coupled transport by the uncouplers of oxidative phosphorylation, 2,4-dinitrophenol (DNP), carboxyl cyanide m-chlorophenylhydrozone (CCCP) or valinomycin plus potassium ions, with no significant effect on D-lactate oxidation. In addition to the above compounds, N-ethylmaleimide (NEM) and p_-chloromercuribenzoate (PCMB) were found to be potent inhibitors of both the D-lactate oxidase-coupled transport and the D-lactate oxidase system. Barnes and Kaback ( 1 9 7 1 ) concluded that since neither the primary D-lactate dehydrogenase nor the cytochrome system appeared to possess a sensitive 13 sulfhydryl s i t e , the site of inhibition of D-lactate oxidation NEM and PCM must occur between D-lactate dehydrogenase and the cytochromes, and probably this corresponded to the site at which transport was coupled to D-lactate oxidation. The conclusions drawn must be considered with some reservation as other investigators have reported that the NADH oxidase (Bragg-and Hou, 1967a), D-lactate-DCIP reductase and D-lactate oxidase (Bennett et a l . , 1966) a c t i v i t i e s of E. c o l i respiratory particles were significantly i n -hibited by PCMB. The failure of PCMB to inhibit the NADH oxidase of the membrane vesicles may be due to an inaccessibility of the PCMB-sensitive site of NADH oxidase i n membrane vesicles. However, as Bennett et a l . , (1966) observed a similar degree of inhibition of D-lactate oxidase and D-lactate-DCIP-reductase, by PCMB, the i n a b i l i t y of Barnes and Kaback (1971) to detect inhibition of the D-lactate-DCIP reductase as well as inhibition of the oxidase i s hard to explain. Extensive investigation of the temperature-dependent parameters of D-lactate oxidase and ^ S-galactoside transport demonstrated the same activ-ation energy for both processes. Determination of the kinetics of temper-ature induced, respiratory inhibitor-induced and uncoupler-induced efflux of j3 -galactoside, and the effect of sulfhydryl reagents on the kinetics of induced efflux, revealed that anaerobiosis, KCN, and HQNO stopped accumulation and also induced efflux of galactosides previously accumulated by the ves-i c l e s . By contrast, PCMB, and oxamate (which inhibits D-lactate dehydro-genase) were shown to block accumulation without inducing efflux. These observations have prompted Kaback and Barnes (1971) to propose a model for the coupling of D-lactate oxidase to transport, in which the 14 "carriers" of the transport systems, in isolated membrane vesicles from E. c o l i . are electron transfer intermediates between D-lactate dehydrogen-ase and cytochrome b^. These carriers possess sulfhydryl groups which undergo reversible oxidation-reduction. In the oxidized state the carrier has a high a f f i n i t y site for the ligand which binds on the exterior surface of the membrane. Electrons coming ultimately from D-lactate reduce a c r i -t i c a l disulfide in the carrier molecule resulting in a conformational change. Concomitantly the a f f i n i t y of the carrier for i t s ligand is mark-edly decreased and the ligand i s released on the interior surface of the membrane. The reduced form of the carrier i s oxidized by the terminal por-tion of the respiratory chain and the conformation and a f f i n i t y for ligand returns to that of the oxidized state. As indicated by Harold ( 1 9 7 2 ) the model proposed by Kaback and Barnes ( 1 9 7 1 ) i s open to criticism on a number of points. F i r s t , the a b i l i t y of the membrane vesicles to oxidize NADH, synthesize phospholipids from ATP, and demonstrate ATPase acti v i t y , which the intact cells do not, suggest that a proportion of the vesicles are inside-out, are open, or are damaged i n some way. Secondly, the failure of NADH or ATP to support transport may simply be due to an i n a b i l i t y of these compounds to reach their proper site of action at the inner surface of competent vesicles. Thirdly, i t i s very d i f f i c u l t to reconcile the u t i l i z a t i o n of redox intermediates as transport carriers with the a b i l i t y of E. c o l i to grow anaerobically at the expense of glycolysis alone, Finally, a major failure of the Kaback and Barnes model i s i t s i n a b i l i t y to account for the striking inhibition of transport by uncouplers, and by valinomycin in the presence of potassium. The fact that these compounds do not inhibit respiration, but only dissociate i t 15 from transport, implies that cyclic oxidation and reduction of electron carriers i s not by i t s e l f sufficient to drive active transport. In this respect, the proposal by Simoni and Shallenberger (1972) 2+ 2+ that Mg (Ca )-ATPase was required for active transport was of particular interest. However, i t appears that the mutant employed by Simoni and 2+ 2+ Shallenberger, in addition to lacking the Mg (Ca )ATPase, was deficient in the coupling factor(s) required to link energy-dependent systems direct-ly to the respiratory chain (Bragg and Hou, 1973). The possible implications of these recent results w i l l be discussed in relation to energy coupling in E. c o l i (section 1.4). 1.2 Amphibolic pathways Subsequent to gaining entry to the c e l l , a l l potential carbon and/or energy sources, directly or after a limited number of preliminary reactions, enter the amphibolic pathways of E. c o l i . The terra 'amphibolic' was intro-duced by Davis (1961) to designate pathways that f u l f i l l both an anabolic and a catabolic function, and includes glycolysis, glucogenesis, the phos-phogluconate pathway, and the tricarboxylic acid cycle (TCA cycle). Prior to discussing some of the controls of the amphibolic pathways of E. c o l i i t i s necessary to point out that investigations into the func-tion and the control of the phosphogluconate pathway of E. c o l i have been very limited. Present evidence with respect to the metabolic function of the oxidative phosphogluconate pathway indicates that the primary role is probably the generation of reduced nicotinamide adenine dinucleotide phos-phate (NADPH) for reductive biosynthesis, rather than the generation of ribose for nucleic acid synthesis (Caprioli and Rittenberg, 1969; Katz and Rognstad, 1967). As to regulation, the opinion generally held is that the 1 g activity of the oxidative phosphogluconate pathway is regulated by the a v a i l a b i l i t y of nicotinamide adenine dinucleotide phosphate (NADP+) (Model and Rittenberg, 1967? Eagon, 1963). The demonstration by Sanwal (1970c) that the f i r s t enzyme of the oxidative phosphogluconate pathway, glucose-6-phosphate dehydrogenase, was a l l o s t e r i c a l l y inhibited by NADH and activated by spermidine,suggests that regulation of this pathway i s pro-bably more complex than originally thought. Since our knowledge of the oxidative phosphogluconate pathway of E. c o l i i s limited, i t i s of some importance to know the relative u t i l i z a t i o n of the Embden-Meyerhof pathway and the oxidative phosphogluconate pathway for glucose metabolism. Reports by Katz and Wood (i960, 1963) indicated that during logarithmic growth of E. c o l i between 20 to 30$ of the glucose is metabolized through the pentose phosphate pathway. On entering the stationary growth phase this proportion may decrease to 5 to 10$ (Model and Rittenberg, 1967). The present state of our knowledge of the reaction sequences and regulatory mechanisms of the amphibolic pathways of E. c o l i , with the exception of the phosphogluconate pathway, has been summarized in Figure 1.5 and table 1.1. For a more comprehensive discussion of the regulation of the amphibolic pathways of bacteria, and i t s importance to bacterial physiology, the reader is referred to a review by Sanwal (1970a), and for regulation of the glyoxylate and the tricarboxylic acid cycles of E. c o l i in particular, to two art i c l e s by Kornberg (1966, 1970). The controls of carbohydrate metabolism in E. c o l i tend to be d i f -ferent from, and more numerous than, those in eucaryotes. This i s probably due to two characteristics of E. c o l i : ( i ) the lack of r i g i d compartmenta-6-P-Gluconate G-6-P * , Glucose F-6-P FDP Fig. 1.5 Amphibolic pathways of E. coli_. (Tarmy and Kaplan, 1968; Kornberg, 1970; Sanwal, 1970; Cunningham and Hager, 1970) Table 1.1 Enzymes of the Amphibolic Pathways of E. c o l i No. Name _____ 1 PEP phosphotransferase 2 Glucose-6-phosphatase 3 Glucose-6-phosphate dehydrogenase 4 Glucose phosphate isomerase 5 Phosphofructokinase 6 Fructose diphosphatase 7 Aldolase 8 Triosephosphate isomerase 9 L-Glycerol-3-phosphate dehydrogenase 10a L-Glycerol-3-phosphate dehydrogenase 10b L-Glycerol-3-phosphate dehydrogenase 11 Glycerol kinase 12 Glyceraldehydephosphate 13 Phosphoglycerate kinase Nature Cofactor P&S Mg Inducer Inhibitor S S 2+ NADP+, Mg2+,ATP Mg , NAD+, FAD, FAD, Mg2+,ATP sugar,con., Activator con., con., con., hexose-1-P, hexose-6-P Reference 164,165,203, 205,289 NADH, spermidine 277 PEP,ATP ADP,AMP FDP ,ADP ,GDP 5,20 97,99 275 275 L-glycerol-3-P L-glycerol-3-P, R:glucose L -gly c e rol -3 -P» R: glucose L-glycerol-3-P, R:glucose con., con., 189 196,319 188 134,340,341 275 275 co No. Name . Nature Cofactor 25 Succinate dehydrogenase P 26 Fumarate reductase P FMN(?) 27 Fumarate hydratase 28a Malate dehydrogenase S NAD+, 28b Malate oxidase P 5NAD+, 29 Isocitrate lyase S 30 Malate synthase S 31 Pyruvate dehydrogenase S TPP,NAD+, complex li p o i c acid 32 Pyruvate oxidase S TPP,FAD 33 Phosphorclastic reaction 34 Acetyl-CoA synthase 55 Acetyl-CoA hydrolase 2+ 36 Acetate kinase S Mg ,ATP 37 Phosphate acetyl- S transferase 38 D-Lactate dehydrogenase S NAD+f Inducer Inhibitor Activator Reference R:glucose R:oxygen NADH R:PEP PEP, pyruvate R:PEP, pyruvate acetyl-CoA, NADH,GTP 185 146 140,274 69,71 148,198 90,148,198 PEP,AMP,GDP, 19,125,284, pyruvate, 285,290 energy charge phospholipid 73,327 269 NADH,ADP,ATP pyruvate 300 pyruvate, 307 H+ No. Name Nature Cofactor Inducer .Inhibitor Activator Reference 14 Phosphoglyceromutase con., 275 15 Phosphopyruvate hydratase con., 275 16a Pyruvate kinase I S Mg2+,ADP DR:glucose FDP,PEP 224,225 16b Pyruvate kinase II S Mg2+,ADP con., AMP, 225,275 17 PEP synthase s Mg2+,ATP ^Cj-cpd., 64,275 18 PEP carboxylase s Mn 2 +,(Mg 2 +) ^j-cpd., aspartate, malate acetyl-CoA, PEP, 2( 5FDP, CDP,GTP) 47,65, 2(278,279,280) 19 PEP carboxykinase s Mn 2 +,(Mg 2 +), ATP ^C^-acids, R:glucose NADH 150, 336 20a Maiate dehydrogenase (d ecarboxylat ing) s NAD+, malate, R:glucose CoA,ATP aspartate 176,231,276 20b Maiate dehydrogenase (decarboxylating) s NADP+, con., CR:acetate & pyruvate R:glucose NADH,NADPH, OAA,acetyl-CoA, c-AMP NH5 176,231,281, 282 21 Citrate synthase s R:glucose, R:anaerobiosis NADH, ctr-KG, ATP,Mg2+ acetyl-CoA, OAA 117,156,320-323, 335 22 Aconitase hydratase 23a Isocitrate dehydrogenase s Mn2+,NADP+ • ATP,ADP ,GTP ' 2(226),275 23b Isocitrate dehydrogenase s Mn2+,NADP+ 2(226),275 24 O-Oxoglutarate P NAD+, a-KG, ro 1,140 3 dehydrogenase No. Name Nature Cofactor Inducer Inhibitor Activator Reference 39 D-Lactate oxidase P (flavin) con., 14,131,191 40 L-Lactate oxidase P (flavin) lactate 14,131,191 41 Pyruvate decarboxylase 42 Alcohol dehydrogenase C^-cpd,: lactate, pyruvate or alanine Salmonella typhimurium Cooperative activation by acetyl Co A and FDP, CDP or GTP C^-acids: malate or succinate Probably the fluorescence assays measured menaquinone (Newton et a l . , 1971) Symbols; P - particulate S - soluble con. - constitutive R - repressed by DR - derepressed by CR - concerted repression by 22 tional controls of the type provided to eucaryotic c e l l s by the presence of mitochondria, ( i i ) the fact that E. c o l i functions largely by anaerobic glycolysis even when grown aerobically on glucose. Control of the amphibolic pathways of E. c o l i i s effected at two levels: (i) induction and repression of enzyme synthesis, and ( i i ) activation and inhibition of enzyme activity. The importance of regulation of enzyme synthesis in the control of the amphibolic pathways of E. c o l i i s readily apparent in the response of enzyme levels to growth conditions, particularly oxygen tension and carbon source. The level of the enzymes of the fermentative pathway of E. c o l i are not significantly influenced by the presence or absence of glucose or oxygen during growth, with the exception of one of the two pyruvate kinases which is derepressed during growth on glucose (Takahashi and Hino, 1968a; Sanwal, 1970a). However, this i s definitely not the case with the enzymes of the tricarboxylic acid cycle. The lowest levels of tricarboxylic acid cycle enzymes are found in anaerobically grown cells (Gray et a l . , 1966a; Takahashi and Hino, 1968a). Amarasingham and Davis (19^5) reported that a-oxoglutar-ate dehydrogenase is absent from anaerobically grown E. c o l i , and detectable in c e l l s grown aerobically on glucose or lactate only after substantial ac-cumulation of metabolites has occurred. These results prompted Amarasingham and Davis to propose that although the tricarboxylic acid pathway functions in a cyclic manner aerobic conditions, under conditions of anaerobic growth i t i s probably modified to a branched noncyclic pathway. An oxidative branch leads from citrate to a-oxoglutarate, serving a purely biosynthetic role. A reductive branch leads from oxaloacetate and malate to succinate via fumarate reductase, serving a biosynthetic role and providing, concurrently, a means for anaerobic electron transport (Hirsch et_ a l . , 1963). These 23 proposals have subsequently received support from investigations by Gray et a l . , (1966a) and Takahashi and Hino (1968a). The repression of TCA cycle enzymes during aerobic growth on glucose is generally considered to be due to the a v a i l a b i l i t y of energy from glycolysis with the result that the TCA cycle functions to generate intermediates for biosynthesis only (Gray et_ a l . , 1966a; Sanwal, 1970a). The elevation of the tricarboxylic acid cycle enzymes during growth on intermediates of the tricarboxylic acid cycle i s considered to be due to the simultaneous generation of energy and production of biosynthetic pre-cursors by this pathway. In addition to the control of the tricarboxylic acid cycle by repression and depression of enzyme synthesis as indicated above, a number of enzymes associated with the tricarboxylic acid cycle are induced in res-ponse to growth on specific carbon sources. These are the enzymes of the anaplerotic pathways of E. c o l i . It i s apparent from Figure 1.1 that catabolic routes which do not give rise d i r e c t l y to intermediates of the tricarboxylic acid cycle enter the cycle as acetyl CoA. Each turn of the cycle results in the complete oxidation of one C2~unit to two units of CO2 and the continued operation of the cycle at an undiminished rate requires that the C^-dicarboxylic acid that i n i t i a t e s each turn i s regenerated in the course of the turn. However, intermediates of the cycle are continually withdrawn from i t for biosynthetic purposes. Consequently, routes ancillary to the cycle must operate to ensure that the intermediates of the cycle are replenished. These are the anaplerotic pathways. Elucidation of the anaplerotic reactions of E. c o l i has been largely due to Romberg and co-workers (Kornberg, 1970). Their results and those of 24 others (References - Table 1.1) may be summarized as follows. Growth on carbon sources which are catabolized via acetyl CoA requires the replen-ishment of the C^-dicarboxylic acid, oxaloacetate, for condensation with acetyl-CoA. This necessitates the induction of different enzymes depend-ent upon the carbon source used. Growth on glucose or glycerol induces the synthesis of phosphoenolpyruvate carboxylase (PEP carboxylase),- while u t i l -ization of pyruvate, lactate or alanine as carbon source results in the induction of phosphoenolpyruvate synthase (PEP synthase) as well as phos-phoenolpyruvate carboxylase. Growth on acetate, on the other hand, requires the induction of the glyoxylate bypass enzymes, isocitrate lyase and malate synthase with the subsequent oxidation of malate via malate dehydrogenase, to oxaloacetate. In addition to the induction of isocitrate lyase and malate synthase, Holms and Bennett ( 1 9 7 1 ) reported that growth on acetate induced the synthesis of a protein which inhibited isocitrate dehydrogenase thus reducing the proportion of isocitrate metabolized by the tricarboxylic acid cycle. Just as oxidation of acetyl-CoA via the tricarboxylic acid cycle requires an adequate supply of oxaloacetate to maintain i n i t i a t i o n of the cycle, oxidation of TCA cycle intermediates requires an adequate means of generating acetyl-CoA, for the same purpose. This is provided by the induction, by C^-dicarboxylic acids, of the enzymes phosphoenolpyruvate carboxykinase and NAD +-specific malic enzyme which catalyze the formation of phosphoenolpyruvate from oxaloacetate, and pyruvate from malate, respec-tivel y . Both phosphoenolpyruvate and pyruvate are readily converted to acetyl-CoA. Whereas the a b i l i t y of a c e l l to synthesize new protein in response 25 to alterations in i t s environment appears to be inversely related to the a b i l i t y of the organism to control i t s environment, the number and complexity of the a l l o s t e r i c controls of enzyme ac t i v i t y as indicated previously, appear to be related to the degree of compartmentation within the c e l l . Thus, although E. c o l i possesses controls identical to those of eucaryotes such as yeast; (i) end product inhibition, ( i i ) precursor activation, and ( i i i ) energy-linked controls, the1 number and complexity of the controls (Table 1.1) particularly of the anaplerotic reactions i s far greater in E. c o l i . In addition to the preceding controls, a fourth type, at the level of enzyme activity, exists in E. c o l i . This i s a l l o s t e r i c regulation by reduced pyridine nucleotides. Thus, while the tricarboxylic acid cycle of eucaryotes is regulated by ATP, or energy charge (Atkinson, 1965), NADH may be the prime control signal for the regulation of the tricarboxylic acid cycle in E. c o l i . Two crucial enzymes of the energy generating path-way, citrate synthase (Weitzman, 1966) and malic dehydrogenase (Sanwal, 1969) are a l l o s t e r i c a l l y inhibited by NADH. Weitzman (1966) and Kornberg (1970) consider NADH as an end product of the tricarboxylic acid cycle and propose that the action of NADH as an allo s t e r i c affector may be thought of as a type of end product inhibition. Sanwal (1970a) states that this is pro-bably not the correct interpretation for the following reasons: ( i ) NADH is generated only at a-oxoglutarate dehydrogenase, and ( i i ) NADH alloster-i c a l l y inhibits several enzymes which have no connection whatsoever with the tricarboxylic acid cycle. It is necessary to question the v a l i d i t y of the i n i t i a l point. If the TCA cycle i s functioning in a cyclic manner, then NADH would be generated at both (X-oxoglutarate and malate dehydrogenases, 26 as both are NAD-requiring enzymes. If during aerobic growth on glucose, the TCA cycle functions as a branched biosynthetic pathway with cx-oxo-glutarate dehydrogenase levels low or absent, u n t i l metabolites have accumulated (Amarasingham and Davis, 1965), one would expect i n i t i a l l y no generation of NADH at CC-oxoglutarate dehydrogenase. Subsequently, with the accumulation of metabolites, there would be generation of NADH by cx-oxoglutarate dehydrogenase and possibly also by malate dehydrogenase, depending on the ac t i v i t y of the cx-oxoglutarate dehydrogenase and the demand for succinate. Consequently, the i n i t i a l argument is valid only under restricted conditions, unless the oxidation of malate to oxaloacetate occurs primarily v ia an NAD^independent malate oxidase (Cox et a l . , 1968b; Newton et a l . , 1971). This does not detract from Sanwal's proposal (Sanwal, 1970a) that the regulation of amphibolic pathways in E. c o l i . by NADH, i s related to the predominant u t i l i z a t i o n of gly c o l y t i c a l l y generated energy by this :organism even during aerobic growth on glucose. E. c o l i possesses an NAD+/NADH ratio of 0.5 when grown aerobically on glucose as compared to a ratio of 1.0 during aerobic growth on succinate. As a result, NADH inhibits citrate synthase and malate dehydrogenase to minimize the production of NADH by CX-oxoglutarate and malate dehydrogenase under conditions i n which the NADH level exceeds the capacity of the repressed electron transport chain. Other enzymes which have been reported to be sensitive to al l o s t e r i c inhibition by NADH include, NADP +-specific malic enzyme (Murai et a l . , 1972; Sanwal and Smando, 1969a,b; Katsuki et a l . , 1967), glucose-6-phosphate dehydrogenase (Sanwal, 1970c), pyruvate dehydrogenase (Hanson and Henning, 1966; Shen and Atkinson, 1970), and phosphotransacetylase (Suzuki et a l . , 27 1969). On the basis of this information the following statements can be made. F i r s t , NADH directly or indirectly regulates a l l NADPH generating enzymes in E. c o l i with the possible exception of the pyridine nucleotide transhydrogenase. Secondly, by regulating the enzymes indicated above, NADH indirectly regulates the a v a i l a b i l i t y of a l l biosynthetic precursors with the exception of those derived from glycolytic intermediates. The levels of NADPH, aspartate and glutamate may in turn influence the form-ation of biosynthetic precursors from glycolytic intermediates. Should this be the case, in E. c o l i , NADH could be a primary coordinator of cel l u l a r function in addition to, or replacing, ATP. Another control mechanism which functions in the amphibolic pathways of E. c o l i i s the existence of multiple enzymes catalyzing the same reaction. This mechanism is u t i l i z e d when a reaction serves more than one purpose i n the c e l l , and particularly when al l o s t e r i c regulation with respect to one of these purposes would be detrimental to the remaining functions. The mech-anism involves the u t i l i z a t i o n of induction, derepression or repression of enzyme synthesis in response to growth conditions. This i s usually combined with d i f f e r e n t i a l a l l o s t e r i c regulation of the enzymes. The enzymes may possess quite distinct kinetic parameters such as K m, Vmaxt and Kg(^. The most thoroughly investigated examples of multiple enzymes in the amphibolic pathways of E. c o l i are the NADispecific and NADpispecific malic enzymes (Sanwal, 1970b; Murai et a l . , 1972; Sanwal and Smando, 1969a,b; Katsuki et a l . , 1967), pyruvate kinases (Maeba and Sanwal, 1968; Malcovati and Romberg, 1969; Sanwal, 1970a), and the particulate and soluble, FAD-linked, L - a -glycerolphosphate dehydrogenases (Roch et a l . , 1964; Werner and Heppel, 1972; K i s t l e r et a l . , 1969) and the NADispecific, L-OC-glycerophosphate 28 dehydrogenase (Kito and Pizer, 1969). In summary, the a b i l i t y of E. c o l i to adapt to i t s environment is reflected in the diversity of controls possessed by this organism. 1.3 Respiratory chain The respiratory chain of E. c o l i manifests the complexity one would expect of an organism possessing the diversity of metabolism indicated above, combined with the a b i l i t y to respire aerobically or anaerobically. Anaero-b i c a l l y , E. c o l i can u t i l i z e n i t r i t e , nitrate or fumarate in place of oxygen as electron acceptor. Electrons can enter the respiratory chain through various dehydrogenases such a3 formate dehydrogenase (Asnis et a l . , 1956; Taniguchi and Itagaki, 1960; Linnane and Wrigley, 1963; Gray et a l . , 1966b; B i r d s e l l and Cota-Robles, 1970), L-a-glycerolphosphate dehydrogenase (Asnis et a l . , 1956; Koch et a l . , 1964; Weiner and Heppel, 1972), D- and L-lactate dehydrogenases (Asnis et a l . , 1956; Haugaard, 1959; Kline and Mahler, 1965; Kidwai and Murti, 1965; Bennett et a l . , 1966; Gutman et a l . , 1968) , succinate dehydrogenase (Asnis et a l , , 1956; Kashket and Brodie, 1963a,b; Kidwai and Murti, 1965; Gray et a l . , 1966b;Gutman et a l . , 1968; Hendler et a l . , 1969? B i r d s e l l and Cota-Robles, 1970; Kim and Bragg, 1971a; Hendler, 1971), malate dehydrogenase (Cox et a l . , 1968b;Gutman et a l . , 1968; Cox et a l . , 1970; Newton et a l . , 1971) and reduced nicotinamide adenine dinucleotide dehydro-genase (Asnis et_ a l . , 1956; Kashket and Brodie, 1960; 1963a,b; Bragg, 1965; Bragg and Hou, 1967a,b,c; Gutman et a l . , 1968; Hendler et a l . , 1969; Birdsell and Cota-Robles, 1970; Hendler, 1971). These enzymes are bound with varying degrees of firmness to the whole enzyme complex of the respir-atory chain. Consequently, the f i r s t stage in the oxidation of substrates i s not necessarily their dehydrogenation by cytoplasmic enzymes to produce 29 NADH. The process may begin with f l a v i n enzymes which transfer an electron directly to the respiratory chain. Information on the membrane bound dehydrogenases is fragmentary due to the d i f f i c u l t i e s associated with the solubilization and purification of membrane bound enzymes. Linnane and Wrigley (1963) extracted a formate dehydrogenase -cytochrome b_-| complex from a particulate fraction of E. c o l i . The formate dehydrogenase was characterized as being a flavoprotein requiring sulfhydryl groups for activity. The nature of the flav i n component was unknown. The possib i l i t y of metal ion involvement was suggested by the inhibition of the dehydrogenase activity of cyanide. Glycerol-3-phosphate dehydrogenase was solubilized from an E. c o l i membrane fraction by Weiner and Heppel (1972). The results of polyacryla-mide gel-electrophoresis in the presence of sodium dodecylsulphate (SDS) indicated that the enzyme was composed of two subunits. The prosthetic group was noncovalently bound FAD. Although the membrane-bound,D-lactate dehydrogenase of E. c o l i has been purified approximately 250-300 fold (Barnes and Kaback, 1971; Kaback, 1972), there has been no report of the purification of the L-lactate dehydrogenase, or of the characteristics of the p a r t i a l l y purified D-lactate dehydrogenase. Results of inhibitor studies reported by Bennett et a l . , (1966) were inconclusive as to the characteristics of these enzymes, although they did suggest that the D-lactate dehydrogenase was a flavo-protein requiring sulfhydryl groups for act i v i t y . Solubilization of the succinate dehydrogenase of E. c o l i has been achieved by Kim and Bragg (1971a). The prosthetic group was not determined 30 but i f i t i s f l a v i n i t must be tightly bound as is the case with the mito-chondrial enzyme. The fact that the membrane bound enzyme was inhibited by PCMB suggested that sulfhydryl groups are probably required for a c t i v i t y . Three distinct NADH dehydrogenase containing systems, designated menadione reductase I, menadione reductase II, and NADH oxidase, have been solubilized from the small particle fraction of the E. c o l i membrane by treatment with deoxycholate and ammonium sulfate (Bragg and Hou, 1967b). Mendione reductase I and II u t i l i z e d both NADH and NADPH as electron donors and required the presence of menadione in order to u t i l i z e oxygen as an electron acceptor. Additional data suggest that the NAD(P)H dehydrogenase of menadione reductase I i s a metalloflavoprotein. Results obtained with menadione reductase II do not permit deductions as to the nature of the prosthetic group of the NAD(P,)H dehydrogenase component. However, the inhibitor data indicate that the dehydrogenase probably does not possess any metal ions essential for a c t i v i t y . The dehydrogenase component of the solubilized NADH oxidase appears to be a metalloflavoprotein. Gutman et a l . , (1968) have investigated two NADH dehydrogenases of E. c o l i , (i) a soluble NADH diaphorase and ( i i ) an NADH dehydrogenase extracted from lyophilized small particles of E. c o l i with d i s t i l l e d water. Very l i t t l e information was reported on the properties of the NADH diaphor-ase but the characteristics reported suggest that i t might correspond to the menadione reductase I of Bragg and Hou (1967b). The NADH dehydrogenase extracted from the membrane fraction was characterized, without further purification, as a metalloflavoprotein containing FMN, PAD, nonheme iron and l a b i l e sulfide. These characteristics are similar to those reported by Bragg and Hou (1967b) for menadione reductase II, suggesting that these may 31 be the same enzyme. Gutman et a l . , . (1968), due to the similarity of the properties of the solubilized NADH dehydrogenase and the membrane bound dehydrogenase of the NADH oxidase system, considered that they had solu-bi l i z e d the NADH dehydrogenase of the NADH oxidase system. However, their data indicated that extraction of 30 to 50$ of the NADH dehydrogenase of the lyophilized' membrane particles did not diminish the NADH oxidase activity of the extracted particles. This indicated either ( i ) that the NADH dehydro-genase extracted was not the dehydrogenase component of the NADH oxidase system, or ( i i ) the function of the NADH oxidase was rate limited at some site between the dehydrogenase and oxygen such that extraction of 30 to 50$ • of the dehydrogenase did not affect the rate of the system. As a result one cannot say with any certainty at this time whether the major NADH dehydro-genase component of the E. c o l i respiratory chain has been solubilized or what i t s characteristics are. The cytochromes of E. c o l i have received more extensive investiga-tion than the dehydrogenases associated with the electron transport chain, the f i r s t report appearing i n 1934 (Keilin, 1934). K e i l i n demonstrated the presence in E. c o l i of cytochromes a^, a^, and b^ but did not detect cyto-chrome _. The presence of cytochromes a-j, » 2-L-j i - n c°li w a s confirm-ed by Yamagutchi (1937) who also detected low levels of cytochrome _. This was disputed by K e i l i n and Harpley who were unable to duplicate these re-sults (Keilin and Harpley, 1941). The presence of cytochrome a^, EU, and b-j was accepted without question. Subsequently Castor and Chance (1959) demonstrated the existence of a second b-type cytochrome in E. c o l i by the photochemical action spectra of cel l s in which respiration had been i n h i b i t -ed by carbon monoxide. This cytochrome, designated cytochrome p_, was shown 32 to function as the terminal oxidase in logarithmic phase ce l l s and shared this function with cytochrome in the stationary phase. It was not u n t i l the work of Wimpenny and coworkers (Gray et a l . , 1963; Wimpenny et a l . , 1963) that the discrepancy of the presence or absence of cytochrome £ was c l a r i f i e d . Gray et a l . , (19^3) discovered that anaero-bic growth of E. c o l i , and related facultative anaerobes,.on minimal salts medium resulted in the synthesis of a soluble c-type cytochrome, subsequently designated cytochrome £-552 (Fujita and Sato, 19^3)• Wimpenny et a l . , (19^3) demonstrated that the synthesis of cytochrome £-552 was repressed by the presence of oxygen. Fujita and colleagues (Fujita, 1966a; Fujita and Sato, 1966a,b, 1967) and Cole (1968) have shown that the synthesis of cytochrome £-552 i s stimulated under anaerobic conditions by the presence of n i t r i t e in the medium and appears concurrently with the development of n i t r i t e re-ductase act i v i t y . However, the exact relationship of cytochrome £-552 to n i t r i t e reductase remains unclear. Fu j i t a and Sato (1963) also reported the existence in anaerobically grown E. c o l i of a second soluble £-type cytochrome, cytochrome £-550, and in anaerobically or aerobically grown E. c o l i , of a soluble b-type cytochrome, cytochrome b-562. Both of these cytochromes have been purified and charac-terized but their function i s unknown (Fujita, 1966b; Itagaki and Hager, 1965; Hager and Itagaki, 1967). Of the membrane bound cytochromes a^, , b-j and £, only cytochrome b-| has been isolated, purified and characterized (Fujita et a l . , 1963; Deeb and Hager, 1964). Generally, i t has been considered that there was only one cytochrome b.| present. However, the results of B a i l l i e et a l . , (1971) and Kim and Bragg (1971) suggest that there are probably two functionally, 33 i f not chemically, distinct cytochrome b^'s in E. c o l i cells grown aerohical-' l y on glucose or succinate. De Moss and coworkers have reported the pres-sence of two distinct cytochrome h^'s in anaerobically grow E. c o l i (Ruiz-Herrera et a l . , 1969). Anaerobic growth in the presence of nitrate resulted in the synthesis of a b-type cytochrome with an alpha band peak at 555 nm (77°K) while anaerobic growth in the absence of nitrate resulted in a cyto-chrome b with an alpha band at 558 nm (77°K). Subsequently, Ruiz-Herrera and De Moss (1969) reported that the cytochrome b-555 (77°K), of nitrate induced E. c o l i consisted of two species differentiable on a kinetic basis. These researchers also reported that the nitrate-specific cytochrome b-555 (77°K) components were distinct from the cytochrome b-555 (77°K) components produced under aerobic conditions. Thus, assuming that the measurement of reduced-minus-oxidized d i f -ference spectra at 77°K results i n a two to three nanometer sh i f t of the absorption maximum to lower wavelength compared to those recorded at room temperature (Shipp, 1972a), i t appeared, u n t i l recently, that the cytochrome complement of E. c o l i at room temperature) consisted of: cytochromes a-] (590), 8^(630), b-557, b-562 and c_(557) under aerobic growth conditions. Under anaerobic growth conditions there were low levels of cytochromes a^ (590), _2(630)» o(557), b-562 and £-550 and depending on the terminal electron acceptor, substantial amounts of cytochrome b-560, or of two cytochrome b-557's, and cytochrome c-552. Recently, however, Shipp (1972a) has applied a fourth order f i n i t e difference analysis technique (Butler and Hopkins, 1970a,b) to low temperature reduced-minus-oxidized difference spectra of aerobically grown E. c o l i . whole cells and respiratory particles. His results indicated that the respiratory chain of E. c o l i may be considerably 34 more complex than anticipated. Shipp proposes that the alpha band pre-viously attributed to cytochrome b^ is a composite of the absorption bands of five or more pigments, cytochromes £-548, £-553, b_-556, b-559 and b-565 (Shipp, 1972b). Associated with the electron transport chain of E. c o l i i s a second type of iron-containing respiration carrier, the iron-sulfur proteins or nonheme iron proteins. Very l i t t l e i s known about the function of respir-atory chain-linked nonheme iron i n E. c o l i . Investigations into this problem have largely been indirect, via the effect of metal chelators on (i) respiration rate; ( i i ) dehydrogenase act i v i t y and ( i i i ) the steady state . reduction of respiratory pigments. Direct measurement of nonheme iron by electron paramagnetic resonance (EPR) has been employed by Hamilton et_ a l . , (1970) and Hendler (1971). Inhibition of NADH oxidase and succinate oxidase by the metal chelators salicylaldoxime, 8-hydroxyquinoline, 2,2'-dipyridyl, thenoyl-trifluoroacetone (TTFA), and £-phenanthroline has been demonstrated (Bragg, in press). Evidence for the involvement of nonheme iron i n the NADH dehydro-genase segment of the respiratory chain has been indicated by Bragg and Hou (1967a) and Gutman et_ a l . , (1968). Bragg and Hou (1967a) reported that 2,2•-dipyridyl inhibited NADH oxidation by E. c o l i respiratory particles and that this inhibition was competitive with the substrate. These results sug-gested that a metal ion, possibly nonheme iron, was close to the NADH binding si t e . Subsequently, Gutman et a l . , (1968) demonstrated that presence of non-heme iron in a solubilized NADH-ferricyanide reductase complex. However, the redox potential of this nonheme iron, which was reducible by ascorbate, must have been considerably more positive than would be expected for nonheme iron 35 associated with the NADH dehydrogenase substrate binding s i t e . Consequently i t must be located closer to oxygen. Although nonheme iron appears to be involved with NADH dehydrogenase, Kim and Bragg (1971a) reported that suc-cinate-PMS reductase was not affected by levels of _-phenanthroline and thenoyltrifluoroacetone sufficient to inhibit the oxidase. It would appear that nonheme iron i s not involved in the succinate diaphorase activity. Further investigations w i l l be needed to establish whether this i s also true for the succinate dehydrogenase associated with the electron transport chain. Bragg (1970) and Kim and Bragg (1971h) have reported the existence, in _. c o l i respiratory particles, of a species of nonheme iron characterized by i t s rate of reaction with c_-phenanthroline (reaction complete within one minute). This nonheme iron constituted approximately 20$ of the total non-heme iron when the reducing equivalents were provided by NADH, ascorbate-PMS or dithionite (Bragg, 1970) but only about 7$ of the total nonheme iron with succinate as electron donor (Kim and Bragg, 1971b). Investigating the loca-tion of the NADH-reducible nonheme iron, Bragg (1970) reported that the reduction of the nonheme iron by NADH was inhibited by HQNO and that although the rate of reduction was decreased, complete reduction of cytochrome b^ could occur without reduction of nonheme iron. The conclusions drawn were: (i) that the NADH-reducible nonheme iron was located between ubiquinone and oxygen. However, since the nonheme iron was f u l l y reducible by ascorbate-PMS and cytochrome b^ was not, the site may be between cytochrome b-j and oxygen. ( i i ) the NADH-reducible nonheme iron probably was not on the main respiratory pathway. Kim and Bragg (1971b) investigated the relationship of nonheme iron to the succinate oxidase system in a membrane fraction from E. c o l i . They reported that succinate was capable of reducing 55 to 70$ of the cytochrome 36 and approximately 35$ of the c_-phenanthroline reacting nonheme iron reducible by either NADH or dithionite. However, in the presence of HQNO both cytochrome b^ and the succinate-reducible nonheme iron reached anaero-bic steady-state values which were equivalent to those given by NADH or dithionite. These results indicated that whereas HQNO inhibited the reduc-tion of nonheme iron and cytochrome b_^  associated with the NADH oxidase system, i t inhibits the oxidation of nonheme iron and cytochrome b-j associ-ated with succinate oxidase. It was not possible, on the basis of the data available, to ascertain the position of nonheme iron i n the succinate oxi-dase system. Hendler (1971) reported on the effect of TTPA on the generation of the EPR signal (g=1.94) associated with reduced nonheme iron. Thenoyl-trifluoroacetone inhibited both the reduction by NADH of the nonheme iron species responsible for the electron paramagnetic resonance signal, and cytochrome b^, but did not prevent the reoxidation of these respiratory chain, components. Prom these results Hendler concluded that the nonheme iron re-ducible by NADH occurred between the NADH dehydrogenase and cytochrome b_^ . Although the results available indicate that nonheme iron may be linked to, or part of the respiratory chain, i t i s impossible to designate the role of nonheme iron in relation to the electron transport chain with any degree of certainty. The function of the quinones of the cytoplasmic membrane, have been, and remain, one of the most baffling aspects of the respiratory chain of E. c o l i . The f i r s t information on the lipoquinones of E. c o l i appeared in 1959 when Lester and Crane reported that aerobically grown E. c o l i possessed a benzoquinone, ubiquinone-8, and a naphthoquinone, while the same organism 37 grown anaerobically contained only the naphthoquinone. Page et a_., (i960) confirmed the identity of the major benzoquinone of _. c o l i as ubiquinone-8. Subsequently, Bishop et a l . , (1962) identified the principal naphthoquinone as vitamin K 2(40). More recently, low levels of demethyl vitamin 1(2(40) (Dolin and Baum, 1965) and the ubiquinone-8 isoprenologues, ubiquinone-5, -6 and -7 have been detected in E. c o l i (Priis et a l . , 1966). • Commencing with the i n i t i a l report by Lester and Crane (1959) there was considerable interest in the influence of growth conditions, particularly aeration rate, on the relative abundance of ubiquinone and menaquinone. Bishop et a l . , (1962) reported that they found no significant difference in the ubiquinone-8 and vitamin K 2(40) levels of aerobically and anaerobically grown E. c o l i . However, subsequent reports by Polglase et a l . , (1966), Y/histance and Threl f a l l (1968) and Whistance et a l . , (1969) supported the results of Lester and Crane (1959), that aerobic conditions favored the form-ation of ubiquinone-8 while anaerobic conditions favored the formation of vitamin 1^(40). The f i r s t indication that lipoquinones might be important in the respiratory chain and energy metabolism of E. c o l i was a report by Kashket and Brodie (i960) that ubiquinone stimulated respiration with NAD^linked substrates, whereas naphthoquinones stimulated both oxidation and phosphor-ylation when added to a near-ultraviolet irradiated system from E. c o l i . Results from the same laboratory (Kashket and Brodie, 1962) sub-sequently demonstrated that cultures of E. c o l i . irradiated with light of 360 nm wavelength, grew aerobically on fermentable carbon sources, with a reduced c e l l y i e l d , and, although retaining the a b i l i t y to oxidize carbon sources such as succinate or malate, were unable to grow on them. These 38 results were interpreted as indicating a role for lipoquinones in the coupling of energy production by the respiratory chain. To further elucidate the possible involvement of the quinones in oxidative phosphylation, Kashket and Brodie (1963a,b) fractionated sonicated E. c o l i , by dif f e r e n t i a l centrifugation, into large membrane particles, small particles and supernate. Results from these systems led them to con-clude that the naphthoquinone, vitamin K'^iAO), functioned in the respiratory chain for NADH oxidation at a position between flavoprotein and cytochrome b^, while ubiquinone-8 was involved in the succinate oxidase pathway in an analogous position. NADH and succinate oxidases were considered to share a common electron transport chain from cytochrome b^ to oxygen (Figure 1.6A). The techniques of destruction of quinones by uv irradiation, such as used by Kashket and Brodie (i960, 1962, 1963a,b), or extraction of quin-ones with l i p i d solvents are subject to a number of criticisms. Brodie (1963) states that benzoquinones are much more slowly destroyed by light at 360 nm than menaquinones. Also, Bragg (197"0 has shown that uv irradiation of E. c o l i respiratory particles results in a marked destruction of cyto-chrome a^. The extraction of membranes with l i p i d solvents must result in damage to the membrane. Moreover, the subsequent addition of quinones i n attempts to restore biological a c t i v i t y i s subject to the point raised by Wagner and Folkers (1963), that i s , are the effects of the compound when add-ed to the system a true effect of that compound because i t i s a component of the system; or an effect because i t i s structurally analogous to a true component of the system? The isolation of mutants unable to synthesize ubiquinone (ubi"), or vitamin K2(men") has provided a technique for investigating the location 39 and function of quinones with respect to the respiratory chain of E. c o l i . This technique is superior to either destruction of quinones by uv i r r a d i -ation or their removal by solvent extraction. Jones (1967) was the f i r s t to exploit this technique, u t i l i z i n g E. c o l i W and a mutant of this strain, isolated by Davis (1952) which was auxotrophic for 4-hydroxybenzoic acid. In the absence of 4-hydroxybenzoate the mutant was unable to synthesize ubiquinone-8, possessed a low respiration rate, and low NADH oxidase and NADH-cytochrome b^ reductase a c t i v i t i e s . Preincubation of respiratory particles from the mutant with ubiquinone-6 largely restored NADH oxidase and NADH-cytochrome b^ reductase a c t i v i t i e s . The succinate oxidase activ-i t y of the mutant respiratory particles was stimulated only about 50$ as compared with a 20 to 50 fold increase i n the NADH oxidase activity. Neither NADH dehydrogenase nor succinate dehydrogenase a c t i v i t i e s were affected by the absence of ubiquinone. These results strongly implicated ubiquinone in the NADH oxidase complex of the auxotroph and located the sit e of ubiquinone between NADH dehydrogenase and cytochrome b^. The results with the succinate oxidase were equivocal. The applicability of these results to the wild type E. c o l i W is uncertain. The NADH oxidase ac t i v i t y of the auxotroph grown with 4-hydroxybenzoate supplementation was destroyed by uv irradiation and subsequently activity was pa r t i a l l y restored by ubiquinone-6. There was no restoration of activity with vitamin K2 isoprenologues. A similar prepara-tion from E. c o l i W was not stimulated by ubiquinone-6 but activity was parti a l l y restored by preincubation with vitamin K2O0), i n agreement with Kashket and Brodie (1963b). The discrepancy between the results with the auxotroph and E. c o l i V/ may be related to the fact that the auxotroph even in the presence of 4-hydroxybenzoate was unable to synthesize normal levels 40 of vitamin K 2 > An extensive investigation of the role of quinones in E. c o l i respir-ation and growth had been carried out by Gibson and coworkers u t i l i z i n g ubi" and men- mutants of E. c o l i K 1 2 . The ubi" and men" mutants grew aerobically on glucose. The u b i " mutants had a growth yield similar to that for anaero-bic growth (Cox e_ a l . , 1 9 7 0 ) , but neither mutant was able to grow on malate or succinate. However, a revertant of the menr mutant was able to grow on malate, succinate and lactate although s t i l l unable to synthesize vitamin K 2 (Cox et a l . , 1 9 6 8 a ) . These results demonstrated that vitamin K2 was not required for oxidation of, or growth on, malate, lactate or succinate and suggested that ubiquinone-8 was essential for electron transport or phosphor-ylation coupled to transport. Subsequently, Cox and Snoswell (1968) and Cox et a l . , ( 1 9 6 8 b ) have shown that ubiquinone was involved in lactate and NADH oxidation, and approximately 5 0 $ of the malate oxidation, but was not involved in the oxidation of L- ct-glycerolphosphate or dihydroorotate. Having demonstrated the involvement of ubiquinone in the respiratory chain, Cox et a l . , ( 1 9 7 0 ) investigated the steady-state reduction levels of f l a v i n , cytochrome b-j and cytochrome a^ of normal and ubi" strains of E. c o l i K 1 2 . The effect of inhibitors on the steady-state reduction levels of these respiratory components of the normal strains was examined also in an attempt to elucidate the functional location of ubiquinone in the electron transport chain. The levels of flavin and cytochrome a^ in the ubi" mutant were sl i g h t -l y higher and the concentration of cytochrome £ s l i g h t l y lower, than in the wild type revertant. Cytochrome b^ levels were identical. The steady-state reduction levels of f l a v i n and cytochrome b-) i n respiratory particles of the 41 ubi~ mutant, and the normal strain inhibited with p i e r i c i d i n A, were i n -creased in both the NADH and lactate oxidase systems. It was also observed that the addition of NaCN to the respiratory particles of the normal strain, at a concentration which caused less inhibition of the NADH oxidase than ubiquinone deficiency, caused a greater increase in the level of steady-state reduction of cytochrome b^. Cox et a l . , (1970) interpreted these results to indicate that ubi-quinone functions at two sites in the NADH oxidase portion of the respir-atory chain. One site was placed before cytochrome bj and one site between cytochrome b^ and oxygen. The results obtained with p i e r i c i d i n A also were consistent with inhibition at two sites, one before and one after cytochrome b^. Ubiquinone appeared to function only after cytochrome b-j in the lactate oxidase system since the steady-state reduction of cytochrome bj in membranes from the ubi~ strain was not markedly different from that in membranes from the normal strain in the presence of cyanide. Subsequently, an investigation of the ubiquinone-8 present in the membranes of the normal strain indicated that approximately 50$ of this quinone (concentration 25 times that of cytochrome b^) was in the reduced form in the absence of added substrates whereas the other electron carriers were essentially f u l l y oxidized. Cox et a l . , (1970) pointed out that i f the high percentage of reduced ubiquinone was due to disproportionation of the semiquinone on extraction into the organic solvent, essentially a l l of the ubiquinone present should have been in the semiquinone form. However, electron paramagnetic resonance measurements (Hamilton et^ a l . , 1970) indicated that only 2$ of the ubiquinone was in the semiquinone form, unless as sug-gested by Cox et a l . , (1970), the low signal might have been due to the 42 binding of the ubisemiquinone to a metal to give a chelate complex which was devoid of an electron paramagnetic signal (Beinert and Hemmerich, 1965). The addition of p i e r i c i d i n A to the membranes eliminated the EPR signal of the ubisemiquinone within 30 seconds and caused oxidation of the reduced ubiquinone within 10 minutes. However, the presence of p i e r i c i d i n A pre-vented the reduction of ubiquinone following the addition of substrate, although i t increased the steady-state reduction of f l a v i n and cytochrome b>|. These results led the authors to conclude that ubiquinone probably did not function as a direct electron carrier. This they considered particularly unlikely at two sites since the redox potential has not been demonstrated to-be markedly changed by environment (Boyer, 1968). As a possible explanation of the observed results, Cox et a l . , (1970) proposed the following. Ubiquinone-8 i s complexed with an electron carrier. The electron carrier alone does not function as e f f i c i e n t l y in electron transport as the carrier-ubiquinone complex, ubiquinone being in the semi-quinone form. Inhibition of electron transport by p i e r i c i d i n A would occur by disruption of the complex. Nonheme iron was suggested as the most l i k e l y electron carrier to form a complex with ubiquinone since nonheme iron can function at different redox potentials (Boyer, 1968). The functions of vitamin K 2(40) are s t i l l unclear, however, Cox et a l . , (1970) state that preliminary investigations with men" mutants indicate that vitamin K 2 i s not involved in aerobic respiration. Newton et a l . , (1971) have demonstrated that vitamin K 2 functions in the anaerobic oxidation of dihydroorotate u t i l i z i n g fumarate as the electron acceptor. A number of proposed models of the respiratory chain of E. c o l i have appeared in the literature (Kashket and Brodie, 1963b; B i r d s e l l and Cota-43 Robles, 1 9 7 0 ; Cox et a l . , 1 9 7 0 ; Hendler, 1971)• The f i r s t model to appear, that of Kashket and Brodie, is presented in Figure 1.6A. It accounted for the experimental results available at the time but, as indicated above, subsequent research (Cox et a l . , 1 9 6 8 a ; Cox et al_., 1 9 7 0 ) has raised consider-able doubt as to the involvement of vitamin K 2 in the respiratory chain of E. c o l i . However, the results of Jones (1967) indicated that the possibility of a strain difference should not be overlooked. Figure 1 . 6 B presents the model proposed by Cox et a l . , (1970), based on their investigations into the respiratory chain of E. c o l i K 1 2 „ As stated by these authors, i t represents a basis for further work taking into account their various observations. In this respect, their model is the simplest linear sequence of carriers that could account for their results. At least one of these observations requires qualification. No attempt was made to evaluate the increased contribution of cytochrome £ to the cytochrome b-| peak following the addition of NaCN to membranes from the normal strain of E. c o l i . Con-sequently, the v a l i d i t y of the statement that NaCN increased the steady-state reduction level of cytochrome b^ above that observed with ubiquinone deficiency i s open to question. However, the fact that reports by other investigators (Jones, 1 9 6 7 ; Lightbown and Jackson, 1 9 5 6 ; Bragg, 1 9 7 0 ) have indicated inhibition by HQNO and p i e r i c i d i n A prior to cytochrome b-) suggests that the conclusions that there are two sites of ubiquinone function i s probably valid. There remains a great number of unanswered questions, in addition to the necessity of verifying the proposals of Cox et a l . , ( 1 9 7 0 ) . Among these questions are the following: (i) What are the relative importances of the cytochromes a^ and £ as terminal oxidases? ( i i ) Where i s cytochrome ai 44 A. NADH dehydrogenase v i t . K 2 ( 4 0 ) , Succinate dehydrogenase *-UQ-8 icyt. b +- cyt. a_2 0 2 B. NADH ^ f p ••Fe Lactate I I -cyt. b.-UQ-8 >Fe-cyt. o cyt. a 2 •0, c. NADH-XPD Fe Succinate »-F. PS Fe eD UQ-8 Fe r S UQ-8 cyt. b Fig. 1.6 Models of the electron transport systems of E. c o l i . 45 involved in the respiratory chain? ( i i i ) Is there more than one function-a l l y and/or chemically distinct cytochrome b^ as results presently suggest ( B a i l l i e et a l . , 1971; Kim and Bragg, 1971; Shipp, 1972a)? (iv) Are the electron transport chains u t i l i z i n g either cytochrome or cytochrome o as terminal oxidases identical with respect to a l l other components? (v) Is ubiquinone involved in the respiratory chain as an electron carrier, as a coupling component, or in a regulatory role? ( v i j What is the function of nonheme iron? With so many degrees of uncertainty in our knowledge of the respir-atory chain of E. c o l i , perhaps the model proposed by Bragg (in press) (Figure 1.6C) is the most appropriate at present. 1.4 Oxidative phosphorylation Historically, bacterial oxidative phosphorylation was f i r s t investi-gated in E. c o l i (Hersey and A j l , 1951; Pinchot and Racker, 195l).Hersey 32 and A j l described the obligatory uptake of P concurrent with succinate 32 oxidation by cell-free extracts of _E. c o l i . The uptake of P was inhibited 32 by 2,4-dinitrophenol and azide while NaF augmented the incorporation of P into acid-labile phosphates. A maximum P:0 ratio of 0.7 was obtained. Pinchot and Racker, also u t i l i z i n g a cell-free system investigated phosphate uptake associated with NAD+-dependent ethanol and acetaldehyde oxidation. Maximum P:0 values of 1.0 and 1.5 respectively were reported. However, as Pinchot subsequently pointed out (1965), due to the presence of the glyco-l y t i c enzymes in the cell-free systems, the phosphorylation observed may have been due to substrate level phosphorylation. Further reports on attempts to elucidate oxidative phosphorylation in E. c o l i did not appear u n t i l 1963. Bragg and Polglase (1963) reported 46 that phosphorylation coupled to NADH oxidation required both particulate and supernatant fractions from sonicated E. c o l i . They were able to obtain P:0 values of 0.2-0.65. Kashket and Brodie (,1963a) demonstrated that a slowly sedimenting membrane fraction (small particles) from sonicated E. c o l i , was capable of oxidizing succinate, ct -oxoglutarate, malate, pyruvate and NADH, and of coupling the oxidation to phosphorylation, when supplemented with a 30 to 60°/o ammonium sulfate precipitated protein fraction from the supernate. Maximum P:0 values of 0.5, 1.0, 1.1, 1.3 and 0,3 were obtained with succinate, a-oxoglutarate, malate, pyruvate and NADH respectively. Phosphorylation associated with malate and succinate oxidation was investi-gated in more detail particularly with respect to the action of uncouplers. Phosphorylation linked to NAD+-dependent malate oxidation was uncoupled by PCMB, lapachol, benzoquinone-6, TTPA, malonate, usnic acid and low concen-trations of dicumarol. Succinate-coupled phosphorylation was uncoupled only by dicumarol, lapachol and benzoquinone-6, while succinate oxidation was inhibited by PCMB and malonate. Neither pathway was sensitive to DNP. Nisman et a l . , (19^3) investigated oxidative phosphorylation by membrane particles from E. c o l i spheroplasts lysed with digitonin. They reported 32 32 the formation of P-ATP and P-ADP concurrently with NADH oxidation in the presence of AMP. Phosphate uptake and incorporation into ATP and ADP was inhibited by DNP, amytal and antimycin. Investigating the component(s) present in the supernate which were essential for, or enhanced oxidative phosphorylation in E. c o l i membrane particles, Ota (1965) isolated three, apparently d i s t i n c t , protein fractions that were essential for phosphorylation coupled to reduction of both oxygen and nitrate. Subsequently, Bragg and Hou (1968) demonstrated that a part-47 iculate fraction of E. co l i capable of NADH-dependent oxidative phosphory-lation was stimulated by a coupling-factor preparation to yield a maximum P:0 of 0.77. These results are characteristic of those obtained for oxidative phosphorylation of bacteria. In general, the properties of fractionated bacterial systems d i f f e r from coupled phosphorylation in intact mitochondria in that the bacterial systems yield lower P:0 ratios and f a i l to exhibit the type of respiratory control observed with mitochondria. These differences may be related to the more drastic procedure required to disrupt bacterial c e l l s . In most bacterial systems soluble components which are necessary for the restoration of oxidation, phosphorylation, or both activities,• of electron transport particles containing a structurally intact respiratory chain, are liberated during c e l l disruption. Bacterial systems also d i f f e r in their sensitivity to uncoupling agents. Some bacteria resemble mitochondrial systems in their response to low concentrations of these agents whereas others are total l y insensitive or require higher concentrations than those normally used with mitochondria (Gel'man et a l . , 1967; Brodie and Gutnick, 1972). Hempfling (1970a) appears to have c l a r i f i e d the uncertainty sur-rounding the P:0 ratio of intact E. c o l i as compared to that of electron transport particles from this organism. He employed a technique whereby i n -tact anaerobic E. c o l i were administered a pulse of oxygen and the concom-itant changes in uptake of ^ 2P and NAD+ levels were measureed. From this data values of P:2e~ were calculated. Hempfling obtained apparent P:2e" values of 2.6-3.0. The vali d i t y of this technique is based on the assumption that due to the rapidity of the terminal electron transport, the process of oxidative 4 8 phosphorylation i s isolated kinetically rather than physically from con-tiguous interfering reactions. Hempfling also points out that energy-linked changes would contribute to an underestimate'of the true P:2e~ ratio. Energy-linked functions in E. c o l i w i l l be discussed later. Subsequently, Hempfling (1970b) investigated the influence of the carbon source and growth stage on the a b i l i t y of intact aerobically grown E. c o l i to couple phosphate esterification to oxygen u t i l i z a t i o n . The results indicated ( i ) that a carbon source could be allocated to one of three cate-gories according to the a b i l i t y of the cells grown on the carbon source to carry out oxidative phosphorylation, and ( i i ) that with c e l l s grown on glucose, the P:2e~ ratio increased from 0.33 in the presence of glucose, to a value of 3.3 within 90 minutes of the exhaustion of glucose. The development of f u l l oxidative phosphorylation, following release from catabolite repression re-quired protein synthesis. There i s considerable sim i l a r i t y between the growth dependence of development of oxidative phosphorylation as reported by Hempfling (1970b), and the development of oxidative phosphorylation associated with anaerobic to aerobic adaptation as described by Cavari e_ a l . , (1968), including the requirement for protein synthesis. Hempfling and Beeman (1971) demonstrated that the catabolite repression of oxidative phos-phorylation could be relieved by the presence of 2-5 mM 3}5'-adenosine mono-phosphate (cAMP) in the growth medium. However, this appeared to alter some characteristic(s) of the system since oxidative phosphorylation by cells grown on glucose in the presence of cAMP was not sensitive to the uncoupler 2,4-dibromophenol (DBP). The mechanism of oxidative phosphorylation i s not known but the reaction responsible for the esterification of phosphate to ADP, to form 49 ATP, i s generally considered to be the reversal of the membrane bound 2+ 2+ magnesium (or calcium) ion dependent, adenosine triphosphatase (Mg (Ca )-ATPase) act i v i t y . Consequently, there i s a great deal of interest in the properties of this enzyme. Recently, due to the efforts of researchers 2+ 2+ from three laboratories the membrane bound Mg (Ca )-ATPase of E. c o l i has been solubilized and characterized in considerable detail (Evans 1969, 1970; Davies and Bragg, 1972; Kobayashi and Anraku, 1972). The reader should consult these papers for a discussion of the properties of this enzyme. 2+ 2+ The f i r s t information on the properties of Mg (Ca )-ATPase deficient mutants of E. c o l i was reported by Butlin et a l . , (1971)- These mutants, designated uncA", were able to grow on glucose but were unable to grow with succinate or D-lactate as the sole source of carbon. A l l membrane components of the respiratory chain and the NADH and succinate oxidase levels of the mutant strain were identical with those of the normal strain. Neither 2+ 2+ Mg (Ca )-ATPase nor oxidative phosphorylation was detectable in the uncA" strain. Kanner and Gutnick (l972a,b) and Simoni and Shallenberger (1972) 2+ 2+ have also described the isolation and characterization of Mg (Ca )-ATPase deficient mutants of E. c o l i . Like the uncA" strains of Butlin et a l . , (1971), these mutants could not u t i l i z e TCA cycle intermediates or D-lactate as sole carbon sources and the aerobic growth yield of the mutants on glucose was intermediate between the aerobic and anaerobic growth yield of the corresponding wild type E. c o l i . However, whereas the anaerobic growth yield of the uncA" strain of Butlin et a l . , was approximately 64% of that for the normal strain, that of the mutant strain isolated by Simoni and Shallenberger was only 45$ of the growth yield of the wild type. 50 The results obtained, particularly those of Butlin e_ a l . , suggest that the Mg(Ca)-ATPase .is essential for oxidative phosphorylation and is not required for growth when ATP is generated by substrate level phos-phorylation. Since the aerobic growth yield of the ATPase deficient mutants was greater than the anaerobic growth yield of the wild type strain, i t 2+ 2+ would appear, as suggested by Kanner and Gutnick (1972b) that the Mg (Ca )-ATPase deficient mutants have retained the a b i l i t y to couple glucose oxida-tion to the generation of some high energy state, or intermediate, which might be u t i l i z e d to drive energy-requiring cellular processes. A possible explanation for the differences in the aerobic growth yields of the ATPase deficient mutants w i l l be discussed later. Interest in energy-linked reactions directly coupled to the electron transport chain of E. c o l i has increased markedly in the last half decade. Energy-linked systems, present in E. c o l i , capable of deriving energy from ATP and/or directly from the respiratory chain appear to include: (i) D-lactate oxidase-coupled transport systems (Kaback, 1972; Schairer and Haddock, 1972); ( i i ) the energy-dependent reduction of NAD+ by succinate (Sweetman and G r i f f i t h s , 1971a); ( i i i ) the energy-dependent pyridine nucleo-tide transhydrogenase (Murthy and Brodie, 1964); and possibly (iv) induced protein biosynthesis (Kovac' and Kuz*ela, 1966; Gal o t t i , Kovac" and Hess, 1968). As the D-lactate oxidase-coupled transport systems have been discussed in section 1.1, they w i l l not be discussed further at this point. The only investigation on the energy-dependent reduction of NAD+ by succinate, in E. c o l i has been by Sweetman and G r i f f i t h s (1971a). The 2+ reaction was dependent on Mg" and succinate, and ATP could only be part i a l l y replaced by ITP. ADP at a concentration equal to that of ATP caused a 5 0 $ 51 inhibition of the ATP-driven reduction of NAD+ by succinate. The proposed stoichiometry for the reaction was one molecule of ATP hydrolyzed per molecule of NADH produced. The reaction was inhibited by the uncouplers 4,5,6,7-tetrachloro-2-trifluoromethyl benzimadazole (TTFB), pentabromo-phenol (PBP) and dicumarol but was insensitive to DNP or oligomycin. Low concentrations of pie r i c i d i n A or HQNO stimulated the reduction of NAD+ by succinate. Based on these results Sweetman and G r i f f i t h concluded that the reduction of NAD+ by succinate involved NADH dehydrogenase, succinate dehydro-genase, and probably nonheme iron since the reaction was inhibited by TTPA. They proposed that high energy intermediates of oxidative phosphorylation provided the necessary energy supply. The energy-dependent transhydrogenase (EDTH) f i r s t described by Murthy and Brodie (1964) has become a popular system for the investigation of the characteristics of energy-linked reactions in E. c o l i because of the ease and rapidity of the spectrophotometric assays. I n i t i a l l y , (Murthy and Brodie, 19^4; Bragg and Hou, 1968) the system was investigated only as the ATP-driven reaction although Bragg and Hou (1968) suggested that since the energy-dependent transhydrogenase was more stable than oxidative phosphor-ylation to ultraviolet irradiation, that these processes probably involved, different coupling factors. Fisher et a l . , (1970), Fisher and Sanadi (1971) and Kanner and Gutnick (1972b) subsequently demonstrated that the energy-dependent transhydrogenase could be driven via ATP or respiration-linked to energy production. Cox et_ a l . , (1971) and Kanner and Gutnick (1972b) have 2+ 2+ shown that mutants deficient in the Mg (Ca )-ATPase lack the ATP-driven transhydrogenase but retain the respiration driven activity. The ATP-driven transhydrogenase did not require ubiquinone-8 or menaquinone-8 for 52 a c t i v i t y , and was inhibited by pi e r i c i d i n A at a site unrelated to the site of inhibition of the electron transport chafn(Cox et a l . , 1971). Both the ATP-driven and respiration linked transhydrogenase were inhibited by TTFB, DNP, dicumarol, PBP and thyroxine while only the ATP-driven transhydrogenase was inhibited by dicyclohexylcarbodiimide (DCCD). Oligomycin had no effect on either the ATP-driven or the respiratory chain-linked energy-dependent transhydrogenase ac t i v i t i e s (Fisher and Sanadi, 1971; Sweetman and G r i f f i t h s , 1971b; Kanner and Gutnick, 1972b). The information available suggests, as proposed by Slater (1953)» that energy-linked reactions are driven by high-energy, nonphosphorylated intermediates of oxidative phosphorylation which can also be generated from, 2+ 2+ and are.in equilibrium with the c e l l u l a r ATP. The Mg (Ca J-ATPase appears to be intimately involved in generating the high-energy intermediate(3) from ATP (Cox et a l . , 1971; Kanner and Gutnick, 1972b; Schairer and Haddock, 1972). 2+ 2+ A coupling factor possessing Mg (Ca )-ATPase activity but without energy-dependent transhydrogenase act i v i t y has been isolated and purified from E. c o l i (Bragg and Hou, 1972). The addition of the purified coupling factor to respiratory particles which had been "stripped", that i s , were devoid of both the respiratory chain-linked and the ATP driven transhydrogen-ase a c t i v i t i e s , resulted in the recoupling of both of these a c t i v i t i e s . This system promises to provide further insight into the mechanism by which energy-linked systems are coupled to the respiratory chain and to ATP. Simoni and Shallenberger (1972) have isolated an ATPase deficient mutant of E. c o l i which lacks the a b i l i t y to transport alanine or proline with either ATP, or respiratory chain-linked D-lactate oxidation as the 53 source of energy. This is contrary to the results previously obtained with the energy-dependent transhydrogenase which, as indicated above, demonstrated that ATPase deficient mutants of E. c o l i lacked the ATP-driven EDTH but retained the electron transport system-linked EDTH (Cox et a l . , 1971; Kanner and Gutnick, 1972b). However, Bragg and Hou (1973), u t i l i z i n g the energy-dependent transhydrogenase as a test system, have demonstrated that the ATPase deficient mutant of Simoni and Shallenberger lacked not only the ATPase, but also the coupling factor responsible for coupling the energy-dependent transhydrogenase directly to the respiratory chain. Thus, i t appears that the same coupling factor could be involved in coupling amino acid transport and the energy-dependent, pyridine nucleotide transhydrogenase to energy generated via the electron transport chain. The lack of direct coupling of energy-linked reactions to the respiratory chain may explain the lower growth yield reported by Simoni and Shallenberger (1972) for the S. c o l i mutant, DL-54, as compared to the growth yield obtained by Butlin et a l . , (1971) for the uncA" mutants. In summary, the preceding discussion of oxidation phosphorylation and energy-linked systems of E. c o l i indicates: (i) E. c o l i i s capable of a maximum P:0 ratio of 3» ( i i ) that the degree of coupling of phosphorylation to oxidation appears to be related to the a b i l i t y of the substrate to maintain an adequate level of, ATP by substrate-level phosphorylation; ( i i i ) that the energy-linked reactions are driven by nonphosphorylated, high-energy intermediates generated d i r e c t l y by the electron transport chain, 2+ 2+ or from ATP via the Mg (Ca )-ATPase; and (iv) that the energy-linked systems of E. c o l i include: (a) transport, 54 (b) NAD+ reduction by succinate, (c) energy-dependent transhydrogenase, and possibly (d) protein synthesis. The control of respiration in bacteria, l i k e the control of other enzymatic pathways could be mediated at two levels: (i) molecular regula-tion of the enzymatic components of the pathway, and ( i i ) the regulation of the biosynthesis of the components of the pathway via induction of repression. The former, as usually expressed in the control of the electron transport system, i s the stimulation of respiration by ADP and inorganic phosphate (P_.) followed by a return to the original rate on the exhaustion of either ADP or P^. This is commonly referred to as respiratory control. As indicated previously, bacteria have been considered to lack respiratory control (Gel'man et a l . , 196"7; Brodie and Gutnick, 1972). However, increased rates of oxygen uptake on the addition of phosphate or phosphate-acceptor have been reported (ishikawa and Lehninger, 1962; Revsin and Brodie, 1967; Scocco and Pinchot, 1968). The presence of respiratory control in intact bacteria has been indicated indirectly by the stimulation of the respiratory rate of whole ce l l s on the addition of uncouplers of oxidative phosphorylation (Bovell and Packer, 1963; Bovell, Packer and Helgerson, 1963; Cavari et a l . , 1967; Hempfling, 1970a,b>. Respiratory control has not been demonstrated conclusively in E. c o l i , either in intact cells or..respiratory particles,nor has the addition of P^ and/or ADP to respiratory particles been shown to stimulate oxygen u t i l i z a -tion. Oishi et a l . , (1970) have reported the occurrence of respiratory control by phosphate, in intact E. c o l i , based on the observed stimulation of respiration by addition of phosphate to phosphate-depleted cells and the subsequent return to the non-stimulated rate on the depletion of P^  from 55 the assay medium. Unfortunately they were unable to eliminate the possi-b i l i t y that the stimulation of respiration was due to the coupling of phos-phate transport to respiration and energy production. Cavari et a l . , (1967) have reported that respiration was stimulated and the incorporation of P was inhibited on the addition of carboxylcyanide-m-chlorophenylhydrazone (CCGP) to suspensions of intact E. c o l i oxidizing glucose. However, iden-t i c a l levels of CCCP inhibited the oxidation of succinate, glutamate and pyruvate. Stimulation of formate and glucose oxidation by intact E. c o l i on the addition of DBP has been reported by Hempfling (l970a,b). However, although DBP abolished phosphorylation associated with the oxidation of endogenous NADH, i t did not increase the rate of endogenous NADH oxidation (Hempfling, 1970a). Although respiratory control has not been demonstrated in E. c o l i , i t has been demonstrated in other bacteria. John and Hamilton (1970, 1971) have shown respiratory control in membrane particles of Micrococcus deni- tr i f i c a n s and i t has also been demonstrated in Azotobacter vinelandii membranes (Silerman et a l . , 1970; Jones et a l . , 1971a,b). Control of the respiration of E. c o l i by those environmental factors which more or less s p e c i f i c a l l y cause alteration of the composition and/or function of the respiratory chain, usually via induction or repression of the synthesis of respiratory chain components, i s of particular interest. The two factors which have been most thoroughly investigated in this res-pect are oxygen tension and the carbon source. The alterations in the levels of ubiquinone-8 and menaquinone-8 in response to aeration have been discussed. The influence of oxygen tension on the cytochrome components of the respira-tory chain and respiratory ac t i v i t y of E. c o l i was f i r s t reported by Moss 56 (1952). His results demonstrated that an increase in the levels of cyto-chrome a^, _2 and b^ occurred on aeration. There was also an increase in the Qo2« The Qo2 was not closely related to the amount of cytochrome _2 present. Subsequently, Gray e_ a l . , (1966b), Hino and Maeba (1966), Takahashi and Hino (1968a,b) and Cavari e_ a l . , (1968) have investigated further the influence of oxygen on the development of the respiratory path-ways. Gray et_ a l . , (1966b) grew E. c o l i K12 under aerobic and anaerobic conditions and reported that higher levels of NADH oxidase, succinate oxidase, succinate dehydrogenase and cytochrome b-j were formed under aero-bic than anaerobic conditions. Levels of cytochromes a^ and _2 were reported not to be influenced by these growth conditions, in contrast to the earlier reports of Moss (1952). Hino and Maeba (1966) observed that there was only a slight (2- to 3- fold) increase in the cytochrome content of anaerobic cells which had been aerated i n a buffer containing casamino acids, while the res-piratory activity had increased 20 to JO f o l d . Inhibition of the develop-ment of increased respiratory a c t i v i t y by chlorophenicol demonstrated that the increase required protein synthesis, probably of some component other than cytochromes. Ishida and Hino (1972) suggested that the slight increase in the level of cytochromes was due to: (i) the increased formation of succinyl CoA; ( i i ) an increased level of an enzyme in a reaction between o*-amino-levu l i n i c acid (ALA) and protoheme; ( i i i ) the activation of preformed enzymes in a reaction between ALA and protoheme; and (iv) the induction of ALA synthase; as a result of the increased oxygen tension. Takahashi and Hino (1968a,b) demonstrated that the markedly higher respiratory activity of the aerated c e l l s was due to: ( i ) the induction of transport systems for intermediates of the TCA cycle, and ( i i ) an approximately 10-fold increase 5 7 in the ac t i v i t y of the tricarboxylic acid cycle enzymes. The mechanism responsible for inducing the increased synthesis of the TCA cycle enzymes i s unknown. However, Wimpenny (1969) has proposed that i t may be related to the c e l l u l a r NAD+/NADH ratio. Cavari et a l . , (1968J have made several interesting observations. F i r s t , NADH dehydrogenase and succinate dehydrogenase a c t i v i t i e s of E. c o l i B were not influenced by the anaerobic to aerobic transition. (Succinate dehydrogenase of E. c o l i K12 was found to increase as previously reported by Gray et a l . , 1966b). Secondly, the NADH oxidase ac t i v i t y of E. c o l i increased rapidly on exposure of the anaerobic cells to aeration, reaching the maximum aerobic level within 10 minutes. Thirdly, succinate oxidase and cytochrome levels increased more slowly and in p a r a l l e l , and approached the levels found in aerobic cells only after one hour of aeration. Fourthly, the P:0 ratio and CCCP sensitivity of cells grown on a carbon source of man-ni t o l and fumarate, began to increase after one hour of aeration, when the succinate oxidase and cytochrome levels had nearly reached the aerobic le v e l . The mechanism responsible for the rapid increase in NADH oxidase ac t i v i t y i s not known, but due.to the short time required i t probably does not involve new protein synthesis. The lack of effect of the anaerobic to aerobic transi-tion on the succinate dehydrogenase activity, and the parallel increase in the succinate oxidase a c t i v i t y and cytochrome le v e l , suggests that the former is a result of the la t t e r . As indicated previously, the increase in the level of cytochromes can probably be explained by the proposals of Ishida and Hino (1972). The development of oxidative phosphorylation and CCCP sensitivity was probably the same phenomenon reported to occur on depletion of glucose in cultures of aerobically grown E. c o l i (Hempfling, 1970b). 58 More recently, Wimpenny and Necklen (1971) and Harrison and Loveless (1971) have reported on the influence of oxygen tension on _. c o l i in contin-uous cultures. Wimpenny and Necklen observed that the levels of cytochrome _2 and h^ increased at low oxygen tension (1-5 mm Hg), while Harrision and Loveless reported increased respiration rates and decreased growth yield under similar conditions. The elevated levels of cytochrome ag and the increased respiration rate combined with a decreased growth yi e l d , observed under condi-tions of low oxygen tension, may be due to a greater a f f i n i t y of cytochrome as? than cytochrome p_ for oxygen (White, 1963), and the uncoupling of respiration from energy conservation (Harrison and P i r t , 1967), respectively. In summary, the growth of E. c o l i under aerobic conditions, in the absence of glucose, results i n : (i) increased levels of TGA cycle enzymes; ( i i ) s l i g h t l y increased levels of respiratory chain cytochromes; ( i i i ) an increased level of ubiquinone-8; (iv) the activation of NADH oxidase; and (v) an increased capacity to perform oxidative phosphorylation; as compared to E. c o l i grown under anaerobic conditions. The influence of the carbon source on the respiration rate is p r i -marily via catabolite repression of the synthesis of TCA cycle enzymes. Halpern et a l . , (1964) demonstrated that aerobic growth on glucose repressed synthesis of the enzymes of the TCA cycle and of the transport systems required for the uptake of TCA cycle intermediates. This effect of glucose was subsequently confirmed, and extended to show that glucose had l i t t l e effect on the cytochrome levels of aerobically grown ce l l s (Hino and Maeda, 1966; Gray et a l . , 1966b; Takahashi and Hino, 1968a,b). 1.5 The influence of s i l v e r ions on growth and enzyme ac t i v i t y Silver ions inhibit the glucose-dependent aerobic respiration of 5 9 E. c o l i (Rainnie and Bragg, 1971 ). The obvious question i s - How? The antibacterial a c t i v i t y of s i l v e r compounds has been known since 1 8 6 9 , and has been differentiated on a basis of concentration; (i) "poisoning" death (silver concentration ^ 6 x 10 g-ions per l i t r e , and ( i i ) "oligodynamic" death (silver concentration ^ -6 x 10~ g-ions per l i t r e ) (Romans, 1957a). The former is believed to occur as a result of the irreversible denaturation and precipitation of the proteins of the bacterial c e l l (Meyers et a l . , 1970). The mechanism of the l a t t e r remains unclear although many proposals have been put forward (Romans, 1957a). The mech-anism of the oligodynamic ac t i v i t y of s i l v e r probably d i f f e r s from that of "poisoning" by s i l v e r only in that a smaller quantity of s i l v e r is bound to proteins with the result that catalytic proteins are reversibly i n a c t i -vated rather than irreversibly denatured. The reaction of s i l v e r with proteins occurs primarily at the a v a i l -able sulfhydryl groups forming insoluble mercaptides, a characteristic which has resulted in the use of s i l v e r salts for the amperometric deter-mination of the sulfhydryl content of proteins (Cecil and McPhee, 1959; Benesch and Benesch, 1962; Leach, 1966). However, the stoichiometry of the s i l v e r ion-sulfhydryl reaction may not be 1:1, nor i s the s p e c i f i c i t y absolute (Cecil and McPhee, 1959; Benesch and Benesch, 1962; Leach, 1966; Burton, 1958; Sluyteiman, 1957; Kolthoff and Eisenstadter, 1961; Cole et a l . , 1958) as s i l v e r has also been shown to form complexes with a histidine residue in invertase (Myrback, 1957) and with free riboflavin (Weber, 1950), and has been suggested to react with carboxyl groups (Dixon and Webb, 1964; Woratz and Thofern, 1955). 60 The reported effects of s i l v e r ions on eucaryotic systems include: (i) the inhibition of numerous enzymes and enzyme systems (Webb, 1966a,b); ( i i ) the stimulation of enzymes (Rapoport and Luebering, 1951; Chappell and Greville, 1954; Cooper, 1960); ( i i i ) the stimulation and inhibition of respiration (Cook, 1926; Chappell and Greville, 1954; Grabske, ^ ^66)^, (iv) alteration of membrane permeability (Romans, 1957a; Brierley et a l . , 1967); and (v) the inhibition of aerobic and anaerobic growth (Romans, 1957a; Takada and Joho, 1964). Included among the enzymes inhibited by s i l v e r ions are a number of enzymes involved in the Hmbden-Meyerhof pathway, the tricarboxylic acid cycle, electron transport, and energy conservation. Glycolytic enzymes from a number of different sources, yeast hexo-kinase (Titova, 1968), aldolase from rabbit skeletal muscle (Herbert et a l . , 1940), 3-phosphoglyceraldehyde dehydrogenase from rabbit and porcine skeletal muscle (Park et a l . , 1961; Boross, 1965; Boross and K e l e t i , 1965), and yeast pyruvate decarboxylase (Stoppani et a l . , 1952), have been demonstrated to be sensitive to inhibition by s i l v e r ions. Silver did not inhibit the catalytic a c t i v i t y of the yeast hexokinase but i t prevented the inhibition of the enzyme by hydrocortisone. The inhibition of 3-phosphoglyceraldehyde dehydro-genase by s i l v e r ions resulted in a complete loss of dehydrogenase activity but had no effect on the transphosphorylation reaction (Park et a l . , 1961). Boross and colleagues have isolated the inhibited 3-phosphoglyceraldehyde dehydrogenase as a crystalline ternary complex consisting of one mole-equivalent of NAD+ and four mole-equivalents of s i l v e r ions per monomer o-f of the enzyme. The concentration of s i l v e r ions required to bring about complete inhibition of these enzymes was in the order of 1 x 10~^  normal (N). 61 In addition to the preceding enzymes a number of glycolytic enzymes have been shown to be sensitive to thiol reagents other than s i l v e r ions and therefore may also be inhibited by s i l v e r ions (Webb, 1966b; Scott et a l . , 1970). ' Of the TCA cycle enzymes, only isocitrate dehydrogenase (Kratochvil et a l . , 1967) glutamate dehydrogenase (Olson and Anfinsen, 1953) and fumarase (Laki, 1942), a l l from mammalian sources, have been shown to be - 7 - 5 inhibited by s i l v e r ions i n the concentration range of 10 to 10 N. However, as in the Smbden-Meyerhof pathway, a number of additional TCA cycle enzymes have been shown to be sensitive to thiol reagents such as p-chloro-mercuribenzoate (PCMB), p-mercuriphenylsulfonate (PMPS),ibdoacetate (IA), iodoacetamide (IAM) and N-ethylmaleimide (NEM) and these may also be inhibited by s i l v e r ions (Barron and Singer, 1945; Webb, 1966b; Slater, 1949; Bernath and Singer, 1962). The only information available on the affect of s i l v e r ions on electron transport is the inhibition of a pa r t i a l l y purified NADH-cytochrome £ reductase from pig l i v e r microsomes, reported by Garfinkel (1957). The enzyme system retained 10$ of i t s i n i t i a l a c t i v i t y at a s i l v e r nitrate con-centration of 3 x 10" 5 N. Slightly more information i s available on the effects of s i l v e r ions on the enzyme systems involved in energy conservation. Chappell and Greville (1954) reported the stimulation of mitochondrial ATPase ac t i v i t y by s i l v e r ions ( l x 10~^ N). Subsequently, Chiga and Plaut (1959) reported the inhibition by s i l v e r ions (5 x 10~^ N), of the ADP«F±ATP and ATP^P^^ exchange reactions catalyzed by the enzyme purified from hog l i v e r mitochondria. Cooper (i960) demonstrated that AgNO* at low concentrations mimicked the 62 action of dinitrophenol and stimulated the ATPase activity of digitonin particles prepared from r a t - l i v e r mitochondria. Silver nitrate levels —8 greater than 4 x 10 moles per milligram of protein markedly inhibited the ATPase act i v i t y . Related to the stimulation of ATPase act i v i t y at low concentrations of s i l v e r ions is the affect of s i l v e r ions on respiration. Cook (1926) reported that the respiration of Aspergillus niger, as measured by CO2 pro-duction, showed an i n i t i a l stimulation in rate, followed by inhibition (silver nitrate concentration of 1 x 10 ^ N), Chappell and Greville (1954) demonstrated a two-fold increase in the respiration rate of rabbit brain mitochondria on the addition of s i l v e r nitrate to f i n a l concentration of -6 5 x 10" N. These results were confirmed by Grabske (1966) with r a t - l i v e r -4 mitochondria at a s i l v e r nitrate concentration of 2.7 x 10 N. The results of Grabske, l i k e those of Cook (1926), demonstrated an i n i t i a l stimulation of respiration followed by inhibition. Thus, s i l v e r ions i n i t i a l l y uncouple respiration of mitochondria in a manner analogous to dinitrophenol, followed by inhibition of respiration. Uncoupling of oxidative phosphorylation and energy transfer by other t h i o l reagents, has also been reported (Kielley, 1963; Boyer et a l . , 1966; Kurup and Sanadi, 1968). Although, as indicated at the beginning of this section, s i l v e r ions have been recognized as bacteriocidal for more than a century, the physiolog-i c a l and biochemical affects of these ions on bacteria remain largely unknown. The influence of s i l v e r ions on E. c o l i include ( i ) the inhibition of aerobic and anaerobic growth which the authors suggested was due to interference with the oxidation mechanism (Grumbach and Wehrli, 1948); ( i i ) the inhibition of succinate transport by E. c o l i membrane vesicles at a 63 s i l v e r nitrate concentration of 1 x 10~^ N (Rayman et a l . , 1972a,"b); ( i i i ) the inhibition of glucose, lactate, succinate and formate dependent reduc-tion of methylene blue (Yudkin, 1937); (iv) the inhibition of citrate, isocitrate, succinate, fumarate and malate dependent reduction of triphenyl-tetrazolium chloride (Woratz and Thofern, 1955); and (v) the inhibition of hydrogenase (Yudkin, 1937; Joklik, 1950), formic hydrogenlyase '(Yudkin, 1937) and catalase a c t i v i t i e s (Woratz and Thofern, 1955). Unfortunately, due to the techniques used for measuring the "dehydrogenase" a c t i v i t i e s (Yudkin, 1937; Woratz and Thofern, 1955) very l i t t l e can be concluded as to the actual site of Ag + inhibition. Also, Yudkin observed that the concentration of s i l v e r ions required to inhibit the enzyme a c t i v i t i e s under his assay con-ditions was 10 to 100-fold greater than the lethal concentration. Although inhibition of the above enzyme systems could certainly result in inhibition of respiration, no investigation has been reported on the influence of s i l v e r ions on the respiration of E. c o l i . 1.6 The influence of iron limitation on respiration and energy conservation Waring and Werkman f i r s t reported, in 1942, on the iron requirement of bacterial growth. They observed that low levels of iron i n the growth media of Aerobacter indologene3, Aerobacter aerogenes, Psuedomonas aeruginosa, Klebsiella pneumoniae, Escherichia c o l i and Serratia marcescens resulted in reduced growth yields. This was subsequently confirmed with E. c o l i (Young et a l . , 1944; Ratledge and Winder, 1964) and has also been reported to occur with Corynebacterium diphtheriae (Pappenheimer and Hendee, 1947), Pseudomonas  fluorescens (Lenhoff et a l . , 1956), Mycobacterium smegmatis (Winder and O'Hara, 1 9 6 2 ) , Neurospora crassa (Padmanaban and Sarma, 1 9 6 5 ) , Mycobacterium phlei (Antoine and Morrison, 1968) and Candida u t i l i 3 (also c l a s s i f i e d as Torulopsis 64 u t i l i s ) (Clegg et a l . , 1969; Clegg and Garland, 1971; Clegg and Light, 1971). The most obvious site for iron involvement in these organisms was cytochromes and consequently research was directed to the influence of iron limitation on enzyme systems associated with the respiratory chain. Effects were found. For example, Waring and Werkman (1944) reported decreased a c t i v i t i e s of lactate, pyruvate and acetate oxidases in A. indologenes grown under iron-deficient conditions. The succinate oxidase activity of _. diph-theriae was also found to be reduced under iron-deficient growth conditions (Pappenheimer and Hendee, 1947; Righelato, 19^9) as was the cytochrome £ oxidase of N. crassa (Nicholas and Commissiong, 1957). Growth of N. crassa and M. smegmatis under conditions of iron limitation resulted in reduced levels of NADH-cytochrome £ reductase (Nicholas and Commissiong, 1957; Winder and O'Hara, 1964)• Nicholas and colleagues have demonstrated lower NADH-nitrate reductase activity in N. crassa (Walker and Nicholas, 1961a; Nicholas and Wilson, 1964), P. aeruginosa (Fewson and Nicholas, 1961a) Micrococcus  denitrificans (Fewson and Nicholas, 1961b) and Pseudomonas denitrificans (Radcliffe and Nicholas, 1970) due to growth under iron-limited conditions. The n i t r i t e reductases of N. crassa (Nicholas et a l . , i960) and P. aeruginosa (Walker and Nicholas, 1961b), and the n i t r i c oxide reductase of the l a t t e r organism (Fewson and Nicholas, 1961c), also were present at reduced levels in iron-deficient cultures. The reduced levels of many of these enzyme systems may be explained on the basis of reduced levels of cytochromes. Decreased levels of cyto-chromes, due to iron limitation, have been reported in cultures of P. fluor-escens (Lenhoff e_ a l . , 1956) Spirillum i t e r s o n i i (Clark-Walker et a_., 1967), M. denitrificans (imai et a l . , 1968), _. diphtheriae (Righelato and van Hemert, 65 1969; Righelato, 1969) and £. u t i l i s (Light and Garland, 1971; Clegg and Garland, 1971; Ohnishi et a l . , 1969). However, iron, as nonheme iron, has been shown to be a component of NADH dehydrogenase and succinate dehydrogenase (Hall and Evans, 1969). Decreased succinate dehydrogenase ac t i v i t y has been reported to occur in A. indologenes (Waring and Werkman, 1944), M. smegmatis (Winder and O'Hara, 1964), N. crassa (Padmanaban and Sarma, 1965) and £. diphtheriae (Righelato and van Hemert, 1969; Righelato, 1969) as a result of growth under iron-limited conditions. Nonheme iron has been implicated as possibly functioning in oxida-tive phosphorylation (Hall and Evans, 1969; Bragg, 1973). Iron limitation has also been u t i l i z e d to study the involvement of iron in oxidative phos-phorylation in £. u t i l i 3 (Clegg et a l . , 1969; Clegg and Garland, 1971; Ohnishi et a l . , 1969; 1971) and M. denitrificans (imai et a l . , 1968) Research by Garland's and Ohnishi's groups has demonstrated that the growth of £. u t i l i s in batch or continuous culture under conditions of iron limitation resulted in the loss of energy conservation at site I as measured either by NADH-dependent P:0 ratios or the energy-dependent rever-sal of electrons from glycerol-1-phosphate to endogenous pyridine nucleo-tide. At the concentration of iron at which site I phosphorylation was lo s t , there was also a loss of the g=1.94 EPR signal of the NADH dehydrogenase region of the respiratory chain. Clegg and Garland (1971) observed that incubating iron-deficient c e l l s aerobically for several hours under non-growing conditions resulted in the recovery of site I energy conservation but not the g=1.94 EPR signal. Incubation in the presence of cycloheximide (360 uM) did not prevent the recovery of site I energy conservation. These 66 results suggested the dissociation of energy conservation at site I from the g=1.94 EPR signal and that d_e novo protein synthesis was not required for the recovery of site I phosphorylation. Additional evidence for the involvement of nonheme iron proteins at site I has been obtained by Clegg and Garland (1971)- Electron trans-port particles from iron-limited cells had lower nonheme iron and acid-l a b i l e sulfide content than those of iron-sufficient c e l l s . The ratio of the decrease in the nonheme iron to the decrease in acid-labile sulfide on transition from iron-sufficient to iron-deficient conditions was 1.2. Since the ratio of iron to sulfide i s usually 1.0 in iron-sulfur proteins (Hall and Evans, 1969)» iron deficiency appeared to result in the loss of iron-sulfur proteins. Aerobic incubation of iron-limited cells in the presence of FeSO^, under non-growing conditions, resulting in the recovery of site I phosphorylation, was accompanied by an increase in the content of both non-heme iron and acid-labile sulfide i n the electron transport particles. Also supporting the idea that iron-sulfur proteins were involved in site I energy conservation was the finding by Haddock and Garland (1971) that sulfate limitation during the growth of C_. u t i l i s resulted in mitochondria lacking site I. In view of these results and having calculated that at least 90fo of the nonheme iron in electron transport particles from C_. u t i l i s grown under iron-sufficient conditions was not required for site I energy conservation, Garland and coworkers have proposed that a small fraction of the mitochondrial nonheme iron proteins which does not show EPR signals may-play a role in site I phosphorylation. Ohnishi and colleagues have attempted to obtain more direct evidence for the involvement of nonheme iron proteins at site I. Ohnishi e_t a l . , 67 (1972a) have restated and reinforced their i n i t i a l proposal, that there i s a close correlation between site I energy conservation and iron sulfur proteins responsible for the g=1.94 EPR signal in the NADH dehydrogenase region of the respiratory chain. In contrast to the results of Clegg and Garland (1971)» their results indicated that there was recovery of site I phosphorylation, and the g=1.94 EPR signal on aeration of intact iron-deficient _. u t i l i s under non-growing conditions. Incubation under the same conditions but in the presence of 100 uM cycloheximide prevented the recovery of both parameters. I f the g=1.94 EPR signal i s definitely associated with site I phosphorylation this would provide conclusive evidence for the involvement of nonheme iron in energy conservation at site I. In contrast to the results with C_. u t i l i s , oxidative phosphorylation in respiratory particles from M. denitrificans was not affected by iron-deficiency although the EPR signal associated with the respiratory chain-linked nonheme iron disappeared (imai et a l . , 1968). It should be noted however, that whereas Clegg and Garland (.1971) obtained a 95$ reduction in nonheme iron levels under conditions of iron limitation, Imai et a l . , reported a decrease of 85$. The decrease obtained by Imai e_ a l . , may have been inadequate to effect oxidative phosphorylation. Thus, i t is not clear whether nonheme iron, as well as being a component of the respiratory chain, has a role in energy coupling in bacteria. It was for this reason that the influence of iron limitation on the respiration of E. c o l i was examined. 1.7 Objectives of the research reported in this thesis It i s apparent from the preceding discussion of the transport systems, amphibolic pathways, respiratory chain and oxidative phosphorylation of E. c o l i that there are major deficiencies in our understanding of each of 6 8 these systems . The lack of knowledge is most marked with respect to the sequence of the respiratory carriers, the regulation of the respiratory chain, and the characteristics of oxidative phosphorylation and/or energy-linked systems. Investigation of the respiratory chain of E. c o l i , and associated energy conservation, has been hampered by the following: (i) the absence of a discrete organelle possessing the respiratory chain and associ-ated energy generating system(s), such that they can be isolated and inves-tigated without complication from the energy u t i l i z i n g systems of the intact c e l l , ( i i ) the lack of a sufficient range of inhibitors of the bacterial respiratory chain to permit an unequivocal determination of the sequence of the respiratory chain components, in contrast to the broad selection of inhibitors of the mitochondrial respiratory chain, and ( i i i ) the conditions required to disrupt the bacterial c e l l may result in the loss or denatura-tion of factors required for respiration and/or oxidative phosphorylation. As a result of the l a t t e r problem, cell-free systems from bacteria generally show much lower P:0 values than those observed in tightly coupled mitochondria (Gel'man et a l . , 1967). Moreover, i t was only recently that ADP-ATP medicated respiratory control has been demonstrated in bacterial cell-free systems (Jones et a l . , 1971b; John and Hamilton, 1971). Thus, cell-free systems were considered to be unsuitable for our studies. However, P:0 values obtained with intact c e l l s also may not accurately reflect the a b i l i t y of the c e l l to generate energy via the respiratory chain. Energy-u t i l i z i n g reactions, other than ATP formation, could compete for the respir-atory chain-generated high energy states or intermediates (Harold, 1972). In mitochondria, that respiration which can be stimulated by ADP and which results in the conversion of ADP to ATP can also be stimulated by uncoupling 69 agents. This has also "been shown with respiratory particles of M, denitrificans (John and Hamilton, 1971)• The stimulation of respiration by uncoupling agents is probably due to the dissipation of a high energy state intermediate (Harold, 1972) and could be used as an indicator of the presence of the high energy state whether or not this could be converted to ATP. This concept has been used in the study of the influence of s i l v e r ions, and the effect of progressive iron limitation such as occurs during growth in an iron-limited batch culture, on the respiration and energy conservation of E. c o l i . The observation that s i l v e r ions released from an oxygen electrode i n i t i a l l y stimulated the respiration of E. c o l i prior to bringing about a complete inhibition of oxygen consumption, suggested that s i l v e r ions might function as an uncoupler as well as an inhibitor of the respiration and prompted an investigation of the site(s) at which s i l v e r ions influenced the aerobic respiration of E. c o l i in the hope that s i l v e r ions might be useable as a tool for further research into the sequence and energy coupling of the respiratory chain of E. c o l i . In view of the observed influence of iron limitation on the site I energy-coupling of Candida u t i l i s (Clegg et al.,'1969; Clegg and Garland, 1971» Ohnishi et a l . , 19^ 9» 1971)» but apparent lack of effect on oxidative phosphorylation in respiratory particles from M. denitrificans (imai et a l . , 1968), the role of iron in the respiratory chain, and in energy-coupling linked to the respiratory chain, in bacterial systems, i s unclear. For this reason an investigation of the involvement of iron in the function of the E. c o l i respiratory chain and in the coupling of energy conservation to the respiratory chain was undertaken. 70 2. MATERIALS AND METHODS 2.1 Materials 2.1.1 Bacteria The organism u t i l i z e d for the majority of the experiments reported in this thesis was Escherichia c o l i , strain 482 of the National Research Council culture collection (E. c o l i NRC 482). This strain was obtained from the Department of Microbiology, University of Bri t i s h Columbia by Dr. W. J. Polglase of the Department of Biochemistry and was subsequently supplied to our laboratory by Dr. Polglase. Additional strains of E. c o l i u t i l i z e d were: (i) E. c o l i B-SG1, which was obtained from R. J . Harvey, Burroughs Wellcame Co., Research Triangle, North Carolina and which was originally isolated by J . Preiss of the Department of Biochemistry, University of California, Davis, California. E. c o l i B-SG1 lacks the enzyme adenosine diphosphate-glucose: a -4-glycosyl transferase and consequently i s unable to synthesize glycogen. Nutritional requirements are the same as E. c o l i B wild type, ( i i ) E. c o l i ATCC 8739 (Crooke's strain) was obtained from the American type culture collection, Washington, D. C. The following strains of E. c o l i were obtained from Dr. Polglase*s laboratory in 19^9. The source from which Dr. Polglase obtained the strain i s indicated in brackets, ( i i i ) E. c o l i B, ATCC 11303 (American type culture collection); (iv) E. c o l i B/r, ATCC 12407 (American type culture collection); (v) E. c o l i K12 (Dr. Stock, Department of Micro-biology, University of Br i t i s h Columbia); (vi) E. c o l i K12 met", a meth-ionine requiring auxotroph of E. c o l i K12 (Dr. R. A. J . Warren, Department of Microbiology, University of Bri t i s h Columbia); ( v i i ) E. c o l i var. com-munis D111 (Department of Bacteriology, Provincial Dairy School, St. Hyacinth, 71 P. Q.')» ( v i i i ) E. c o l i UL 10.1 (Department of Bacteriology, University of Laval, Quebec, P. Q.); (ix) E. c o l i CRX-7(04863-P.D.) (Laboratory of Hygiene, Ottawa, Ontario); (x) E. c o l i W3110 (R. Sommerville, University of Michigan, Ann Arbor, Michigan); (xi) E. c o l i W1485 (R. Sommerville, University of Michigan, Ann Arbor, Michigan); ( x i i ) E. c o l i B, strep-tomycin dependent (isolated in Dr. Polglase's laboratory from E. c o l i B, ATCC 11303); ( x i i i ) E. c o l i UL 10.1, streptomycin dependent (isolated in Dr. Polglase's laboratory from E. c o l i UL 10.1) and (xiv) E. c o l i BRA (origin unknown). 2.1.2 Chemicals The following chemicals were obtained from Calbiochem: N-tris (hydroxymethyl) methyl-2-aminoethane sulfonic acid (TES), A grade; morpho-linopropane sulfonic acid (MOPS), A grade; N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES), A grade; piperazine-N,N'-bis (2-ethane sulfonic acid), monosodium salt , monohydrate (PIPES), A grade; tris-(hydroxymethyl) aminomethane (Tris), A grade; triethanolamine hydrochloride, A grade; DL-glyceraldehyde-3-phosphate diethylacetal, barium s a l t , B grade; nicotin-amide adenine dinucleotide, disodium sal t , trihydrate, reduced (NADH), A grade; nicotinamide adenine dinucleotide, free acid, tetrahydrate (NAD+), A grade; phenazine methosulfate; D- and L-lactate, lithium salts, A grade; and bovine serum albumin, crystalline, A grade. Glycine hydrochloride, 2,4-dibromophenol (DBP) and 2,6-dichlorophenol-indophenol monosodium salt (DCIP) were purchased from Eastman Organic Chemicals. Fisher S c i e n t i f i c Co., supplied: glycine; monoethanolamine; o-phenanthroline; and s i l v e r nitrate, Fisher c e r t i f i e d ACS grade. Ultrapure grade tris-(hydroxymethyl) aminomethane, free base (Tris) 72 and ammonium sulfate were obtained from Schwarz-Mann Division of Becton, Dickinson and Company. Glycylglycine, chloride free, was obtained from Nutritional Bio-chemicals and glutathione, reduced (dSH), was purchased from C. F. Boehringer and Soehne GmbH. Heart infusion broth, dehydrated, was supplied by Fisher S c i e n t i f i c and Difco Laboratories, while the l a t t e r company also supplied special agar. Nitrogen gas, L grade (99.97$ N 2) was purchased from Canada Liquid Air Ltd. The following reagents were prepared i n the laboratory: 3-phospho-glyceraldehyde, free acid; monoethanolamine hydrochloride; monoethanolamine; triethanolamine; and iron-free sodium citrate. The procedures used are described in brief below. The conversion of DL-glyceraldehyde-3-phosphate diethylacetal, barium salt,to DL-glyceraldehyde-3-phosphate was carried out by the tech-nique of Meloche and Wood (1966), with a slight modification. Dowex AG-50 resin (H + form) was added to DL-glyceraldehyde-3-phosphate diethylacetal, barium salt u n t i l the l a t t e r dissolved and the pH of the solution was approximately 2.5. The solution was f i l t e r e d to remove the AG-50 resin and the f i l t r a t e was incubated at 35°C for 48 hours to hydrolyze the acetal. The resulting solution was diluted to the required concentration. Ethanolamine hydrochloride was prepared by the careful addition of a slight excess of concentrated hydrochloric acid to monoethanolamine. The resulting solution was allowed to cool and was then diluted with d i s t i l l e d water. The monoethanolamine hydrochloride was precipitated at 4°C by the addition of absolute ethanol to the aqueous solution. The crystals were 73 collected by f i l t r a t i o n and purified by twice recrystallizing from aqueous ethanol. A melting point of 81-83°C was obtained. Ethanolamine and triethanolaraine solutions were prepared from solutions of ethanolamine hydrochloride and triethanolamine hydrochloride respectively, by passage through Dowex AG1-X4 (0H~ form) resin columns. The ethanolamine and triethanolamine solutions obtained were tested for the presence of chloride by the addition of s i l v e r nitrate. Iron-free sodium citrate solutions were prepared by passage of the sodium citrate solutions through a Chelex 100 column (Na + form). A l l other chemicals were reagent grade. 2.1.3 Equipment The bacteria were harvested with either a Sorval centrifuge, model RC2-B, with GSA and SS-34 rotors, or a Beckman Spinco model J-21 centrifuge with J-10 and J-20 rotors. The cell-free extract was prepared from disrupted E. c o l i by u l t r a -cent rifugation i n a Beckman Spinco, model L2-65B, ultracentrifuge using a number 65 rotor. The absorbance of bacterial cultures was measured with a Beckman model B, single beam spectrophotometer or a Coleman (Hitachi) model 124 double beam spectrophotometer. Quartz cuvettes of 10 mm path length were employed with the former instrument, while 10 mm path length quartz cuvettes with a 9 mm quartz path reduction bar, reducing the effective path length to 1 mm, were used with the l a t t e r instrument. A Coleman model 124 spectophotometer coupled to a Sargent model SRG recorder was used for 3-pbosphoglyceraldehyde dehydrogenase, succinate dehydrogenase and NADH oxidase assays, and for the determination of total 74 iron and protein. Quartz cuvettes of 10 mm path length were used in the enzyme assays and total iron determinations. Protein determinations were performed with optical glass cuvettes of 10 mm path length. Dithionite-reduced minus hydrogen peroxide-oxidizing difference spectra were determined with a Cary model 15 spectrophotometer u t i l i z i n g the zero to 0.1 absorbance scale and quartz cuvettes of 10 mm path length. A Techtron IV atomic absorption spectrophotometer (Cary Instruments) with a hollow cathode lamp (serial no. AC761), and air-acetylene flame, was used for the determination of s i l v e r . Bacterial cells were disrupted with either a Branson Sonifier, model W-185-C, or with an American Instruments Co., Inc., French pressure c e l l , 4-338 and French pressure c e l l press, 5-598A. Oxygen consumption of E. c o l i c e l l suspension was monitored with: (i) an American Instrument Co., vibrating platinum electrode, C151-62081, coupled to a Varian s t r i p chart recorder, model G-14A-2; ( i i ) a Gilson Medical Electronics Inc., Oxygraph model K-1C, with a Yellow Springs Instru-ment Co., Inc., model 4004 Clark-type oxygen electrode; or ( i i i ) a Yellow Springs Instrument Co., oxygen monitor model 55, with a Yellow Springs Instrument Co., model 5533 Clark-type oxygen electrode, coupled to a Varian model G-14A-2 recorder. The la t t e r instrument and electrode combination, without the coupled recorder, was used to monitor the oxygen level of growing cultures of E. c o l i (section 2.2.6.1). Measurements of hydrogen ion concentration were made with a Fisher Sci e n t i f i c Accumet pH meter, model 310, in combination with one of the following electrodes. For routine buffer preparation and for monitoring the pH of the bacterial cultures, a Fisher S c i e n t i f i c standard combination 75 pH electrode, 13-639-90, was used. A Radiometer microcombination pH electrode, GK2321C, modified for \ise with the Fisher Accumet pH meter, or a Fisher Sc i e n t i f i c microprobe combination pH electrode, 13-639-92, was used to adjust the pH during total iron determinations. The la t t e r elec-trode was also employed to measure acid production concurrent with oxygen consumption in E. c o l i c e l l suspensions. The Accumet pH meter was coupled with a Varian model G-14A-2 s t r i p chart recorder for these determinations. The redox potential of growing cultures of E. c o l i was measured with a Photovolt pH meter, model 115, with a Beckman platinum redox elec-trode 3 9 2 7 3 . The Ag+/AgCl h a l f - c e l l of the Fisher S c i e n t i f i c standard combination pH electrode, 13-639-90 was employed as the reference electrode. The rate of aeration of the growing bacterial cultures was moni-tored with a Rogers Gilmont Instruments, size 4, f l u i d flowmeter. Aera-tion of the culture was provided v i a a Kimax 12C sintered glass sparger. A Buchler Polystaltic pump R was employed to deliver 5 N H C 1 , or 5 N NaOH, to the culture vessel for the regulation of the pH of the bacterial culture. Circulation of the culture through the electrode vessel (Figure 2 . 1 ) was provided via a Gorman-Rupp Industries 60 cycle o s c i l l a t i n g pump. Media were f i l t e r e d through 0.45 u MF-Millipore membrane f i l t e r s (47 "im diameter) and pre f i l t e r s of glass fiber bound with a starch binder, in a pyrex Millipore f i l t e r holder. Either a New Brunswick S c i e n t i f i c Co., Inc., reciprocal water bath shaker, model R 7 6 , or a Labline Inc., reciprocal water bath shaker 3581 was used for growth of the inoculating bacterial cultures. 2.1.4 Media The following media were used during the research described in this 76 thesis. Medium A corresponds to the medium of Davis and Mingioli (195C1) and consists of 40.2mM K^HPO^, 22.0 mM KH 2P0 4, 0.8 mM MgS04, 7.6 mM (NH 4) 2S04 and 1.7 mM sodium citrate. Medium B differed from medium A in the deletion of sodium citrate. Medium C differed from medium A in four respects: (i) sodium citrate was deleted; ( i i ) the (NH^^SO^ used in the medium was ultrapure grade; ( i i i ) the concentration of (NH^^SO^ was 22.7 mM, and (iv) the medium was fi l t e r e d through a 0.45 u MF-Millipore membrane f i l t e r to reduce iron content, prior to the addition of MgSO^. This medium w i l l be referred to as iron-deficient medium. Medium D corresponds to medium C to which f e r r i c citrate was added to a f i n a l concentration of 6 uM. This medium w i l l be referred to as iron-sufficient medium. The media for growth of strains of E. c o l i K12 were supplemented with 500 ug of thiamine per l i t r e , while E. c o l i K12 met" was grown with 2 mg methionine per l i t r e . Streptomycin at a concentration of 1 g / l i t r e was added to the media for growth of the streptomycin dependent strains of E. c o l i . The pH of a l l media was adjusted to 6.9 to 7.1. Heart infusion broth-agar for petri plates and slants consisted of 2.5$ dehydrated heart infusion broth and 1.5$ agar. Minimal salts-glucose-agar slants were prepared from medium B with the addition of glucose (2 g / l i t r e ) , and agar (15 g / l i t r e ) . 2.2 Methods 2.2.1 Culturing procedures for bacteria 77 2.2.1.1 Maintenance of stock cultures Stock cultures of E. c o l i were maintained at 4°C on minimal sal t s -glucose-agar slants, or heart - infusion broth-agar slants in the case of E. c o l i K12 met". Cultures were transferred on a monthly basis. 2.2.1.2 Growth of cultures for inoculation Growth of an inoculum was commenced 48 hours prior to starting an experiment. Bacteria of the desired strain of E. c o l i were aseptically removed from a stock culture slant with a platinum or Nichrome wire inocu-lating loop, and were inoculated into a tube containing 10 ml of medium A or B (section 2.1.4) with a glucose concentration of 0.4$ (w/v). The culture was incubated without shaking at 35-37°C for approximately 24 hours at which time 2.5 ml were inoculated into 25 ml of the desired medium (section 2.1.4), in a 100 ml Erlenmeyer flask, which contained the same carbon source, at the same concentration as was to be used in the f i n a l growth. The cultures were incubated in a reciprocating water bath shaker at 37°C for approximately 12 hours. A sufficient number of 250 ml Erlenmeyer flasks containing 100 ml of s t e r i l e medium of the desired type and carbon source were inoculated with 2.5 ml of the immediately preceding culture, to provide a volume of inocu-lating culture equivalent to one-tenth to one-twentieth the volume of the f i n a l culture. The inoculating cultures were incubated at 37°C for 12 to 13 hours in a reciprocating water bath shaker. 2.2.1.3 Culture of the bacteria Due to variations in the culturing conditions, they w i l l be des-cribed in the appropriate sections, 2.2.2 Demonstration of s i l v e r release from the Aminco oxygen electrode 78 2.2.2.1 Growth and harvesting Five hundred to 700 ml of freshly prepared, s t e r i l e medium A, containing glucose at a f i n a l concentration of 0.4$ (w/v), in a two l i t r e Erlenmeyer flask was inoculated with 50 to 70 ml of inoculating culture. The culture was grown at 37°C with an a i r flow rate of 6.7 l/min. Evapor-ation and cooling were minimized by bubbling the a i r through d i s t i l l e d water at 37°C prior to i t s entry into the culture. Growth of the culture was monitored v i a the absorbance at 420 nm and the cells were harvested in mid-logarithmic growth phase at an absorbance of 3.5 to 4.5. Cells were harvested in stainless steel contrifuge bottles at 5,800 x __ for 10 minutes at 4°C. The medium was decanted and the cells were suspended in 20 ml of 0.85$ NaCl per centrifuge bottle. The resuspended cel l s were transferred to 50 ml cellulose nitrate centrifuge tubes and were centrifuged at 4,300 x _ for 10 minutes at 4°C. The cells were resuspended in 10 ml of 0.85$ NaCl per tube and were transferred to two weighed 50 ml cellulose nitrate centrifuge tubes. The c e l l suspensions were recentrifuged at 4,300 x __ for 10 minutes at 4°C, the supernate was decanted, the tubes were drained, wiped dry and weighed. The wet weight of the bacteria was calculated by difference. In order to reduce the endogenous respiration the ce l l s were resus-pended in 0.85$ NaCl at a dilution of 1:80 (w/v) and incubated at 37°C with aeration for 15 to 60 minutes. At the end of this time the c e l l suspension was centrifuged at 4,300 x for 10 minutes at 4°C, the supernatant f l u i d was decanted and the c e l l pellet was kept on crushed ice u n t i l required. When required, the cells were resuspended to the desired dilution in cold 300 mM glycylglycine-KOH buffer of pH 7.0. 79 2.2.2.2 Measurement of oxygen consumption The oxygen consumption of the bacterial suspensions was measured with an Aminco oxygen electrode system consisting of a vibrating cathode of platinum wire sealed in a glass capillary, except for the t i p . A bare s i l v e r wire was the stationary anode. The electrode was operated with a polarizing voltage of approximately 0.6 V. The current after amplification, was recorded continuously. The assay system u t i l i z e d for the investigation of the effect of c e l l concentration on respiration rate consisted of air-saturated, 270 mM glycylglycine-KOH buffer, pH 7.0, 1.8 mM, potassium phosphate buffer, pH 7.0, 0.5 mM KC1, 25 mM glucose and 200 u l of bacterial c e l l suspension, of increasing dilutions from 1:6 (w/v) to 1:26 (w/v), in a f i n a l volume of 4.00 ml. The assay system used subsequently for the investigation of the non-linear relationship between c e l l concentration and respiration rate was composed of air-saturated, 281 mM glycylglycine-KOH buffer, pH 7.0, 1.8 mM potassium phosphate buffer, pH 7.0, 0.5 mM KC1, 26 mM glucose and 40-200 u l of bacterial c e l l suspension at a dilution of 1:10 (w/v). The f i n a l volume of the assay system was 3.8 ml to 4.00 ml. A l l determinations were carried out at room temperature (22°C). The Aminco oxygen electrode was found to be inadequately grounded when used separate from the Aminco-Chance dual wavelength spectrophotometer. Also, s t i r r i n g in addition to the vibration of the platinum electrode was found to be essential in order to maintain a homogeneous suspension and a stable diffusion current when measuring the respiration of intact c e l l s . Adequate s t i r r i n g was accomplished by means of a Teflon-coated magnetic 80 f l e a and magnetic s t i r r e r . The s t i r r i n g rate was regulated via a Powerstat variable autotransformer. High s t i r r i n g rates were avoided in order to minimize the back diffusion of oxygen into the system. The oxygen level was expressed as a percentage of the oxygen content of the air-saturated buffer. Oxygen consumption rate was expressed as the decrease in percent oxygen saturation per minute per milligram-of bacterial wet weight. 2 . 2 . 2 . 3 Determination of c e l l v i a b i l i t y The determination of the v i a b i l i t y of E. c o l i was performed accord-ing to the "pour-plate" method as described by Harrigan and McCance (1966). A 1.0 ml aliquot was removed at the desired time intervals after the addition of c e l l suspension to the assay system. The samples were s e r i a l l y diluted to a f i n a l c e l l concentration of 30 to 300 bacterial cells per m i l l i l i t r e in one-quarter strength Ringer's solution. One m i l l i l i t r e aliquots of the two highest dilutions were inoculated onto heart infusion broth-agar medium. Plates were prepared i n t r i p l i c a t e . The sample was spread and then overlayed with molten T/o agar ('—'45°C). The petri plates were incubated at 35-37°C for 24 hours and the colonies marked and counted. The petri plates were re-incubated, under the same conditions for an addi-tional 24 hours and were examined for the appearance of additional colonies. The number of viable cells was calculated for each sample, and these were analyzed for significant differences v ia the "unpaired t-test". 2.2.3 Measurement of s i l v e r release from the Aminco oxygen electrode 2.2.3.1 Sample preparation Samples for s i l v e r analysis were collected under operational con-ditions identical to those used in measuring oxygen tension. Both the 81 anode and the cathode were par t i a l l y immersed in 4.0 ml of buffer solution in a 5 nil v i a l which had previously been cleaned by soaking in 7.5 N HNO^  and thoroughly rinsed with glass d i s t i l l e d water. The buffer solution consisted of air-equilibrated buffer, pH 7.0, and potassium chloride at a f i n a l concentration of 0.5 mM. In the preparation of buffers, glycylglycine and HEPES -were adjusted to the required pH with KOH. Phosphate buffer was prepared from potassium phosphates, and Tris was adjusted to the required pH with HC1, Samples were obtained by removing the oxygen electrode from the buffer solution after a predetermined period of time and subsequently adding 10 u l of 7.5 N n i t r i c acid (final concentration of 18 .7 milliequivalents per l i t r e ) to reduce the tendency of s i l v e r ions to adhere to the glass v i a l s . For the same reason the samples were assayed within 4 to 5 hours of their collection. 2.2.3.2 Atomic absorption spectrophotometry The s i l v e r content of the samples was determined by atomic absorption spectroscopy at a wavelength of 3280.7 A using a Techtron IV atomic absorp-tion spectrophotometer. Standard solutions of s i l v e r nitrate were prepared in the appropriate buffers with n i t r i c acid present at a f i n a l concentration of 18.7 milliequivalents per l i t r e . 2.2.4 The influence of pH, buffer ion and buffer concentration on the respiration of E. c o l i 2.2.4.1 Growth and harvesting E. c o l i NRC 482 was grown and harvested under conditions identical to those described in section 2.2.2.1. However, the procedure to reduce the endogenous respiration differed s l i g h t l y from that described previously (section 2.2.2.1). The c e l l pellet was suspended 1:80 (w/v) in 0.85$ NaCl and the c e l l suspension was incubated at 37 C without aeration for 60 minutes. At the end of the incubation, 20 ml aliquots of c e l l suspension were trans-ferred to weighed 50 ml cellulose nitrate centrifuge tubes and were centri-fuged at 7>700 x __ for 5 minutes at 4°C. The supernates were decanted, the tubes were drained, were wiped dry and then were weighed. The wet weight of the E. c o l i c e l l pellet was determined by difference. The c e l l pellet was retained in crushed ice u n t i l needed and then was resuspended at a dilution of 1:10 (w/v) in the appropriate buffer. 2.2.4.2 Measurement of oxygen consumption A Yellow Springs Instruments oxygen monitor, equipped with a Clark-type oxygen electrode and operating at a polarizing voltage of approximately 0.8 V, was employed to determine the oxygen consumption. The assay system consisted of 5.0 ml of air-saturated buffer, 0.5 mM KC1, 9.6 mM glucose, and 50 ul of c e l l suspension i n a total volume of 5.2 ml. The measurement of oxygen consumption was carried out at 37°C. The isotonic buffers were prepared by mixing the isotonic solutions of the salt-form of a buffer with the isotonic solution of the corresponding free acid or free base, in the required proportions to obtain the desired pH. With the exception of the isotonic solutions of KB^PO^ and K^HPO^ whose concentrations were obtained from the Merck Index, the concentrations of the salt form, and the free-acid and free-base form of the buffer corresponding to isotonicity were calculated according to the Van't Hoff equation: <7T = CRT where 7f-is the osmotic pressure, C - the molar concentration, R - the gas constant and T - the absolute temperature. The following assumptions or approximations were made in performing 83 these calculations: (i) the salt-form of the buffer was considered to be f u l l y dissociated; ( i i ) the addition of a solution of the free-acid, or free-base form of the buffer to the salt-form was assumed to have an insigni-ficant affect on the degree of dissociation of the salt-form of the buffer; and ( i i i ) the influence of the hydrogen ion concentration on tonicity was considered negligible. The acid-base pairs used in the investigation of the influence of pH and buffer ion on the respiration of E. c o l i were: c i t r i c acid (0.29 N)-sodium citrate (0.1 N); Tris hydrochloride (0.13 N)-Tris (0.26 N); sodium phosphate (monobasic) (0.18 N)-sodium phosphate (dibasic) (0.12 N); MOPS (0.26 N)-M0PS-Na+ (0.13 N); HEPES (0.26 N)-HEPES«Na+ (0.13 N); glycylglycine (0.26 N)-sodium glycylglycinate (0.13 N); triethanolamine hydrochloride (0.13 N)-triethanolamine (0.26 N); ethanolamine hydrochloride (0.13 N)-ethanolamine (0.26 N); and glycine (0.26 N)-sodium glycinate (0.13 N). The concentrations in brackets are those of the isotonic stock solutions used in the preparation of the isotonic buffers of desired pH. The buffers were prepared to give the specified pH at 37°C. 2 . 2 . 4 . 3 Protein Determination Aliquots of 0.5 ml of c e l l suspension in buffer (1:10 w/v) were added to 4.5 nil of 1.1 N NaOH and were mixed thoroughly. Protein deter-minations were performed on 0.5 ml aliquots of this solution by the method of Lowry et a l . , (1951). Crystalline bovine serum was used as protein standard. The standard protein solutions were prepared i n the appropriate buffer solutions as the buffer ion and concentration had a marked influence on the color development (Appendix B) (Turner and Manchester, 1970). 2.2.5 The influence of s i l v e r on the respiration of E. c o l i 84 2.2.5.1 Growth and harvesting E. c o l i NRC 482 was grown and harvested according to the conditions described in section 2.2.2.1. The carbon sources used were 0.4$ glucose, 0.4$ glycerol, 0.8$ DL-lactate, 0.4$ succinate and 0.4$ fumarate. Two procedures for the reduction of endogenous respiration were used, dependent upon the parameters to be measured subsequently. When only oxygen consumption was to be measured the endogenous respiration was reduced by resuspending the c e l l s 1:80 (w/v) in 0.85$ NaCl and incubating at 37°C for 5 minutes with gentle aeration. Twenty m i l l i l i t r e aliquots were removed to weighed 50 ml cellulose nitrate centrifuge tubes. The c e l l suspension was centifuged at 4,300 x __ for 10 minutes at 4°0, the supernates were decanted and the tubes were then drained, wiped dry and weighed. The weight of the c e l l pellet was calculated by difference. The c e l l pellet was kept in crushed ice u n t i l required, at which time the c e l l s were resuspended 1:10 (w/v) i n 300 mM glycylglycine-KOH buffer, pH 7 . 0 . However, i f acid production and oxygen consumption were to be measured simultaneously, the endogenous respiration was reduced by resus-pending the c e l l pellet 1:80 (w/v) in medium B. The c e l l suspension was incubated at 37°C for 90 minutes with gentle aeration. On completion of the incubation, 40 ml aliquots of c e l l suspension were transferred to weighed 40 ml screw-cap, polycarbonate centrifuge tubes (Oak Ridge type, wide mouth) and were centrifuged immediately at 7,700 x _ for 10 minutes at 4°C. The supernatant f l u i d was decanted, the tubes were drained, were wiped dry, and were weighed. The weight of the c e l l pellet was calculated by difference. The c e l l pellets were maintained at room temperature, with the 85 centrifuge tube tightly capped to prevent dehydration of the pellet, u n t i l required. The duration at room temperature, prior to the assay of oxygen u t i l i z a t i o n and acid production was kept as short as possible (ca. 1 hour). The c e l l pellet was resuspended in 3 mM glycylglycine-KOH buffer, pH 7.5 to a f i n a l c e l l concentration of 0.1 g wet weight of E. c o l i per ml of c e l l suspension. 2.2.5.2 Measurement of oxygen consumption The conditions used for the measurement of respiration only were the same as those specified in section 2.2.4.2. The buffer component of the assay system was 300 mM glycylglycine-KOH, pH 7 . 0 . Substrates used in addition to glucose, were glycerol, D-lactate and L-lactate as lithium salts, potassium formate, potassium acetate, disodium succinate, and pot-assium fumarate, a l l at a f i n a l concentration of 9.6 mM. Silver nitrate was added to the "test" samples to yield a f i n a l concentration of 86 uM (450 nmoles of AgNO^ were added to the assay system). Appropriate controls were performed. 2.2.5.3 Concurrent measurement of oxygen consumption and protein production The oxygen consumption was measured with a Yellow Springs Instruments oxygen monitor, with Clark-type oxygen electrode. Hydrogen ion concentration was monitored with a Fisher Accumet pH meter, employing the expanded scale mode of operation, coupled with a Fisher microprobe combination pH electrode. The concurrent measurement of oxygen u t i l i z a t i o n and acid production by E. c o l i NRC 482 was carried out in separate, identical assay systems. El e c t r i c a l interaction of the two electrode systems necessitated this arrangement. The components of the assay system were 2.9 mM glycylglycine-KOH 06 buffer, pH 7«5» 9.6 mM glucose, and 50 to 200 ul of c e l l suspension. The total volume was 5.2 ml. The "test" samples also received the addition of 125 nmoles of AgNO^  (final concentration - 24 >iM) subsequent to establishing the substrate-dependent rate of oxygen u t i l i z a t i o n and the rate of respira-tion-associated acid production. A l l determinations were performed at 37°C. 2.2.5.4 Assay for glyceraldehyde-3-phosphate dehydrogenase B. c o l i NRC 482, grown and harvested as described in section 2,2.2.1, were resuspended 1:5 (w/v) in 30 mM Tris-HCl buffer, pH 8.0. The c e l l sus-pension was disrupted with a French press at a pressure d i f f e r e n t i a l of 20,000 p.s.i. The disrupted c e l l s were centrifuged at 95»000 x g_ for two hours. The cell-free supernate was carefully removed with a Pasteur pipette and was kept in an ice bath. The assay system was comprised of, in order of addition: 12.1 mM Tris-HCl buffer, pH 8.0, 1.7 mM glyceraldehyde-3-phosphate, 0.3 mM NAD+, 4.9 mM cysteine, 20.6 mM sodium arsenate, 10 ul of enzyme source. The f i n a l volume was 2.56 ml. The assay was carried out at room temperature (22°C). The progress of the reaction was monitored v i a the increase in absorbance of the assay system at 340 nm. Due to the insol u b i l i t y of s i l v e r arsenate (Ksp = 1 x 10" " mole3 / l i t r e ^ ) , s i l v e r could not be added directly to the assay system. The pro-cedure adopted was to preincubate the cell-free supernate with an equal volume of 0.5 mM or 5 mM AgNO^ solution. Ten microlitre aliquots were removed at recorded time intervals during the incubation and the glyceralde-hyde-3-phosphate dehydrogenase activity was measured. The s i l v e r concentra-tions during the preincubation at room temperature were 0.25 mM and 2.5 mM. The corresponding concentrations in the f i n a l assay system were 1 uM and 87 10 uM, respectively. 2.2.6 The influence of iron limitation on respiration of K. c o l i 2.2.6.1 Growth and harvesting The bacteria were grown on medium C or D, with either 0.6$ succinate or 0.4$ glucose as the carbon source. Additions of f e r r i c citrate during growth, were of the required volume of a stock solution to yield a concen-tration of 6 uM in the culture medium. The culture was grown in the apparatus shown in Figure 2.1. The culture vessel (12) was a brown glass jar with a capacity of 2500 ml. The culture was continuously circulated through a second sealed vessel (8) (50 ml capacity) at a rate of 200-250 ml/min by means of a 60 cycle o s c i l -lating pump (9). This vessel (8) contained a combination glass pH electrode (4)» a platinum redox electrode (5)> a Clark-type oxygen electrode (6), a small, Teflon-coated magnetic s t i r r i n g bar (7) and was supported on a mag-netic s t i r r e r ( 10) . The s t i r r i n g rate was regulated v i a a Powerstat variable autotransformer. Growth was initiated by inoculation of 1500-1800 ml of medium with 100 ml of inoculating culture (section 2 . 2 . 1 . 2 ) . The culture was grown at 37°C with aeration through a sintered glass sparger (15) at an a i r flow rate of 4.4 l/min/l of culture. Growth was monitored by following the absorb-ance of the culture at 420 nm. The pH, oxygen tension and oxidation-reduc-tion potential of the culture were determined at intervals of 15 or 30 min, depending on the rapidity of change in these parameters. The pH of the culture was maintained at pH 7.00 - 0.15 by the addition of 5 N HC1, or 5 N NaOH v i a a pe r i s t a l t i c pump. The cells were harvested through the sampling port (11) at specific / / u \ i it n 88 1. 2. 3. 4. 5. 6. 7. - - - 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Fig. 2.1 Culture apparatus. 1. Air Input 2. F i l t e r 3. Flow Meter 4. Combination pH Electrode 5. Platinum Redox Potential Electrode 6. Clark Oxygen Electrode 7. Magnetic Flea 8. Electrode Vessel 9. Oscillating Pump 10. Magnetic S t i r r e r 11. 3-Way Stopcock, Sampling Port . 12. Culture Vessel (2.5 Litre) 13. Water Bath 14. Air Vent, and Addition Port 15. Sparger 16. Evaporation-Control Flask 17. Stirrer 89 times throughout growth and were sedimented immediately by centrifugation of the c e l l suspension at 12,000 x _ for 10 min at 2°C. The cells were washed twice by sedimentation from a wash medium at 12,000 x g_ for 10 min at 2°C. The wash medium used depended upon the assays to be performed sub-sequently. I f only the cytochrome contents were to be measured, the cells were washed with medium C. However, i f total iron was to be determined in addition to the cytochrome levels, the c e l l s were washed with glass d i s -t i l l e d water to minimize the retention of adsorbed iron by the c e l l s . I f , in addition, enzyme a c t i v i t i e s were to be assayed, then the cells were washed with 0.1 M Tris-HCl buffer, pH 7«5» prepared from ultrapure- Tr i s . The c e l l pellet obtained after washing was kept at 0°C u n t i l required. 2.2.6.2 Estimation of the efficiency of succinate conversion to c e l l mass An approximate measurement of the efficiency with which the c e l l culture could convert disodium succinate to c e l l mass was obtained by calculating the increase in absorbancy of the culture at 420 nm over a specified time interval relative to the increase in pH, produced by the u t i l -ization of succinate, for the corresponding time period. Since the change in pH was small (0.04 to 0.15 pH units) and over the same pH range, pH 7»00 i 0.15, i t was proportional to the amount of succinate oxidized. The absorb-ance at 420 nm was s t r i c t l y proportional to wet c e l l mass (Appendix A). "Efficiency" was defined.as the change in the absorbance of the culture at 420 nm during a time interval (generally 30 min) divided by the change in the pH of the culture during the same time interval. 2.2.6.3 Measurement of the effect of dibromophenol and s i l v e r nitrate on oxygen consumption Respiration of intact E. c o l i NRC 482, in the presence or absence of 90 dibromophenol (DBP) or si l v e r nitrate, was determined at 22°C using an oxygraph equipped with a Clark-type oxygen electrode. The assay procedure i s as follows. Forty microlitres of c e l l sus-pension in 0.1 M Tris-HCl buffer, pH 7 .5 , (1:10 w/v) were added to 3.9 ml of air-saturated Tris-HCl buffer, pH 7 .5, and the endogenous respiration was recorded for 3 minutes. At the end of this time 20 u l of M glucose was added (final concentration, 5 mM). When the glucose-dependent respiration . rate was established, DBP or AgNO^  were added to the desired f i n a l concen-tration. 2 . 2 . 6 . 4 Respiratory control ratio Hempfling (1970b) has defined the ratio of the rate of oxygen u t i l -ization in the presence of DBP to the rate of oxygen u t i l i z a t i o n in the absence of DBP as the respiratory control ratio (RCR). This definition has been adopted in this thesis and i t s application extended to experiments in which DBP has been replaced by AgNO^. 2 . 2 . 6 . 5 Determination of total iron and nonheme iron The washed c e l l pellet was suspended 1:10 (w/v) in glass distilled., water and a 2.0 or 3 .0 ml sample was transferred to a labelled micro^Kjeldahl flask, calibrated to contain 10.0 ml. The sample was frozen in a dry ice-ethanol freezing mix and subsequently lyophilized. An appropriate blank of glass d i s t i l l e d water was carried through the entire procedure. The total iron content of the lyophilized c e l l suspension was deter-mined according to the method of King e_ a l . , (1964). The procedure was as follows. One m i l l i l i t r e of concentrated ^SO^ was added to each micro-Kjeldahl flask. The flasks were heated gently on Kjeldahl digestion heaters and rotated periodically u n t i l a l l c e l l material had gone into solution. 91 The temperature then was increased and heating was continued u n t i l the samples became viscous. The flasks were allowed to cool and 0.5 ml of 30$ hydrogen peroxide was added dropwise to each sample. The procedure of heat-ing, cooling and adding 30$ hydrogen peroxide was repeated u n t i l a clear colorless solution was obtained. After cooling the following reagents were added to the digested sample in the order indicated: 1.0 ml of 0.25$ £-phenanthroline (aqueous solution), 0.5 ml of 1.0$ hydroquinone (freshly prepared), 2.5 ml of 25$ sodium citrate. The mixture was adjusted to pH 3.8 to 4.3. (pH meter) by the addition of 28$ NH^ OH and made up to a volume of 10.0 ml with glass d i s t i l l e d water. The solutions were incubated at room temperature for 60 min, the absorbance at 510 nm was measured and the content of iron was -1 -1 calculated using an extinction coefficient of 11.1 mM cm . Nonheme iron was calculated as the difference between the total iron and the heme iron. The value used for the level of heme iron was the cyto-chrome b^ content unless the level of cytochrome in the cells was suf-f i c i e n t l y high to be quantitated in which case the sum of the levels of cytochrome b-j and cytochrome was used as the content of heme iron. 2 . 2 . 6 . 6 Preparation of c e l l extracts The washed c e l l pellet was suspended 1:10 (w/v) in medium C, glass d i s t i l l e d water or 0.1 M Tris-HCl buffer, pH 7 .5, depending upon the deter-minations to be performed (section 2 . 2 . 6 . 1 ) . The c e l l suspension was di s -rupted at 0°C by two pulses of sonication, each of 30 sec duration, separ-ated by a 30 sec period, unless the c e l l extract was to be used for assaying enzyme activity in which case a single period of sonication of 30 sec dura-tion was used. 2 . 2 . 6 . 7 Determination of cytochromes a-, and b. 92 The levels of cytochrome a^ ^2.-] ^ n c e l l extracts were deter-mined from dithionite-reduced minus hydrogen peroxide-oxidized difference spectra. Three successive scans were carried out on each sample to ascer-tain that complete reduction and oxidation of the contents of the respec-tive cuvettes had been obtained. The content of cytochrome a^ was calculated from the height (in absorbance units) of the peak at 630 nm as measured along a vertical line drawn from the spectral recording at 63O nm to an extrapolation of the baseline at this point as shown in Figure 2.2. The level of cytochrome b_-j was calculated in an analogous manner from the height (in absorbance units) of the peak at 560 nm. The peak height was measured along the vertical line drawn from the spectral trace at 56O nm to a point of intersection with a second line drawn through the spectral recording at 540 nm, and tangent to the spectral trace in the region of 580 nm as shown in Figure 2.2... The extinction coefficient used in the calculation of cytochrome ^ levels was -1 -1 8.51 mM cm (Jones and Redfearn, 1966) while the extinction coefficient —1 —1 used for cytochrome b^ was 16,0 mM" cm"" (Deeb and Hager, 1964). 2.2.6.8 Assay for succinate dehydrogenase The succinate dehydrogenase present in the c e l l extract was "activated" by incubating 0.5 ml of c e l l extract and 0.5 ml of 0.1 M disodium succinate, pH 7.5, at 37°C for 10 min prior to assaying. To a cuvette containing 1.0 ml of reaction mix, consisting of 2 mM KCN, 68 uM DCIP, 27.3 mM disodium succinate and 41 mM potassium phosphate buffer, pH 7.5, 5 ul of "activated" c e l l extract was added followed by the addition of 20 ul of 9 mM PMS. The succinate dehydrogenase activity was calculated from the rate of decrease in the absorbance at 600 nm. The assay 0.9 0 4 1 ' ' 1 ' ' 1 1 ' 1 1 1 1 1 550 600 650 Wavelength (nm) Fig. 2.2 Dithionite reduced-minus-oxidized difference spectrum, (.method: sec. 2.2.6.7). 94 was performed at 2?°C, Calculation were performed using an extinction coefficient for DCIP of 21.8 m!vf1cm~1. 2 . 2 . 6 . 9 Assay for succinate oxidase Succinate oxidase activity was determined from the rate of oxygen consumption on the addition of 200 ul of "activated" c e l l extract (sec.2.2.6.8) to 5.0 ml of 0.1 ml potassium phosphate buffer, pH 7 . 5 , containing 19.6 mM disodium succinate. Oxygen consumption was measured at 37°C with a Clark-type oxygen electrode. The micromoles of oxygen consumed were calculated from an oxygen s o l u b i l i t y of 0.199 umoles/ml for air-equilibrated, t r i -ethanolamine buffered mitochondrial medium, pH 7 .2, at 37°C (Lessler, M.A., 1972). 2 . 2 . 6 . 1 0 Assay for NADH oxidase NADH oxidase was measured spectrophotometrically at 22°C by measur-ing the decrease in absorbance at 340 nm following the addition of 50 ul of ce l l extract to 2.0 ml of 0.1 ml potassium phosphate buffer, pH 7 . 5 , contain--1 -1 ing 0.3 mM NADH. An extinction coefficient for NADH of 6.22 mM cm was used for the calculations. 2.2.6.11 Glucose determination Ten m i l l i l i t r e aliquots of c e l l suspension were removed as required and centrifuged at 0°C for 10 min at the maximum speed of a c l i n i c a l centri-fuge (approximately 2,000 x g_). The supernate was removed carefully and used directly, or after dilution with 50 mM phosphate buffer, pH 7 . 0 , for determination of the glucose content via the micro-Glucostat procedure. 95 3 . PART I: SILVER IONS AND THE RESPIRATION AND ENERGY-COUPLING OF E. COLI ' 3.1 Results 3.1.1 Release of s i l v e r from the Aminco oxygen electrode During preliminary experiments on factors influencing the respira-tion rate of E. c o l i , an investigation of the respiration rates of E. c o l i as a function of the quantity of c e l l s in the assay system was undertaken (Figure 3.1). (The data has been replotted from continuous traces for con-venience of presentation.) Curves A to E correspond to 33 mg, 18 mg, 12 mg, 8 mg, and 2 mg cells wet weight, respectively. Nearly linear oxygen u t i l i z -ation traces, and a linear relationship between the quantity of cells and the respiration rate had been expected. However, the results demonstrated: (i) a greater decrease than expected in the respiration rate with increasing d i l u -tion of the c e l l suspension; ( i i ) with decreasing c e l l concentration the shape of the oxygen u t i l i z a t i o n trace changed from linear (Curve A) to sigmoid (Curve D); and ( i i i ) at a s u f f i c i e n t l y low c e l l concentration there was a complete cessation of respiration prior to depletion of oxygen from the sys-tem (Curve E). A number of alternative explanations of the observed results were apparent: (i) an increasing loss of c e l l v i a b i l i t y , during the course of the assay, with increasing dilution of the c e l l suspension; ( i i ) the existence of respiratory control; or ( i i i ) an inhibition of respiration. Of primary con-cern was the v i a b i l i t y of the c e l l s which had ceased to respire prior to the depletion of oxygen from the assay system. Viable c e l l counts as determined by the "pour-plate" technique (sec.2.2.2.3) (Table 3.1) indicated that there was no significant difference between the number of viable c e l l s present in the assay system prior to the cessation of respiration, and the number 10 Minutes Fig. 3.1 Effect of c e l l concentration on oxygen consumption by E. c o l i c e l l suspensions as measured with the Aminco oxygen electrode. A: 33 mg; B: 18 mg; C: 12 mg; D: 8 mg; E: 2 mg; (method: sec.2.2.2.2). ON 97 Table 3.1 The v i a b i l i t y of E. c o l i before and after the cessation of oxygen consumption. Time1 Viable Cells " t " test (per ml) value  Before (6.69 ± 0.44)x108 1.19 After (6.31 - 0.27)x108 ( &.= 0.05, d.f. = 4) relative to the cessation of oxygen u t i l i z a t i o n (with reference to curve E of Figure 3.1, "before" corresponds to a time of about 1 minute while "after" would correspond to a time of approximately 8 minutes.) 98 present when respiration had ceased. These results suggested that the ces-sation of respiration prior to the depletion of oxygen from the assay system was due to either respiratory control or inhibition of respiration. Prior to further examination of the cessation of respiration at low oxygen concentrations, the ubiquity of the phenomenon in strains of E. c o l i was examined. The following strains of E. c o l i : E. c o l i ATCC 8739, E. c o l i B (ATCC 11303), E. c o l i B str-D, E. c o l i B/r, E. c o l i BRA, E. c o l i K12, E. c o l i MK12, E. c o l i W1485. E. c o l i W3110. E. c o l i CRX, E. c o l i 33111, E. c o l i UL 10.1, and E. c o l i UL 10.1 str-D ceased to respire prior to the depletion of oxygen from the system when assayed under conditions identical to those which gave rise to the cessation of respiration with E. c o l i NRC 482. Due to the generally accepted usage of unshielded, platinum wire cathode-silver wire ajiode oxygen electrodes in the investigation of oxygen u t i l i z a t i o n by biological systems prior to the development of the Clark-type oxygen electrodes, metal ion inhibition of respiration was not suspected at f i r s t . However, the discovery that: (i) the addition of 1 umole of reduced glutathione to the assay system prior to the addition of the c e l l suspension prevented the cessation i n oxygen consumption (Figure 3.2) and maintained a rate of oxygen consumption equal to that of the control (Figure 3 . 3 ) ; and ( i i ) that the addition of 2 umoles of reduced glutathione subsequent to the cessation of respiration resulted in a stimulation of respiration (Figure 3 . 4 ) , prompted us to consider this p o s s i b i l i t y . To investigate the p o s s i b i l -i t y that the Aminco oxygen electrode system was responsible for the observed cessation of the respiration of the E. c o l i c e l l suspensions, the measure-ment of oxygen consumption by E. c o l i c e l l suspensions was performed with the Clark-type oxygen electrode of a Gilson oxygraph under conditions iden-GSH C Glu Minutes Fig. 3.2 Prevention of cessation of respiration of E. c o l i by the addition of reduced glutathione. O2 uptake measured with Aminco electrode; GSH: 1 umole GSH; C: 4 mg c e l l s ; Glu: 100 umoles glucose; (method: sec. 2.2.2.2). C Glu Minutes Fig. 3.3 Respiration of E. c o l i as measured with Clark-type oxygen electrode. C: 4 mg c e l l s ; Glu: 100 umoles glucose; (method: sec. 2.2.2.2). 1 C W C Glu J i GSH 50 10 20 Minutes 3 0 40 Fig. 3.4 Stimulation of respiration v i a the addition of reduced glutathione. 02 uptake measured with Aminco electrode; C: 4 nig ce l l s ; Glu: 100 umoles glucose; GSH: 2 umoles GSH; (method: sec. 2.2.2.2). 102 t i c a l to those under which a cessation of respiration was observed when oxygen consumption was measured with the Aminco oxygen electrode. The results obtained demonstrated a linear uptake of oxygen subsequent to the addition of glucose, and no cessation in oxygen consumption (Figure 3 . 3 ) . This i n -dicated that the Aminco oxygen electrode was responsible for the observed cessation of oxygen consumption but gave no indication of the mechanism involved. During investigation of the mechanism by which the Aminco electrode system produced an inhibition of the respiration of E. c o l i i t was observed that i f the cessation of respiration was f i r s t obtained with the Aminco oxygen electrode, the Aminco electrode system removed, and the oxygen level subsequently monitored with a Clark-type oxygen electrode (Figure 3 . 5 ) , "the oxygen level did not alter. This indicated that the continued presence o£ the Aminco oxygen electrode was not required for maintenance of the cessa-tion of respiration. These results suggested that an inhibitor of oxygen consumption was produced by, or released from, the Aminco oxygen electrode. This p o s s i b i l i t y was tested by performing a blank run with the Aminco oxygen electrode of approximately the same duration required to obtain the cessa-tion of respiration but with no ce l l s present. The Aminco oxygen electrode was then replaced with the Clark-type oxygen electrode and cells were added. The results (Figure 3.6) demonstrated that under these conditions cessation of respiration did occur in the same pattern as was characteristic of the Aminco oxygen electrode. This result demonstrated conclusively that the Aminco oxygen electrode system was responsible for the production or release of an inhibitor of the respiration of E. c o l i . As indicated, there were two possible sources of an inhibitor in C Glu C Minutes Fig. 3.5 Retention of the cessation of respiration following the replacement (arrows) of Aminco electrode by Clark-type electrode. C: 4 mg c e l l s ; Glu: 100 uxnoles glucose; (method: sec. 2.2.2.2). o 0 10 20 3 0 4 0 Minutes Pig. 3.6 Inhibition of the oxygen consumption pf E. c o l i by a substance released by the Aminco electrode; 0 2 uptake measured with Clark-type oxygen electrode. C: 4 mg ce l l s ; (method: sec. 2.2.2.2). 105 the Aminco oxygen electrode assay system: (i) an inhibitor produced from the medium components by the operation of the oxygen electrode; or ( i i ) an inhibitor released from the Aminco oxygen electrode system. The release of an inhibitor was favored as a result of the observed action of reduced glutathione in preventing and reversing the inhibition, suggesting the involvement of a metal ion. There are only two major metal components of the Aminco oxygen elec-trode, the platinum of the cathode, and the s i l v e r of the anode. Since plat-inum is chemically very inert the most l i k e l y candidate for the inhibition of respiration was s i l v e r . Atomic absorption spectrophotometry of the assay medium from a blank run with the Aminco oxygen electrode indicated the pre-sence of significant amounts of s i l v e r (sec.3 . 1 . 2 ) . Further confirmation of the a b i l i t y of s i l v e r to inhibit respiration was obtained when the addition of 125 nmoles of AgNO^ to respiring suspensions of E. c o l i was shown to rapidly inhibit oxygen consumption (Figure 3.7). 3.1.2 Factors influencing the release of s i l v e r from the Aminco oxygen electrode A further investigation of the release of s i l v e r from the anode of the Aminco oxygen electrode was decided upon for two reasons: (i) to deter-mine why the inhibition of respiration by s i l v e r released from the naked anode of an oxygen electrode had not been cautioned against in the l i t e r a -ture, although this type of oxygen electrode has been in use for some time, and ( i i ) the information would aid in evaluating the results reported by other researchers using this type of equipment. Figure 3.8 demonstrates the influence of buffer ion on time depend-ent release of s i l v e r from the Aminco electrode system u t i l i z i n g the four buffer systems: glycylglycine-KOH, HEPES-HC1, Tris-HCl, and potassium C Glu A g \ 1 I Minutes Fig. 3.7 Inhibition by s i l v e r of the respiration of E. c o l i as measured with Clark-type oxygen electrode. C: 10 mg cells; Glu: 50 umoles glucose; Ag: 125 nmoles AgN0^;#: control; J_: test; (method: sec. 2.2.5.2). 25 Minutes Fig. 3.8 Buffer dependence of the release of s i l v e r from the Aminco oxygen electrode. Glygly: 0.3 M glycylglycine-NaOH, pH 7 . 0 ; HEPES: 0.3 M HEPES-NaOH, pH 7 . 0 ; T r i s : 0.3 M Tris-HCl, pH 7 . 0 ; 0.3 M potassium phosphate buffer, pH 7 .0; (method: sec. 2 . 2 . 3 ) . 108 phosphates, a l l 0.3 M with a pH of 7.0. The quantity of s i l v e r was both time- and buffer-dependent. The very minimal release in phosphate buffer —18 was probably due to the ins o l u b i l i t y of s i l v e r phosphate (Ksp = 1 .6 x 10 molest/litre^). In order to obtain some insight into the mechanism of s i l v e r re-lease, the time course of the release of s i l v e r from the anode .in 0 .3 M glycylglycine-KOH buffer, pH 7.0, in the presence or absence of the polar-izing voltage was determined (Table 3 . 2 ) . Although the presence of the polarizing voltage f a c i l i t a t e d the release of s i l v e r from the anode i t was not essential. Since the release of s i l v e r from the anode was relatively independ-ent of the presence or absence of the polarizing voltage, this suggested that the s i l v e r might be removed primarily by chelation in which case the amount released should be dependent upon the concentration of the chelating agent, presumably in this case the buffer. Table 3.3 demonstrates that the influence of buffer concentration on the release of s i l v e r from the oxygen electrode was quite marked. 3.1.3 The influence of pH, buffer ion and buffer concentration on the respiration of E. c o l i Prior to proceeding with further investigations on the influence of s i l v e r on the respiration of E. c o l i , i t was considered important to obtain a buffer system of a pH and concentration which consistently supported; (i) a high respiratory rate, ( i i ) a constant rate of oxygen u t i l i z a t i o n , and (,iii) which would maintain these characteristics of the E. c o l i c e l l sus-pensions for a considerable period of time. The results of the determination of the respiratory rates of E. c o l i c e lls suspended in nine isotonic buffers (.sec.2.2.4.2), covering the pH 109 Table 5.2 Time course for the release of s i l v e r from the Aminco oxygen electrode, with presence or absence of polarizing voltage Silver Release, (ug)  Time Polarizing Voltage (min. ) Present Absent Difference 0 0 0 0 10 7.6 4.2 ' 3.4 20 13.6 13.4 0.2 40 23.2 17.2 6.0 60 30.8 23.2 7.6 Table 3.3 Effect of concentration of buffer on the release of s i l v e r from the Aminco oxygen electrode Silver Released 1, (ug)  Buffer 3 mM Buffer 50 mM Buffer 500 mM Buffer Glycylglycine-KOH 0.52 Tris-HCl 0.44 Phosphate 0.48 HEPES-K0H 0.40 0.72 0.88 0.72 0.80 50.8 6.8 1.8 10.0 1 amount released in one hour. 110 range of 4 to 10, are presented in Figure 3 . 9 . The respiration rate i s expressed as the decrease in the percent oxygen saturation per min per mg wet c e l l weight. Determinations were performed in duplicate and the mean rate was plotted. Of the buffers investigated high respiratory rates between pH 6 and 8 were obtained with glycylglycine, phosphate, Tris, citrate, MOPS and HEPES buffers. However, of these buffer systems only phosphate in the pH range 5.5 to 5 . 7 , and glycylglycine over the pH range of 7.0 to 8.5 supported linear rates of oxygen consumption, as shown for glycylglycine buffer, pH 7 . 0 , in Figure 3.10. The oxygen consumption traces of the other buffer systems indicated decreasing rates of oxygen u t i l i z a t i o n with decreasing oxygen l e v e l , as shown for HEPES buffer, pH 7 . 0 , in Figure 3 .10. Of the two buffers, glycylglycine and phosphate, glycylglycine was the obvious choice for.experiments involving the addition of s i l v e r due to the ins o l u b i l i t y of s i l v e r phosphate. The influence of the concentration of glycylglycine-KOH buffer, pH 7 . 0 , on the respiration rate (Figure 3 . 1 1 ) » and on the short term s t a b i l i t y of the respiration rate of the c e l l s suspended i n the buffers (Table 3 .4) was determined. Although the results presented in Figure 3.11 are from a single experiment they were reproducible. The respiration rate, expressed as the decrease in the percent oxygen saturation per min per mg of protein, was maximal with a buffer concentration of 100 to 300 mM (Figure 3 . 1 1 ) » while the short term s t a b i l i t y of the respiration rate was optimal with buffer concentrations in the range of 100 to 500 mM (Table 3 . 4 ) . The long term s t a b i l i t y of respiration of ce l l s suspended in 300 mM glycylglycine-KOH buffer, pH 7 . 0 , (Table 3 .5) demonstrated fluctuations in 111 Fig. 3.9 Buffer ion and pH dependence of the respiration of B. c o l i . Glygly: glycylglycine-NaOH; P i: Na2HP04-NaH2P04; T r i s : Tris-HCl; Trieth: triethanolamine-HCl; Git: citrate-NaOH; MOPS: MOPS-NaOH; HEPES: HEPES-NaOH; Gly: glycine-NaOH; Eth: ethanolamine-HCl; a l l buffers were isotonic (method: sec 2.2.4.2); Rate: the decrease in the O2 saturation/min/mg c e l l wet weight. 112 Pig. 3.9 Fig. 3.10 The influence of buffer ions on the oxygen consumption traces of E. c o l i . Glygly: isotonic glycylglycine-NaOH, pH 7 .0; HEPES: isotonic HEPES-NaOH, pH 7 . 0 ; C: 5 mg cells; Glu: 50 umoles glucose; (method: sec. 2 . 2 . 4 . 2 ) ; (data from single determinations). 4 0 10 1 0 0 Millimolar 1 0 0 0 Fig. 3.11 The influence of the concentration of glycylglycine-KOH buffer, pH 7.0 on the respiration rate of E. c o l i . (method: sec. 2.2.4.2); Rate: the decrease in O2 saturation/min/mg protein. 115 Table 3 .4 The influence of the concentration of glycylglycine-KOH buffer, pH 7 .0, on the respiration rate of E. c o l i Respiration Rate  Concentration (at times after suspension in buffer) (mM) 0 15' 30' 0 4.0 3.0 2.2 1 6.4 5.9 5.5 3 13.5 8.5 8.4 5 11.6 7.8 7.8 10 15.0 11.5 10.6 30 19.6 15.6 16.6 50 22.1 19.1 18.2 100 30.8 28.2 26.4 300 25.5 26.8 28.3 500 23.3 23.4 • 23.4 1 respiration rate i s expressed as: percent decrease in oxygen satur-ation per min per mg of protein. 116 Table 3.5 The influence of the duration of suspension in 300 mM glycylglycine-KOH buffer, pH 7.0, at 0°C, on the respiration of K . c o l i Time Rate of Respiration ihi) : 1 14.6 3 14.9 5 16.0 11 15.6 17 12.7 23 14.0 29 14.1 38 14.1 47 14.2 The regression l i n e for: y = 15.05 - 0.03 (x) where y: is the rate of respiration, and x: is the time respiration rate expressed as: percent decrease in oxygen saturation per min per mg protein. values reported are the average of values obtained from duplicate experiments. 117 the respiration rate with a slight decrease in the rate of respiration over a 47 hour period. In this case the fluctuations were probably due to problems in stab i l i z i n g the temperature of the aluminum block incubation chamber of the Yellow Springs Instruments oxygen monitor. Thus, 300 mM glycylglycine-KOH buffer, pH 7.0 (or pH 7.5) was selected as the most suitable buffer of those examined since i t possessed the desired characteristics of supporting (i) a high respiratory rate, ( i i ) a constant rate of oxygen u t i l i z a t i o n , and ( i i i ) which would maintain these characteristics of the E. c o l i c e l l suspensions for a considerable period of time. 3.1 .4 Inhibition of the respiration of E, c o l i by added s i l v e r nitrate Having established that s i l v e r metal (ie., nascent silv e r ) or s i l v e r ions were responsible for the inhibition of the respiration of E. c o l i , the mechanism of their action was investigated. In an attempt to localize the possible site(s) of inhibition by si l v e r , E. c o l i were grown on different carbon sources to induce or repress specific enzyme systems of carbohydrate metabolism and/or transport (sec.1). The effect of AgNO^on substrate-dependent, or endogenous respiration then was tested (Figures 3.12 to 3.18 inclusive). In a l l figures the dotted line connecting the solid circles represents the control respiration of 5 mg of cells (wet weight) ( c ) plus 50 umoles of the substrate indicated. The solid line connecting the solid triangles represents the results of the test system, identical to the control system with the exception that 0.5 to 2 minutes after the addition of 50 umoles of substrate, 450 nmoles of AgNO^ were added (Ag). The substrate dependent respiration in a l l cases i s essentially linear. However, the addition of AgNO^  rapidly inhibited the substrate dependent respiration in a l l cases with the exception of formate Ag C C C 100 S> 50 >» x O 20 Minutes Pig. 3.12 Inhibition of the endogenous respiration of E. c o l i by s i l v e r . C: 5 mg cel l s ; Ag: 450 nmoles AgNO^; (method: sec. 2.2.5.2); carbon source: 0.4$ glucose. C GluAg C C C ForAg Minutes Fig 3.13 Inhibition of the glucose-dependent (A) and formate-dependent (B) respiration of E. c o l i by silver. C: 5 mg cells; Glu: 50 umoles glucose; For: 50 umoles formate; Ag: 450. nmoles AgNOj; •:control;A:test; (method: sec.2.2.2.5); carbon source: 0.4$ glucose. ^ C A c A g C C C C C C 11111111 1 Minutes Fig. 3.14 Inhibition of the acetate-dependent respiration of _. c o l i by sil v e r . C: 5 nig cells; Ac: 50 umoles acetate; Ag: 450 nmoles AgNO^j • : control; A : test; (method: sec.2 . 2 . 5 . 2 ) ; carbon source: 0.4$ glucose. ro O 10 20 0 Minutes 10 20 Fig. 3.15 Inhibition of the glycerol-dependent (A) and glucose-dependent (B) respiration of E. c o l i by silver. C: 5 cells; Gly: 50 umoles glycerol; Glu: 50 umoles glucose; Ag: 450 nmoles AgNO^; • : control; A • test; (method: sec.2 . 2 . 5 . 2 ) ; carbon source: 0.4$ glycerol. Pig. 3.16 Inhibition of the D-lactate-dependent (A) and glucose-dependent (B) respiration of E. c o l i by silver. C: 5 mg ce l l s ; D-Lac: 50 umoles D-lactate; Glu: 50 umoles glucose; Ag: 450 nmoles AgNOj; • : control; A : test; (method: sec,2 . 2 . 5 . 2 ) ; carbon source: 0 . 8 $ DL-lactate. ^ ro C L - L a c A g C C S A g C C Minutes Pig. 3.17 Inhibition of the L-lactate-dependent (A) and succinate-dependent (B) respiration of E. c o l i by silver. C: 5 mg cells; L-Lac: 50 umoles L-lactate; S: 50 umoles succinate; Ag; 450 nmoles AgKO^; • : control; A : test; (method: sec,2.2.5.2); carbon source: A, 0 . 8 $ DL-lactate; B, 0.4$ ^ succinate. ^ 10 20 0 Minutes 20 Fig. 3.18 Inhibition of the fumarate-dependent (A) and glucose-dependent (B) respiration of E. c o l i by silver. C: 5 mg cells; Fum: 50 umoles fumarate; Glu: 50 umoles glucose; Ag: 450 nmoles AgNO^; • : control; A : test; (method: sec.2 . 2 . 5 . 2 ) ; carbon source: 0.4$ fumarate. 125 (Figure 3.UB). The peculiar stepwise nature of the traces of the test system i s due to the successive additions of 5 mg of c e l l s . This experimental pro-cedure was used in an attempt to tit r a t e the amount of AgNO^  added, and to routinely determine that the oxygen electrode response to zero oxygen cor-responded to zero on the st r i p chart reading. The response of endogenous respiration of E. c o l i to the addition of AgNO^ i s indicated in Figure 3.12. The results presented were obtained with E. c o l i grown on glucose as the carbon source, however, analogous results were obtained with E. c o l i grown on succinate. The response of the endogenous respiration of E. c o l i grown on other carbon sources to.the addi-tion of AgNO^  was not determined. The sensitivity of glucose-dependent or glycerol-dependent respir-ation to inhibition by s i l v e r i s indicated by Figures 3.13A, 3.15A, 3.15B, 3.16B and 3.18B. By comparison, the respiration of E. c o l i u t i l i z i n g D-lactate (Figure 3.16A), L-lactate (Figure 3.17A), succinate (Figure 3.17B) or fumarate (Figure 3.18A) as substrate appeared less sensitive to in h i b i -tion by s i l v e r than glycerol-dependent or glucose-dependent respiration. The responses of D-lactate, L-lactate, succinate and fumarate to inhibition by s i l v e r were very similar. Acetate-dependent oxygen consumption was very sensitive to inhibition by AgNO^ (Figure 3 . 1 4 ) . Formate oxidation (Figure 3.13B) was the least sensitive to inhibition of the substrates examined. In general, the growth of E. c o l i on different carbon sources did not appear to have much influence on the response of any particular sub-strate-dependent respiration to s i l v e r inhibition. In order to determine i f glycolysis was more sensitive to in h i b i -126 tion than the combined function of the tricarboxylic acid cycle and the electron transport system as suggested by a comparison of the results of Figures 3.13A, 3.15A, 3.15B, 3.16B and 3.18B with those of Figures 3.16A, 3.17A, 3.17B and 3.18A, the effects of AgNO^ on acid production and oxy-gen consumption with glucose as substrate were monitored concurrently, in separate but identical systems. Figure 3.19A indicates the results obtained with the control system of 20 mg wet weight of E. c o l i ( c ) and 50 umoles of glucose (Glu). Oxygen consumption is indicated by the solid li n e and acid production, as pH, i s plotted as the broken l i n e . The results from the test system (Figure 3.19B) demonstrated that at a f i n a l siver nitrate concentration of 24 uM (125 nmoles of AgNO^ added), -acid production was inhibited immediately and completely, while oxygen consump-tion was only pa r t i a l l y inhibited. These results suggested that there was at least one site in glycolysis which was more sensitive to inhibition by siver than the site(s) of the TCA cycle and the respiratory chain. An indication of the location of the silver-sensitive site of glycolysis was provided by the similarity of the results obtained for s i l v e r inhibition of glucose-dependent, and glycerol-dependent respiration of c e l l s grown on glycerol (Figure 3•15) • The similarity of the pattern of response suggested that the si t e , in the glycolytic pathway, which poss-essed the high sensitvity to s i l v e r inhibition must occur in that portion of glycolysis which is common to the metabolism of both substrates, that i s , between glyceraldehyde-3-phosphate and pyruvate. A possible candidate in this region of the glycolytic pathway was glyceraldehyde-3-phosphate dehydrogenase, an enzyme which i s well known as possessing a thiol reagent sensitive s i t e . As indicated in table 3 . 6 , s i l v e r did inhibit glyceralde-Minutes Fig. 3.19 Inhibition of the respiration and acid production of j_. c o l i by s i l v e r . C: 20 mg ce l l s ; Glu: 50 umoles glucose; Ag: 125 nmoles AgNO^ ; • : oxygen;A: pH; (method: sec. 2 . 2 . 5 . 3 ) . ' ro 128 Table 3.6 The influence of s i l v e r nitrate and reduced glutathione on glyceraldehyde-3-phosphate dehydrogenase activity Additions Final Cone. Duration of With Ag+ Preincubation With GSH (min) Activity 340/min none — — 0 . 3 . 6 9 5 ul 0.50 mM AgNO^ 1 uM AgNO^  5 — O.169 13 — 0 . 3 1 8 . 20 — 0.224 5 ul 5 .0 mM AgNO^ 10 uM AgNO^ 5 — 0 . 1 3 6 13 — 0.050 22 — 0.026 5 p.1 5 .0 mM AgNO, 1 0 uM AgNOj 5 2 0.124 100 u l 0.1 M G S i r 4 mM G S H 5 10 0.204 5 18 0.142 values reported are from single determinations 129 hyde-3-phosphate dehydrogenase and the inhibition was pa r t i a l l y reversed by reduced glutathione. However due to the requirement for arsenate in the assay medium, the assay for s i l v e r inhibition was somewhat less than satisfactory since s i l v e r arsenate is insoluble. During the determination of the inhibition of the respiration of _. c o l i by s i l v e r (Figures 3.12 to 3.1B inclusive) i t was observed that the addition of AgNO^  stimulated the rate of oxygen consumption prior to i n -h i b i t i n oxygen u t i l i z a t i o n . This is even more apparent in table 3.7 where the i n i t i a l rates of oxygen consumption in the presence and absence of AgNO^ are tabulated. This suggested the po s s i b i l i t y that s i l v e r might function as an uncoupler prior to inhibiting respiration. 3.1.5 Selection of a carbon source for the growth of E. c o l i to be used for the investigation of the uncoupling of respiration by s i l v e r nitrate In order to investigate the possible action of s i l v e r nitrate as an uncoupler of E. c o l i respiration i t was essential to obtain E. c o l i c e lls which derived the majority of their energy requirement from the respiratory chain and which were "highly coupled". Of some interest to this problem was the report by Hempfling (1970b) that the levels of oxid-ative phosphorylation of E. c o l i , grown on a complex medium with glucose as the carbon source, was dependent upon the growth stage at which the cells were harvested. As a result i t was decided to grow E. c o l i in batch cultures (sec. 2 . 2 . 6 . 1 ) using a number of different carbon sources in an attempt to -evaluate the most suitable carbon source and growth phase to obtain the "highly coupled" cells required. The oxygen level of the growing culture was monitored to provide some information on the physiological character of the c e l l at any particular time during growth, while the redox potential Table 3.7 The influence of the addition of s i l v e r nitrate on the i n i t i a l rate of oxygen consumption. I n i t i a l Rate of Oxygen Consumption'' Carbon Source Substrate AgNO^  Absent AgTTO^  Present RCR Glucose endog. 3.8 3.9 1.03 glucose 18.9 30 .0 1.59 formate 9.9 13o5 1.36 acetate 3.8 6.5 1.71 Glycerol glycerol 24 .0 31 .0 1.29 glucose 15.7 26.7 1.70 DL-Lactate D-lactate 31.8 45.5 1.43 L-lactate 46.0 49.6 1.08 glucose 10.8 18.7 1.73 Succinate succinate 25.3 40.5 1.60 Fumarate fumarate 33.6 46.7 1.39 glucose 12.4 24 .4 1.97 rate of oxygen consumption expressed as: the decrease in the percent oxygen saturation per min per mg c e l l (wet weight), values reported are from single determinations. 131 was measured to investigate the correspondence between the measured redox potential and the oxygen l e v e l . Prior to presenting the results of this section i t is necessary to discuss a particular aspect of the design of the culture apparatus. The electrodes were separated from the bulk of the culture medium since the vigorous aeration resulted in unstable measurements. Consequently, the culture in the electrode vessel could be at a lower oxygen tension than the main vessel. However, as there was a dead space of only 2 ml between the main culture vessel and the electrode chamber, and since the liquid in the chamber was continually renewed at the rate of once every 15 sec, i t i s l i k e l y that the physical conditions in the electrode vessel were not greatly different from those in the main culture vessel. To check this point, the circulation of a mid-exponential phase culture between the two vessels was stopped. In 15 sec the oxygen tension in the electrode vessel dropped by 5$. This would probably be close to the maxium difference to be expected between the main culture and the electrode vessel. Batch cultures of R. c o l i NRC 482 with 0.4$ glucose as the carbon source, supplemented with f e r r i c citrate (6 uM) (Figure 3.20) demonstrated three distinct phases of oxygen u t i l i z a t i o n . Two phases of growth were observed. During the f i r s t phase of oxygen u t i l i z a t i o n the level of oxygen saturation decreased at an accelerating rate from 0.5 to 4.75 hr, after inoculating the culture, at which time the culture became oxygen-limited and remained oxygen-limited u n t i l a l l the glucose had been exhausted at about 5.25 hr. The redox potential of the culture decreased essentially in parallel with the oxygen level, during the f i r s t phase of oxygen u t i l i z a t i o n , u n t i l the culture became oxygen-limited. Under the oxygen-limited conditions the 132 I I I I 1 I I 0 6 12 Hours 20 Growth of E. c o l i on 0.4$ glucose in medium containing 6 uM f e r r i c citrate. Absorbance at 420 nm; units: redox potential, mV x 10~2 (methods: sec. 2.2.6.1). 133 redox potential continued to f a l l u n t i l the glucose content of the medium was depleted. The i n i t i a l phase of growth was essentially exponential and corresponded to the f i r s t phase of oxygen u t i l i z a t i o n . There appeared to be l i t t l e i f any effect of the oxygen-limited conditions on the growth rate. The relationship between the glucose concentration in the medium, redox potential and the growth of the culture, for a similar experiment i s indicated in Figure 3.21. Measurement of the oxygen level was not perform-ed during this experiment but the abrupt increase in the redox potential (corresponding to an increase in the level of oxygen saturation (Figure 3.20)), and cessation of growth corresponding to the depletion of glucose from the medium (8.5 hr) i s demonstrated clearly. On depletion of the glucose (Figure 3.20), the level of oxygen sat-uration increased for 30 min, and then proceeded to decrease at a linear rate for approximately one hour. This constituted the second phase of oxygen u t i l i z a t i o n and was accompanied by a low, essentially linear, growth rate. There was a close correspondence between the oxygen level and the redox po-tential of the culture during the second phase of oxygen u t i l i z a t i o n . Sub-sequent to the second phase of oxygen u t i l i z a t i o n , the oxygen level increased rapidly to approximately 70$ of saturation, then dropped to 65$ of saturation and remained constant at this value for 1.25 hr before returning to close to saturation. During this third phase, the c e l l mass of the culture declined slowly. The redox potential increased only s l i g h t l y compared to the i n -crease in oxygen level during the i n i t i a l portion of the third phase of oxygen u t i l i z a t i o n , then remained constant at about 280 mV for the majority of this phase, f a i l i n g to demonstrate an increase corresponding to the increase in oxygen content of the medium to close to the saturation l e v e l . Pig. 3.21 Growth of E. c o l i on 0.4$ glucose. Absorbance at 420 nm; units: redox potential, mV x 1 0 ~ 2 ; glucose concentration, mg/ml. (methods: sec. 2.2.6.1 and 2.2.6.11) 135 The absolute values of the oxygen levels in Figures 3.20, 3.22, 3.23 and 3.24 are in some doubt due to an unexpected, large residual electrode current in the absence of oxygen. A l l data used in these figures' were corrected using an "average" residual current value. However, the magnitude of the residual current as determined in subsequent experiments varied sl i g h t l y , thus the uncertainty. Growth of E. c o l i NRG 482 on 0.4$ glycerol as carbon source (Figure 3.22) demonstrated a single distinct phase of oxygen u t i l i z a t i o n and growth comparable to the f i r s t phase of oxygen u t i l i z a t i o n of the culture grown with glucose as carbon source (Figure 3.20). The oxygen-limited period of growth was of greater duration in the glycerol grown culture than in the culture grown on glucose, and had a greater influence on the growth. The growth data show a definite decrease in growth rate corresponding to the oxygen-limited conditions. As in the case of the culture grown on glucose there was a close correlation between the level of oxygen saturation and the redox potential of the culture u n t i l the culture became oxygen-limited. Under the conditions of oxygen limitation the redox potential decreased markedly, reached a minimum, and increased rapidly prior to any large i n -crease in the oxygen saturation. Subsequently, the level of oxygen satura-tion increased from 10 to 87$ and remained essentially constant. During the same interval, however, the redox potential demonstrated a slight i n i t i a l increase (7.25 to 7.75 hr), followed by a period of relatively rapid increase (7.75 to 8.5 hr) succeeded by a period of comparative constancy (9.0 to 11.0 hr). Thus, there was very poor correlation between changes in the oxygen level and the redox potential of a culture grown on glycerol, during and subsequent to the period of oxygen limitation. 136 137 With 0.8$ DL-lactate as the carbon source, E. c o l i NRC 482 cultures demonstrated oxygen u t i l i z a t i o n and growth phases (0 to 7.5 hr, and 7.5 to 10.0 hr) (Figure 3.23) similar to the f i r s t two phases of oxygen u t i l i z a t i o n and the two growth phases of the E. c o l i culture u t i l i z i n g glucose as the carbon source (Figure 3.20). The growth, however, showed the same sensi-t i v i t y to oxygen limitation as was observed in cultures grown.on glycerol as carbon source (Figure 3.22). The phases of redox potential and oxygen level show a reasonably close correspondence with the usual continued decrease in the redox potential during conditions of oxygen limitation. However, the second phase of oxygen u t i l i z a t i o n , consisting of a considerable decrease in the level of oxygen saturation, corresponded to a plateau in the redox potential. A comparison of Figures 3.20, 3.22 and 3.24 indicates sim i l a r i t i e s in the redox potential measurements of cultures of E. c o l i grown with glucose, glycerol or DL-lactate as the carbon source. With each of these carbon sources the most predominant feature of the redox potential curve i s the change in redox potential corresponding to the large f i r s t phase of oxygen u t i l i z a t i o n . However, glucose and DL-lactate grown cultures demon-strate distinct second phases of oxygen u t i l i z a t i o n with corresponding changes in redox potential. The slight shoulder (7.25 to 8.25 hr) during the rise in the redox potential, of a culture grown on glycerol (Figure 3.22), sub-sequent to the interval of oxygen limitation, possibly corresponds physio-l o g i c a l l y and biochemically to the more disti n c t second phases of the cultures grown on glucose (Figure 3.20) or DL-lactate (Figure 3 . 2 3 ) . Cultures of E. c o l i NRC 482 grown on 0.8$ acetate (Figure 3.24) demonstrated a single phase of oxygen u t i l i z a t i o n and a smooth continuous 140 growth curve with no obvious discontinuities of the type apparent in the growth curve obtained from cultures grown on glucose (Figure 3.20) or DL-lactate (Figure 3 . 2 3 ) . A considerable portion of the growth curve indicates a linear growth rate (8.5 to 12.0 hr) although there does not appear to be an obvious correlation with the level of oxygen satur-ation. The growth rate of B. c o l i NRC 482 with acetate as the carbon source was the slowest of the growth rate for the carbon sources investigated. The correspondence between the oxygen level and the redox potential was good with respect to general characteristics. The significance of the shoulders on the oxygen level and redox potential curves at 3.0 and 6.0 hr respectively is not known. Growth of E. c o l i NRC 482 on 0.6$ succinate in a medium supplemented with f e r r i c citrate (6 juM) (Figure 3.25) demonstrated a single phase of oxygen u t i l i z a t i o n and growth. The oxygen level decreased rapidly and became limiting at approximately 5.5 hr at a level of 7 to 8$ of saturation, con-siderably higher than expected. During the period of oxygen limitation growth was linear. Changes in the redox potential did not coincide completely with changes in the oxygen level and growth. The redox potential of the culture remained constant for the f i r s t 3.0 hr of growth by which time the oxygen saturation had decreased to 80$. The redox potential decreased linearly for the greater portion of the period of oxygen limitation but not during the i n i t i a l 0.5 to 0.75 hr. No secondary phases of oxygen u t i l i z a t i o n or growth were observed in cultures which were allowed to proceed further into the stationary phase of growth. Based on the results presented in Figures 3.20 to 3.25 inclusive, Pig. 3.25 Growth of E. c o l i on 0.6$ succinate in medium containing 6 uM f e r r i c citrate. Absorbance at 420 nm; units: redox potential, raV x 10-2. (methods: sec. 2 . 2 . 6 . 1 ) . 142 the data reported by Hempfling (1970b), the relative s e n s i t i v i t i e s of glycolysis and respiration to inhibition by sil v e r nitrate (sec.?.1.4), and the requirement that the majority of the energy be obtained from the respiratory chain, succinate was chosen as the most suitable carbon source for the growth of cells for the investigation of the action of AgNO^  as a uncoupler of the respiration of _. c o l i . 3.1.6 Uncoupling of the respiration of E. c o l i by added s i l v e r nitrate Figures 3.26 and 3.27 demonstrate the uncoupling action of DBP and AgNO^, respectively, on the respiration of E. c o l i grown under "iron-suf-f i c i e n t " conditions. The concentration dependence of DBP and AgNO^  as uncouplers of the respiration of E. c o l i , and the importance of the pres-ence of adequate iron in order to obtain "highly coupled" E. c o l i i s further demonstrated in Figure 3.29 and table 3.8. Growth of E. c o l i under "iron-sufficient" conditions does not appear to alter the sensitivity of the respiration to uncoupling by AgNO^, only the degree of uncoupling possible. On the other hand, E. c o l i grown under "iron-sufficient" conditions and "iron-limited" conditions appear to d i f f e r in sensitivity to the uncoupler DBP. A comparison of the concentration dependence of the RCR values obtained with the two uncouplers, DBP and AgNO^  (Figure 3.29) indicates that AgNO^ i s the more effective uncoupler irrespective of whether the cells were "iron-sufficient" or "iron-limited". 3.2 Discussion 3.2,1 The measurement of oxygen tension with an oxygen electrode Electrode measurements of oxygen consumption and evolution offer several advantages over manometric precedures. These are: (i) complete determinations are usually of short duration; C Glu DBP Minutes Fig. 3.26 Stimulation of the respiration of E. c o l i "by 2,4-dibromophenol. C: 4 mg cel l s ; Glu: 20 umoles glucose;- DBP: 50 umoles of DBP; (method: sec. 2.2.6.3); temperature: 22°C. C Glu Ag Minutes Fig. 3.27 Stimulation of the respiration of E. c o l i by s i l v e r nitrate. C: 20 mg ce l l s ; Glu: 20 umoles glucose; Ag: 1 umole AgNO^ ; (method: sec 2.2.6.3); temperature: 22°C. 0 5 10 15 20 Minutes Fig. 3.28 The influence of potassium nitrate on the respiration of E. c o l i . C: 4 mg c e l l s ; Glu: 20 umoles glucose; N: 200 nmoles of KNO3; (method: sec. 2.2.6.3); temperature: 22°C. 146 Fig. 3.29 The dependence of the respiratory control ratio (RGR) on the uncoupler concentration. A: DBP; B: AgN03;#, iron-deficient; •, iron-sufficient; (methods: sec.2.2.6.3 and 2.2.6,4). 147 Table 3.8 The stimulation of the respiration of E. c o l i by 2,4-dibromophenol and s i l v e r nitrate. Uncoupling Agent Rate of Oxygen Consumption^ RCR (uM) Uncoupler Absent Uncoupler Present  Iron deficient c e l l s : DBP 500 2.44 3.87 1.59 250 3.21 4.28 1.33 125 2.82 4.87 1.73 50 2.96 . 3.81 1.29 25 3.09 3.54 1.15 12 3.30 3.73 1.13 Iron sufficient c e l l s : DBP 500 2.94 250 3.20 6.01 1.88 125 3.04 4.22 1.39 50 3.06 3.87 1.26 25 3.01 3.66 1.22 12 3.09 3.63 1.17 Iron deficient c e l l s : AgNO, 480 3.23 7.07 2.19 7 240 2.76 3.55 1.28 120 2.01 50 2.90 5.37 1.86 25 3.31 5.44 1.64 12 3.10 5.70 1.84 5 3.06 5.51 1.80 2 3.17 4.90 1.55 Iron sufficient c e l l s : AgNO, 480 2.20 5.81 2.64 240 2.62 5.64 2.15 120 2.56 5.35 2.09 50 1.99 5.57 2.80 25 2.04 5.11 2.50 12 1.98 4.86 2.45 5 2.12 3.69 1.74 2 2.45 3.25 1.33 1 rate of oxygen consumption expressed as: the decrease in the present oxygen saturation per min per mg c e l l (wet weight). values reported are single determinations. 148 ( i i ) some economy on the use of expensive substrate reagents is achieved since small reaction volumes are used; ( i i i ) multiple additions of substrates and/or inhibitors or effectors are readily made within a single experiment of short duration; (iv) calculation of results and calibration procedures are much less time consuming; (v) measurements made reflect oxygen exchange by chemical reaction and are not limited by the rate of equilibration of oxygen across a l i q u i d - a i r interface; (vi) measurements are made without the removal of carbon dioxide which in i t s e l f may be important to the cel l u l a r metabolism; (v i i ) measurements are generally obtained as continuous recordings of changes in the level of oxygen saturation, permitting accurate determinations of changes in the rate of oxidation, and ( v i i i ) circuits have been designed which permit the recording of the f i r s t derivative of the output from an oxygen electrode, a method which is often useful in detecting small changes in the rate of oxidation. Two of the major disadvantages of oxygen electrode measurements are (i) the t ime of the reaction i n an oxygen electrode chamber is limited by the amount of oxygen which can be dissolved in the incubation medium; and ( i i ) experiments are performed in a medium of constantly decreasing oxygen tension. The former generally has not proven to be a disadvantage in investi-gations with mitochondrial suspensions, although the oxygen limitation may become a d i f f i c u l t y when estimation of changes in inorganic phosphate (P^) or substrate concentration i s necessary for direct calculations of P:0. 149 However, the number of energy-linked systems of the mitochondrion, a c e l l organelle which in vivo is buffered against major environmental fluctuations by the cytoplasmic membrane, are probably considerably fewer than those of the intact bacterium which must deal directly with a l l environmental stresses. Consequently, since researchers investigating the respiration of intact bacteria with oxygen electrodes generally u t i l i z e c e l l concentrations which are sufficient to deplete the assay medium of oxygen within a relatively short time (^10 min), in order to avoid errors due to the back diffusion of oxygen, the amount of oxygen available per c e l l may be insufficient to gen-erate adequate energy to raise the bacterium to a high energy steady-state. This may be one reason why i t has not been possible to demonstrate respira-tory control in intact bacterial systems. The fact that the measurement of respiration or oxidation is per-formed in a medium of constantly decreasing oxygen tension generally has not been considered a problem in the measurement of the respiration of mitochon-d r i a l or bacterial suspension where the "Km" of the cytochrome oxidases for -8 -6 oxygen is usually of the order of 10~ to 10 M (Longmuir, 1954). However, White (1963) has demonstrated that the sensitivity of the respiration of Hemophilus parainfluenzae to decreasing oxygen tension can vary considerably dependent upon the conditions used for growth of the organism. Clark and Sachs (1968) and Pietra and Cappelli (1970) have also reported on the sensi-t i v i t y of the metabolism of cells to oxygen tension. Metabolic shifts encountered when cells are sensitive to changes in oxygen tension complicate the interpretation of data, and consequently l i m i t the usefulness of the assay to those conditions under which the metabolism is independent of the oxygen concentration. 150 Both the problem of the limited amount of oxygen available and the problem of the constantly decreasing oxygen tension could be eliminated by the use of an "oxystat" procedure which would maintain the oxygen satura-tion of the electrode chamber at a predetermined level by the injection of small volumes of an oxygen-saturated solution. During the course of the assay the oxygen saturation of the system and the rate of addition of oxygen-saturated solution would be recorded simultaneously, permitting an accurate determination of the rate of oxygen u t i l i z a t i o n . Such oxystats have been designed for use with isolated tissues (Clark and Sachs, 1968; Garrison and Ford, 1970) but as yet have not been u t i l i z e d with c e l l suspensions or subcellular particles. No attempt w i l l be made to discuss the operational sources of error of oxygen electrode measurements, such as back diffusion of oxygen (or a i r ) , temperature, agitation and electrode aging. The necessary precautions were taken in performing experiments to eliminate or minimize the introduction of errors due to these parameters. For a thorough discussion of these problems, the reader i s referred to the following reviews on oxygen elec-trode measurements: Beechey and Ribbons, 1972; Lessler, 1972; and Lessler and Brierley, 1969. 3.2.2 A proposed mechanism for the release of s i l v e r from the anode of the Aminco oxygen electrode . During the presentation of the preceding sections of the thesis no indication has been given of the suspected mechanism by which the s i l v e r i s released from the oxygen electrode, or of the form of the si l v e r in solution. If the s i l v e r was released e l e c t r o l y t i c a l l y from the anode of the oxygen electrode: ( i ) the quantity released should have been independent of 151 the type of buffer (Figure 3.8), and the buffer concentration (Table 3.3) provided that the conductivity of the solutions were of the same order of magnitude; ( i i ) s i l v e r release into phosphate buffer should have been maximal due to the continuous removal of s i l v e r from solution as the i n -soluble s i l v e r phosphate (Figure 3.8 and Table 3.3); and ( i i i ) the quantity of s i l v e r released should have been dependent upon the presence of the pol-arizing voltage (Table 3.2). None of these characteristics applied to the. release of s i l v e r from the Aminco oxygen electrode. The concentration dependence of the si l v e r release from the anode of the oxygen electrode (Table 3.3) and the s t a b i l i t y of the s i l v e r complex of Tris ( K a 3 S 0 C > = 2.75 x 10~^) (Benesch and Benesch, 1955), suggested that s i l v e r was probably chelated from the anode and existed in solution as a sil v e r ion chelate of the buffer ion. This would explain the low quantity of s i l v e r released into the phosphate buffer. Although i t i s hard to recon-c i l e the formation of a si l v e r ion chelate of HEPES with the reported negli-gible metal binding constants of this buffer (Good et a l . , 1966), the data are consistent with the removal of s i l v e r from the anode as s i l v e r ion chelates. As indicated previously (sec.3.1.2) one of the reasons for investi-gating the factors influencing the release of s i l v e r from the oxygen electrode was the apparent failure of previous researchers to be confronted with this problem. An examination of the data on the dependence of s i l v e r release on the type of buffer (Figure 3.8), and on buffer concentration (Table 3.3), indicates that the probable reason that the inhibition of respiration by s i l v e r released from the naked s i l v e r anode of an oxygen electrode had not been reported previously i s that the conditions used by most researchers, 50 to 100 mM Tris buffers, would have resulted in a much lower level of 152 s i l v e r release than was obtained with 300 mM glycylglycine-KOH buffer. Unfortunately, 300 mM glycylglycine-KOH buffer, pH 7.0, which of the four buffer systems examined resulted in the maximal release of s i l v e r , was the buffer system that was chosen for the i n i t i a l investigation of the respir-ation of E. c o l i using the Aminco oxygen electrode. Consequently a large amount of experimental data was unuseable in this thesis. 3.2.3 The influence of pH, buffer ion and buffer concentration on the respir-ation of E. c o l i Although the volume of the bacterial c e l l and the bacterial proto-plast, and the respiration rates of intact bacterial cells or spheroplasts have been demonstrated to be sensitive to the osmotic pressure and the pH of the suspending medium (Henneman and Umbreit, 1964a,b; Packer and Perry, 1961; Smith, 1962; Knowles and Smith, 1971a,b; Knowles, 1971) researchers primarily interested in the characteristics of the respiration of bacteria generally have not considered an investigation of the influence of the buffer ion, pH and concentration on the respiration to be important. However, the intact bacterium must deal directly with a l l environmental stresses, natural or experimental, to which i t i s exposed, and possesses the mechanism to do so. At present our knowledge i s insufficient to even speculate as to what proportion of these mechanisms are directly or indirectly linked to energy production via the electron transport chain. Thus, the more closely the "optimum" conditions are approximated the less the likelihood that respir-ation w i l l be influenced by unknown energy demands on the respiratory chain. For this reason, an investigation of the influence of buffer ion, pH and concentration on the respiration of B. c o l i was performed. The major problem of such a project i s the specification of the c r i t e r i a one should employ to select an "optimum" buffer-pH combination. The c r i t e r i a used in 153 the research reported in this thesis were the c r i t e r i a of enzyme kinetics: (i) a high rate of oxygen consumption; ( i i ) a linear rate of oxygen u t i l i z -ation; and ( i i i ) maintenance of these characteristics of the E. c o l i c e l l suspension over a greater period of time than required to complete an experiment. During the i n i t i a l investigation of the influence of buffer ion and pH on the respiration of E. c o l i , isotonic buffers were used to avoid the po s s i b i l i t y of transport-linked energy demands or distortion of the cyto-plasmic membrane resulting from differences in the osmotic pressure of the c e l l and the suspending medium. Data obtained subsequently (Figure 3.11 and Table 3.4) confirmed this concentration as "optimal" under the c r i t e r i a used. It i s apparent from Figure 3.9 that high rates of glucose-dependent respiration can be obtained with several buffer ions, over a wide pH range. Due to the complexity of the system i t i s impossible to rationalize the rates of respiration obtained with the different buffer ions, or at the different pH values. Of the buffers investigated only phosphate and gly-cylglycine buffers f u l f i l l e d the second criterion of supporting a linear rate of oxygen u t i l i z a t i o n (Figure 3.10). Cells suspended in any of the remaining buffer systems demonstrated decreasing respiration rates with decreasing oxygen saturation (Figure 3.10). This has previously been attributed to the possession of a higher "K^ " for oxygen by the cells sus-pended in these buffers (Longmuir, 1954; White, 1963). However, since the c e l l s u t i l i z e d in these investigations were grown under identical conditions, this would require that the buffer ions were influencing the "Km" of the cytochrome oxidase for oxygen. Although this would be a pos s i b i l i t y in 154 c e l l extracts, i t would appear unlikely to occur in the intact bacterial c e l l due to the impermeability of the l a t t e r to charged compounds. An alternative explanation for the two classes of oxygen consump-tion traces obtained with the different buffers was suggested by the results obtained by Oishi and Aida (1970), during investigation of the control of bacterial respiration. These authors observed that the respiration of phos-phate-starved bacteria was stimulated by the addition of inorganic phosphate and that this stimulated rate of respiration was maintained u n t i l either the inorganic phosphate was depleted from the medium or the level of the phosphate-pool of the c e l l was replenished. On the basis of the data presented the authors were unable to eliminate the p o s s i b i l i t y that the stimulation of res-piration resulted from an energy demand for the active transport of phosphate rather than via the classical respiratory control of ADP, P^ and ATP. Thus, perhaps the linear rates of respiration obtained with c e l l s suspended in phosphate or glycylglycine buffers were due to the stimulation of respiration as a result of the imposition of an energy demand on the c e l l , while the pro-gressively decreasing respiration rates obtained with c e l l s suspended in other buffer systems might indicate the approach of the c e l l to a high energy steady-state. Irrespective of the cause(s) of the oxygen saturation independent, and the progressively decreasing respiration rates, the problem of minim-izing unknown energy demands on the respiratory chain i s one that must be dealt with before the characteristics of respiration, with associated respir-atory control, can be investigated in the intact bacterial c e l l . 3.2.4 The inhibition of the respiration of K. c o l i by s i l v e r ions One of the primary objectives of the research reported in this thesis 155 was to investigate the site(s) at which s i l v e r ions influenced the respira-tion of E. c o l i . As indicated in the introduction the aerobic respiration of an intact organism, as measured by oxygen consumption, reflects the function and kinetic characteristics of a l l pathways and systems which pro-vide reducing equivalents and oxygen to the terminal oxidase(s) of the elec-tron transport chain. Consequently, the inhibition of the respiration of E, c o l i could occur via: (i) inhibition of transport of the substrate; ( i i ) inhibition of one or more enzymes of the amphibolic pathways (Figure 1.5); ( i i i ) inhibition of respiratory chain enzymes; and/or (iv) inhibition of energy coupling ( i . e . the inhibition of the u t i l i z a t i o n of energy generated by the respiratory chain). Irrespective of the number of sites of inhibition by s i l v e r in the systems indicated above, the demonstrated characteristics of the inhibition of res-piration w i l l be those of the site most sensitive to s i l v e r inhibition and which cannot be by-passed. On examining the structure of the gram-negative c e l l (Costerton, 1970) i t i s possible to subdivide the interaction of s i l v e r ions with the c e l l into three stages: ( i ) at the c e l l wall; ( i i ) at the cytoplasmic membrane; and ( i i i ) in the cytoplasm or cytosol. The c e l l wall is believed to function primarily as a molecular sieve, preventing the entrance of large molecules, and on this basis would be expected to have l i t t l e i f any effect on s i l v e r ions. However, Ca + +, Mg + +, K + and phosphate are concentrated to high levels in the c e l l walls of gram-negative bacteria (Eagon, 1969). Consequently a large proportion of the s i l v e r ions interacting with an 156 E. c o l i c e l l might be precipitated or bound in the c e l l wall as s i l v e r phos-phate. Under these circumstances the effective concentration that reaches the cytoplasmic membrane would be drastically reduced. However, due to the relatively nonspecific reactivity of s i l v e r with available sulfhydryl groups and possibly also histidine (sec.1.5), those enzyme systems in the cyto-plasmic membrane should be among the most vulnerable. The results obtained on the influence of s i l v e r ions on the respir-ation of E. c o l i can be roughly classified into four groups according to the apparent sensitivity to inhibition: ( i ) endogenous and acetate-dependent respiration (Figures 3.12 and 3.14, respectively); ( i i ) glucose- and glycerol-dependent respiration (Figures 3.13A, 3.15B, 3.16A, 3.18B, and 3.15A, respec-ti v e l y ) ; ( i i i ) D-lactate-, L-lactate-, fumarate- and succinate-dependent respiration (Figures 3.16A, 3.17A, 3.18A and 3.17B, respectively); (iv) formate-dependent respiration (Figure 3.13D). Considering f i r s t of a l l the po s s i b i l i t y that the transport of sub-strates (glucose, glycerol, D-lactate, L-lactate, fumarate, succinate and acetate) into E. c o l i i s the site of s i l v e r inhibition of respiration, insufficient data are available to draw a conclusion as to whether this was the mechanism involved. With respect to this problem, however, Rayman et^ a l . , (l972a,b) have reported that the ascorbate-PMS-dependent, and the D-lactate-dependent active accumulation of succinate by membrane vesicles of 3. c o l i were inhibited by s i l v e r . They did not indicate whether s i l v e r also inhibited the oxidation of D-lactate and ascorbate-FMS. Although their results indicated that D-lactate oxidation by the membrane vesicles was inhibited by the thiol reagents NEM and PCMB, ascorbate-PMS oxidation was not inhibited by NEM and was only s l i g h t l y inhibited by P C i M B . Succinate trans-157 port, by the membrane vesicles, driven by either system was not inhibited by NM (Rayman et a l . , 1972b), while D-lactate-dependent succinate uptake and D-lactate oxidation by intact E. c o l i K12 was inhibited by NEM (Lo e_t a l . , 1972b). The results obtained with respect to NEM inhibition of D-lactate oxidase but lack of NEM inhibition of the accumulation of succinate are. d i f f i c u l t to comprehend and are in disagreement with the characteristics of D-lactate coupled transport systems as described by Kaback (1972). These discrepancies render it'impossible to deduce whether :the inhibition of suc-cinate transport by s i l v e r was due to: (i) inhibition of the transport sys-tem; ( i i ) inhibition of the function of the respiratory chain; or ( i i i ) the inhibition or uncoupling of the mechanism of energy coupling. The data presented in Figure 3.16A demonstrate that oxidation of D-lactate by intact E. c o l i was rapidly inhibited by s i l v e r nitrate at a concentration of 86 uM. This i s comparable to the concentration (100 uM) reported by Rayman et a l . , (1972b) to cause 100$ inhibit ion of succinate transport by membrane vesicles. This suggests that the observed inhibition of succinate transport may have occurred via the inhibition of D-lactate oxidation. Kinetic studies of the inhibition of succinate transport and D-lactate oxidation by s i l v e r ions are required to refute or verify this argument. The action of s i l v e r ions as an uncoupling agent (sec.3.1.6; Figure 3.27; Table 3.8) could also inhibit the accumulation of succinate since this is presumably driven by a high energy intermediate generated by the respir-ation chain (Hong and Kaback, 1972; Simoni and Shallenberger, 1972; Kashket and 7/ilson, 1972; Bragg and Hou, 1973). The preceding discussion of the s i l v e r ion inhibition of succinate transport i s equally applicable to fumarate transport (Kay and Kornberg, 1971; Lo et a l . , 1972b; Rayman et a l . , 1972b). 158 No experimental information is available on inhibition of glucose transport by s i l v e r ions. However, enzyme I of the PEP phosphotransferase system (sec . 1 . 1 ) is sensitive to inhibition by the t h i o l reagents NEM, PCMB and dithionitrobenzene (DTNB) (Kundig and Roseman, 1971a). The in h i -bition by PCMB and DTNB can be reversed by reduced glutathione, cysteine and ^-mercaptoethanol. Anraku (1968) demonstrated the inhibition of glucose uptake into E. c o l i by Zn + +, a heavy metal ion also believed to react with, thiols (Kashara and Anraku, 1972), and similar results were reported by Eagon and Asbell (1969) with Pseudomonas aerugenosa. These results suggest that glucose transport i s probably sensitive to inhibition by s i l v e r ions. There is no information available on the inhibition by Ag +'of trans-port of glycerol, D-lactate, L-lactate and acetate into E. c o l i . These trans-port systems have been insufficiently characterized to permit speculations as to their sensitivity to thi o l reagents. It i s questionable to attempt to deduce the site of action of s i l v e r ions from the action of other thiol-reacting agents. Brierley and co-workers investigated the influence of mercurical reagents and heavy metal ions on the permeability and ATPase act i v i t y of bovine heart mitochondria (Brierley et a l . , 1967, 1971; Scott et a l . , 1970, 1971). They have found that the extent of reaction of these reagents with the mitochondrial membrane depends on the polarity of the reagent, the anionic composition of the suspending medium, the pH, and to some degree on the metabolic status of the mitochon^ drion. Thus, at present s i l v e r ions are only known to inhibit the accum-ulation of succinate and fumarate. Since the oxidation of formate (Figure 3 . 1 3 B ) , a compound to which E. c o l i i s freely permeable (Bovell et a l . , 159 1963), was much less sensitive to inhibition by s i l v e r ions than respira-tion dependent on glucose, glycerol, D- or L-lactate, succinate, fumarate or acetate, the conclusion could be made that transport of the other sub-strates into E. c o l i was inhibited by s i l v e r and that this was the site at which respiration was inhibited. However, the fact that the endogenous res-piration of cells grown on glucose (Figure 3.12) or succinate demonstrated stimulated and inhibited phases following the addition of AgNO^, and a sen-s i t i v i t y to inhibition by Ag + equal to or greater than exogenous substrate-dependent respiration, suggests that i t i s unlikely that the observed s i l v e r ion inhibiton of respiration with exogenous substrates was due to the i n h i - • bition of transport of the substrate into the c e l l . This suggested that s i l v e r inhibition at a site in the amphibolic pathways or the respiratory chain was probably responsible for the observed inhibition of respiration. In the subsequent discussion of the possible i n -hibition of the amphibolic pathways and the respiratory chain, the literature pertinent to the results w i l l be dealt with concurrently with the discussion of the results. Prior to this discussion a comment on the technique of successive additions of cells subsequent to the inhibition of respiration i s in order. This unusual procedure was used i n i t i a l l y in an attempt to t i t r a t e the number of s i l v e r ions present in solution to determine the approximate number of s i l v e r ions per c e l l required to completely inhibit the respiration. Yudkin (1937) had indicated that a single E. c o l i c e l l was capable of binding in the 8 7 7 order of 5 x 10 s i l v e r ions, while approximately 1.3 x 10 and 1.7 x 10 s i l v e r ions per c e l l were required to bring about 5<y/o inhibition of glucose and succinate diaphorase a c t i v i t i e s respectively, Although the desired values 160 could have been more accurately determined from the concentration dependence of the Ag + inhibition of respiration, or by reducing the amount of AgNO^ originally added to the assay system, i t was found that the pattern of res-ponse of oxygen consumption in the presence of s i l v e r ions to successive addi-tions of E. c o l i provided a good indication of the sensitivity of substrate-dependent or endogenous respiration to inhibition by Ag +. A lower concentra-tion of AgNO^  would have been insufficient to completely inhibit the D-lactate-, succinate-, and fumarate-dependent respirations (Figures 3.16A, 3.17A, 3.17B and 3.18A respectively). The similarity of the response patterns obtained with the s i l v e r i n h i -bition of endogenous respiration (Figure 3.12), and of the acetate-dependent respiration (Figure 3.14) of E. c o l i was unexpected. However, as the E. c o l i c e lls were grown on glucose as the carbon source and harvested during the late exponential phase of growth,"Ithe acetate transport system, and enzymes of the TCA and glyoxylate cycles would be repressed (sec.1.1 and 1.2 and Table 1.1). Consequently, the respiration, even with acetate present, vrould probably be largely endogenous and quite different from the response of cells which had been grown on acetate as carbon source. Figure 3.14 clearly demon-strates the influence of the t i t r a t i o n (removal) of the s i l v e r ions on the rate and extent of oxygen u t i l i z a t i o n of each succeeding addition of E. c o l i . Due to the unknown nature of the endogenous substrates i t i s impossible to propose a specific site(s) of s i l v e r inhibition in the metabolism prior to the introduction of reducing equivalents into the respiratory chain. Subsequent experiments indicated that the addition of AgNO^  (24 pM) to cells metabolizing glucose immediately inhibited the decrease in pH while respiration continued at less than 10$ of the rate prior to the addition of 161 s i l v e r nitrate (Figure 3.19). There are three possible explanations for these results. The f i r s t p o s s i b i l i t y is that glycolysis was inhibited at a site between glucose and pyruvate. At a s i l v e r nitrate concentration of 24 uM, when acid production ceased, respiration continued due to the oxidation of accumulated acids (lactate, acetate and formate). This seems unlikely since the increase in pH subsequent to inhibition by Ag + does not appear adequate to account for the continued respiration on the basis of the oxidation of accumulated acids. The second alternative i s that the cessation in the decrease in pH does not represent a complete inhibition of acid production at a site between glucose and pyruvate, but that acid production was pa r t i a l l y inhibited such that the rate of acid production was balanced by i t s rate of oxidation. The results (Figure 3.19) interpret~^ed in terms of this proposal are consis-tent with glycolysis being more sensitive to inhibition by s i l v e r ions than the TCA cycle and/or the respiratory chain. The third postulated mechanism also involves the inhibition of glycol-ysis between glucose and pyruvate. However, at a s i l v e r nitrate concentration of 24 uM the inhibition at this site was approximately 90$. Pyruvate produc-tion continued at 10$ of the rate prior to the addition of the AgNO^, account-ing for the 10$ respiration remaining. The complete inhibition of acid pro-duction could be accounted for by postulating the complete inhibition of a reaction between pyruvate and the excreted acid(s). The reactions involved could be the soluble D-lactate dehydrogenase catalyzed conversion of pyruvate to D-lactate (reaction 38, Figure 1.5) and/or the phosphorclastic reaction (reaction 33, Figure 1.5). 162 There is insufficient data available to make a choice between the second and third proposals. Neither of these proposals exclude the possi-b i l i t y of their being less sensitive sites of Ag + inhibition in the TCA cycle. Isocitrate dehydrogenase (Kratochvil et a l . , 1967) and fumarate (Laki, 1942) from mammalian sources have been shown to be inhibited by Ag + while aconitase (Krebs and Eggleston, 1944), 06-oxoglutarate dehydrogenase (Baron and Singer, 1945; Gonda et a l . , 1957), and succinate dehydrogenase (Kim and Bragg, 1971a) have been shown to be inhibited by thiol-reacting reagents. The similarity of the glucose-dependent and glycerol-dependent patterns of oxygen consumption (Figure 3.15) led to the conclusion" that the glycolytic enzyme which possessed the high sensitivity to s i l v e r inhibition must occur between glyceraldehyde-3-phosphate and pyruvate as this portion of glycolysis i s common to both substrates (Figure 1.5). Based on this assumption the inhibition of glyceraldehyde-3-phosphate dehydrogenase by Ag + was examined (Table 3.6) as i t i s an enzyme in the desired portion of the Smbden-Meyerhof pathway which i s known to possess a th i o l reagent-sensitive s i t e . The glyceraldehyde-3-phosphate dehydrogenase from rabbit and porcine skeletal muscle have been shown to be inhibited by Ag + (Park et a l . , 1961; Boross, 1965; Boross and Keleti, 1965). A concentration of Ag + of 10 pM. resulted in a 94$ inhibition of the rabbit skeletal muscle enzyme which i s comparable to the observed inhibition of glycolysis at a AgNOj concentration of 24 uM. Aldolase from rabbit skeletal muscle also i s com-pletely inhibited by a Ag + concentration of 20 uM. The results (Table 3.6) indicated that E. c o l i glyceraldehyde-3-phosphate dehydrogenase was inhibited b y Ag +. The observed variation in the activity of the enzyme at a f i n a l 163 assay concentration of 1 uM is probably due to the nonspecific binding and release of Ag + by other protein components of the 95*000 x g_ supernate and/or the precipitation of Ag + as s i l v e r arsenate. A higher concentration of AgNO^  (10 uM) produced a progressive decrease in the enzyme act i v i t y . The observed increase in enzyme acti v i t y as a result of incubation of the inhibited enzyme with 0.1 M reduced glutathione may be due to the reversal of Ag + inhibition of the enzyme or to an activation of previously inactive but uninhibited enzyme. The presence of 4.9 mM cysteine in the assay medium suggests that the former explanation i s more probable than the lat t e r . Due to the problems of Ag + precipitation as Ag^AsO^, and the nonspecific binding of Ag + to proteins present in the supernate, i t i s impossible to evaluate s i l v e r ion concentra-tion. The specific site(s) of s i l v e r inhibition of glucose-dependent and glycerol-dependent respiration could be obtained by determining the levels of glycolytic and TCA cycle intermediates in the absence and presence of s i l v e r nitrate at concentrations which are insufficient to inhibit the res-piratory chain but sufficient to inhibit glucose-dependent or glycerol-dependent respiration. The similarity of the results obtained for s i l v e r inhibition of res-piration with D-lactate, L-lactate, succinate and fumarate as substrates (Figures 3.16A, 3.17A, 3.17B and 3.18A respectively) suggests either that: the respective sites of s i l v e r inhibition have the same degree of sensitiv-i t y to Ag + or that there i s a single site of s i l v e r inhibition common to the respiration of these four substrates. Of these substrates, the f i r s t three undergo an i n i t i a l dehydrogenation via membrane bound dehydrogenases which introduce reducing equivalents directly into the respiratory chain (Figure 164 1.5 and Table 1.1). Fumarate i s only one reaction removed from the intro-duction of reducing equivalents into the electron transport system via the membrane bound malate oxidate system, or two reactions removed via the soluble NAD+-dependent malate dehydrogenase and the membrane bound NADH de-hydrogenase of the respiratory chain. Consequently there are no, or at most two reactions prior to the entry of reducing equivalents from the substrate into the respiratory chain. Therefore, in a l l probability the site at which s i l v e r ions inhibit E, c o l i respiration on these substrates i s in the elec-tron transport chain i t s e l f . Kasahara and Anraku (1972) have investigated the inhibition of the respiratory chain, in an E. c o l i membrane fraction, by zinc ions. • They observed that the presence of Z n + + (1 mM) decreased the succinate-dependent aerobic steady-state reduction level of cytochrome b-j, and that Z n + + at a concentration of 100 uM inhibited succinate dehydrogenase completely and D-lactate dehydrogenase by 69$. However, Barnes and Kaback (1971) reported that neither the D-lactate-DCIP reductase of E. c o l i membrane vesicles nor the partially purified D-lactate dehydrogenase was sensitive to the thi o l reagents NEM or PCMB. In agreement with the results of Kasahara and Anraku (1972), Bennett ejt a l . , (1966) have reported that D-lactate-DCIP reductase of E. c o l i respiratory particles was inhibited by PCMB. L-lactate-DCIP reductase was only s l i g h t l y inhibited. Barnes and Kaback (1971) also reported that NADH oxidation by membrane vesicles of E. c o l i was insensitive to i n h i -bition by NEM and that the slight inhibition observed by PCMB was not reversed by dithiothreitol. However, Bragg and Hou (1967a) have demonstrated the inhibition of NADH oxidase by PCMB, and Kim and Bragg (1971a) found a 33$ inhibition of E. c o l i succinate dehydrogenase by PCMB (0.5 mM) while at the 165 same concentration succinate oxidase activity was completely inhibited. Although i t i s impossible to conclude, from this conflicting information, whether the respiratory chain-linked dehydrogenases of _. c o l i are a l l sen-s i t i v e to inhibition by thi o l reagents i t would appear safe to assume that they w i l l not a l l have the same degree of sensitivity to s i l v e r inhibition. Reactive thiols have been demonstrated in the respiratory chain—linked dehydrogenases of mitochondria (Slater, 1949; Bernath and Singer, 1962; Singer and Gutman, 1971)» If one considers Ag + inhibition at a respiratory chain component present in each of the branches of the electron transport system prior to the convergence of these branches at cytochrome b_^  (Figure 1.6B or 1.6C) the same requirement of equal sensitivity to s i l v e r ion inhibition arises. Consequently, i t seems more reasonable to propose that the site of s i l v e r inhibition of D-lactate-, L-lactate-, succinate- and fumarate-dependent respirations of E. c o l i i s located in the respiratory chain between cyto-chrome b_i and oxygen. The site(s) at which Ag + inhibits the D-lactate-, L-lactate-, suc-cinate- and fumarate-dependent respiration could be determined with a greater degree of certainty by determining the influence of increasing Ag + concen-tration on the level of the steady-state reduction of the respiratory chain components with each of these substrates. These determinations would be more readily performed using E. c o l i membrane vesicles or respiratory par t i -cles, in which case i t would not be possible to examine the s i l v e r ion inhi-bition of the fumarate-dependent respiration. The failure of formate-dependent respiration to be completely i n h i -bited by a AgNO^  concentration of 86 uM (Figure 3.13B) suggests that the 166 entire formate oxidase system was less sensitive to Ag + inhibition than the oxidation of any of the other substrates examined. This requires that the formate dehydrogenase be resistant to Ag + inhibition at the concentration employed and that the electron transport system of the formate oxidase must be distinct from the respiratory chain of the D-lactate, L-lactate, and succinate oxidases, at least in the region of s i l v e r ion inhibition of the l a t t e r oxidases. No information i s available on the sensitivity of the E. c o l i formate dehydrogenases to inhibition by s i l v e r ions. However, Yudkin (1937) reported that the formate diaphorase activity of E. c o l i was an order of magnitude less sensitive to inhibition by s i l v e r than the corresponding glucose and succinate diaphorase a c t i v i t i e s . This is in agreement with the results reported in this thesis. With respect to the p o s s i b i l i t y that the electron transport system of the formate oxidase i s distinct from the respiratory chain of the D-lactate, L-lactate and succinate oxidases, investigations by Birdsell and Cota-Robles (1970) have led them to propose a model of the respiratory chain of E. c o l i in which the formate oxidase system i s distinct from the succinate and NADH oxidase systems except for the terminal oxidases, cytochromes a^ and o_. The site at which formate oxidation i s inhibited by Ag + could be determined by investigating the influence of higher concentrations of Ag + on the formate-dependent respiration and on the steady-state level of reduction of the respiratory chain components. To increase the sensitivity of the l a t t e r measurements i t might be necessary to grow the cells in the presence of formate to induce higher levels of the formate oxidase system. The author has described means by which the s i l v e r ion inhibition of 167 the various substrate dependent respirations could have been determined with . a greater degree of accuracy. The reason that such experiments were not per-formed was that our primary interest in the influence of Ag + on the respira-tion of E. c o l i was i t s apparent uncoupling action. Although silve r ions have been shown to inhibit the regulation of yeast hexokinase (Titova, 1968) , the author has not discussed the possible implications of s i l v e r ion inhibition of enzyme regulation on the results obtained as such influence would be essentially impossible to ascertain in an intact c e l l system. 3.2 . 5 Selection of a carbon source for the growth of E. c o l i to be used for the investigation of the.uncoupling of respiration by s i l v e r nitrate In evaluating the data presented in this section i t is important to remember that the culture was aerated continuously. Therefore, the oxygen level at a particular time represents the balance between the rate of u t i l -ization of oxygen and the rate at which oxygen dissolves in the medium. The investigation of the possible action of a substance as an un-coupler requires the u t i l i z a t i o n of a test system which generates the majority of i t s energy via the electron transport chain, and e f f i c i e n t l y conserves this energy (i.e.. a system which i s "highly coupled"). These are essential pre-requisites. I f energy is generated by a system other than the respiratory chain, and/or i f energy conservation i s not "highly coupled", then the action of a substance as an uncoupler, causing a stimulation of respiration, at best w i l l be minimal and at worst w i l l be undetectable. Thus, as indicated in section 3 . 1 . 5 , in order to investigate the action of s i l v e r nitrate as an uncoupler of E. c o l i respiration i t was essential to obtain E. c o l i cells which derived the majority of their energy requirement from the respiratory chain, and which were "highly coupled". 168 In addition to the preceding requirements with respect to energy-production and conservation, i t was of practical importance to be able to harvest cells possessing uniform physiological and biochemical properties. A f i n a l consideration was the greater sensitivity of the glycolytic pathway than respiration to inhibition by AgNO^ (sec.3.1.5 and 3.2.4). The i n h i b i -tion of respiration by AgNO^  when glycolytic intermediates are used as sub-strates might complicate the interpretation of the uncoupling data. Growth of E. c o l i NRC 482 on glucose as carbon source resulted in three phases of oxygen u t i l i z a t i o n and two phases of growth (Figure 3.20) as described in section 3.1.5. The f i r s t phase was demonstrated to be due to glucose oxidation (Figure 3.21). The continued decrease i n the redox poten-t i a l during the interval when the oxygen electrode indicated no detectable oxygen i s presumably due to the greater sensitivity of the redox electrode to low oxygen tensions but also could be due to the accumulation of redox active products (Jacob, 1970). The second phase of oxygen u t i l i z a t i o n and growth could correspond to the oxidation of lactate (Butlin et a l . , 1973) or pyru-vate (Raunio, 1966) accumulated under these conditions. The third phase of oxygen u t i l i z a t i o n commencing approximately 2 hr after the depletion of glucose i s believed to correspond to the induction of the glyoxylate cycle enzymes and the oxidation of acetate (Holms and Bennett, 1971). The second and third phases of oxygen u t i l i z a t i o n are more pronounced when fe r r i c citrate has been added to the medium. The reason for the apparent decline in the absorbance during the period of acetate oxidation i s unclear but may be related to a decrease in c e l l size and the dependence of light scattering on particle size and shape. There was no evidence to indicate that the de-crease in absorbance was due to c e l l l y s i s ( i . e . , increased foaming). The 169 significance and the value of the redox measurements w i l l be discussed later. Glucose was considered undesireable as the carbon source for the growth of E. c o l i which were "highly coupled" to energy production via the electron transport chain for the following reasons: (i) during exponential growth on glucose the major proportion of the energy u t i l i z e d by E. c o l i i s generated by glycolysis; ( i i ) Hempfling.'s data (1970b) indicated that the coupling of energy pro-duction to the respiratory chain developed after approximately $0 min of incubation subsequent to the depletion of glucose from the medium; ( i i i ) the second and third phases of oxygen u t i l i z a t i o n would make i t d i f -f i c u l t to harvest cells with constant biochemical and physiological proper-ties and; (iv) glycolysis is more sensitive to inhibition by AgNO^ than respiration. E. c o l i can grow only aerobically with glycerol as carbon source. Consequently the development of oxygen limited conditions between 5.5 and 7.25 hr demonstrated a greater effect on growth (Figure 3.22) than was apparent with cultures grown on glucose (Figure 3.20), which can be oxidized fermentatively. The interpretation of the continued decrease in the redox potential under oxygen limited conditions i s the same as that given for the glucose grown culture, that i s , the greater sensitivity of the redox measure-ments at low oxygen tensions. The significance of the shoulder in the redox potential curve, between 7.25 and 8.25 hr i s not known but may be related to the second phase of growth and oxygen u t i l i z a t i o n demonstrated by the cul-tures of E. c o l i grown on glucose (Figure 3.20), that i s , oxidation of accum-ulated lactate or pyruvate, although there was no marked change in the oxygen 1 7 0 level during this interval of growth. Although cells grown on glycerol obtain the majority of their energy from the respiratory chain and consequently could be harvested during the exponential phase of growth thus providing cells with constant characteris-t i c s , glycerol was considered to be unsuitable as the carbon source due to the greater sensitivity of glycolysis than respiration to inhibition by AgN05. The cultures of E. c o l i NRC 482 grown with 0.8$ DL-lactate as carbon source demonstrated two phases of oxygen u t i l i z a t i o n and growth (Figure 3.23) similar to the f i r s t two phases observed in the culture of E. c o l i grown on 0.4$ glucose. The f i r s t phase of growth and oxygen u t i l i z a t i o n was inter-preted as resulting from the oxidation of D-lactate and L-lactate, as E. c o l i has been shown to possess both D- and L-lactate oxidases when grown on DL-lactate (Bennett et a l . , 196"6). D- and L-lactate, l i k e glycerol, can not be oxidized fermentatively by E. c o l i . Consequently, there was a marked influence of the oxygen limited conditions on the growth rate of these cultures of E_. c o l i . During the interval of oxygen limitation growth was essentially linear and the redox potential of the culture also decreased linearly. The substrate for the second phase of oxygen u t i l i z a t i o n i s possibly excess pyruvate which would possibly accumulate under the conditions of oxygen lim-itation, as has been reported to occur with cultures grown on glucose (Raunio, 1966). However, as catabolite repression of the glyoxylate cycle enzymes would probably be much reduced with DL-lactate rather than glucose as the carbon source, the p o s s i b i l i t y that the second phase of oxygen u t i l i z -ation i s due to the oxidation of acetate can not be eliminated. Due to the second phase of oxygen u t i l i z a t i o n and the resulting 171 d i f f i c u l t y in harvesting cells with uniform biochemical and physiological properties, DL-lactate was considered to be unsuitable as the carbon source for the growth of "highly coupled" E. c o l i . Growth of S. c o l i on 0.8$ acetate v/as slow with a large proportion of the growth curve demonstrating a linear rate of growth (during the inter-val 8.0 to 12.0 hr) (Figure 3 . 2 4 ) . The reason for the linear rate of growth is uncertain. It would not appear l i k e l y that i t was due to oxygen li m i t a -tion, since the oxygen level was 35 to 40$ of saturation when the growth rate f i r s t became linear. Perhaps the respiratory chain of these c e l l s have a low a f f i n i t y for oxygen. This is a distinct p o s s i b i l i t y as the c e l l s have a slow growth rate and were grown with an a i r flow rate of 4 .4'l/min/l of medium and consequently may possess a cytochrome oxidase system with a high Kj^ for oxygen as has been reported for Hemophilus parainfluenzae and Azotobacter vinelandii (White, 1963; Nishizawa et a l . , 1971). As was observed in the culture of E. c o l i grown on 0.8$ DL-lactate the decrease in the redox potential of the culture was linear during the period of linear growth. There are a number of other characteristics which are unusual. The smooth nature of the growth curve as i t approaches the stationary phase, and the failure of the oxygen level to return to near saturation when growth has ceased suggests that the culture may have been limited by some component, other than the carbon source, possibly nitrogen, and that while growth has ceased metabolism of the carbon source has not. No significance has been attached to the shoulders in the oxygen level and redox potential curves at 3.0 and 6.0 hours respectively. E. c o l i grown on succinate in the presence of 6 uM f e r r i c citrate possessed the desired characteristics: 172 ( i ) of obtaining the majority of their energy requirements from the respiratory chain; ( i i ) were "highly coupled" (sec. 4 .2 .2 ) ; ( i i i ) of no secondary phases of oxygen u t i l i z a t i o n or growth so that cells of constant biochemical and physiological properties could be harvested during either the logarthmic or the stationary growth phase; and (iv) interpretation of uncoupling data would not be complicated by the sensitivity of the glycolytic pathway to inhibition by AgNO^. Although the B . c o l i grown on acetate as carbon source also possessed these desired characteristics succinate was chosen in preference to acetate as the most suitable carbon source for the growth of E. c o l i "highly coupled" to energy production by the.electron transport chain on the basis of the more rapid growth rate and the shorter adaptation procedure with succinate. 3.2.6 The uncoupling of the respiration of E. c o l i by added s i l v e r nitrate While determining the characteristics of s i l v e r inhibition of the endogenous and substrate-dependent respiration of E. c o l i (Figures 3.12 to 3.18 inclusive) i t was observed that the respiration immediately following the addition of s i l v e r nitrate, or immediately after the addition of E. c o l i to the assay medium containing substrate and AgNO^, was greater than the control rate. The stimulation was particularly marked with cells grown on glucose as carbon source but u t i l i z i n g acetate as the respiratory substrate, reaching a respiration rate 4-fold greater than that of the control (Figure 3.14) . There are two alternative explanations for these observations: (i) that the rate of respiration was stimulated by the presence of the nitrate ion which was acting as an alternative electron acceptor; or ( i i ) that s i l v e r ions were acting as an uncoupling agent of E. c o l i respiration. 173 Figure 3.28 demonstrates that the addition of 40 uM KNO^  to the assay system had l i t t l e i f any stimulatory effect on the respiration rate. This eliminated the f i r s t p o s s i b i l i t y . A comparison of the stimulation of the respiration of E. c o l i by AgNO^ (Figure 3.27), and by a known uncoupler of mitochondrial respiration, 2,4-dibromophenol (Figure 3.26), supports the second interpretation. Additional support for the action of si l v e r ions as uncouplers of E. c o l i energy conservation was obtained from Figure 3.29 and table 3.8. These results indicate that the respiration of highly coupled E. c o l i (grown under iron-sufficient conditions) (sec.4.2.2) was stimulated to a greater extent by AgNO^  than the less highly coupled cells (grown under iron-limited conditions). The uncoupling of mitochondrial respiration and energy conservation by s i l v e r ions has been reported by Chappell and Greville (1954) and Grabske (1966). Chappell and Greville were able to correlate the increased respira-tion rate with a five-fold increase in ATPase activity. Cooper (i960) has subsequently examined the influence of Ag on the Mg -stimulated ATPase of submitochondrial particles prepared from rat l i v e r mitochondria. A two-fold stimulation of ATPase activity was observed when approximately 50$ of the total free, readily available, sulfydryl groups had reacted. Further addition of Ag +, resulted in loss of ac t i v i t y . Similar results were obtained with HgCl 2 and PCMB as complexing agents. Thus, the observed stimulation of the respiration rate appears to be directly related to the stimulation of the ATPase by thiol reagents. Cooper (i960) proposed that the stimulating action of Ag (Hg or PCMB) was due to the blockage of an inhibitory grouping at the active s i t e . Kielley (1963) has compared the loss of oxidative phosphorylation, 174 inhibition of the ATP - P^ exchange reaction and disappearance of DNP stim-ulation of the ATPase during t i t r a t i o n with PCMB and suggests that these . sens i t i v i t i e s are related to t h i o l groups of very similar reactivity, i f not the same groups. These observations raise the p o s s i b i l i t y that the observed inhibition of the D-lactate, L-lactate-, succinate- and fumarate-dependent + + respirations by Ag , may be due to Ag inhibition of the u t i l i z a t i o n of res-piratory chain generated energy. 3 . 2 . 7 Redox potential as an indicator of the oxygen level of batch cultures of E. c o l i During the experiment to select the most suitable carbon source, the usefulness of the redox potential as an indicator of the oxygen level and/or in providing additional information about the bacterial culture was evaluated. Jacob (1970) has discussed the measurement of the redox potential of microbial cultures in considerable d e t a i l . Compared to a limit of sensitivity of approx-imately 1 mm Hg for the measurement of the oxygen tension of the medium with an oxygen electrode, the dependence of the. redox potential of the medium on the - 9 oxygen tension has been reported down to oxygen pressures of about 10 atm (Jacob, 1970) . Thus, the redox potential of the culture i s a much more sen-si t i v e indicator of the oxygen content of the medium at low oxygen tension, that i s below oxygen tensions of approximately 10 atm. However, since the redox potential of the culture i s a function of the log of the oxygen tension i t i s a less sensitive indicator of the changes in oxygen tension between 10 atm and 10 1 atm than the oxygen electrode which responds directly to the oxygen tension. In addition, the redox potential measured during the c u l t i -vation of microorganisms could be the result of metabolic products with redox character, and/or by the ac t i v i t y of different enzymes (oxidases, dehy-drogenases) in the c e l l . 175 On closely examining Figure 3.20, and 3.22 to 3.25» inclusive, with respect to the relationship between the redox potential and the oxygen level of the bacterial culture, the following observations can be made. (i) Generally there is a considerable similarity in the shape of the redox potential curve and the oxygen saturation curve to the point at which the oxygen level becomes undetectable by the oxygen electrode. However, the redox potential changed much more rapidly than the log of the oxygen tension. ( i i ) During the interval when the oxygen tension was not detectable with the oxygen electrode the redox potential continued to decrease presumably due to the greater sensitivity of the redox electrode to low oxygen tension. How-ever, during the period of oxygen-limited growth on carbon sources which had to be oxidized aerobically, the redox potential decreased linearly over essentially the same interval that the growth was linear (Figures 3.22, 3.23, 3.24 and 3.25). This suggested a closer relationship between the redox poten-t i a l and the c e l l concentration or the accumulation of some product, ( i i i ) The redox potential and oxygen saturation demonstrate the same phase part i -cularly in the cultures grown on glucose and DL-lactate (Figures 3.20 and 3.23 respectively) with the redox potential demonstrating the lower sensi-t i v i t y that one would expect of a logarithmic function of the oxygen concen-tration. However, the complete opposite was observed with E. c o l i cultures u t i l i z i n g glycerol as the carbon source (Figure 3.22). Approaching the end of the interval of oxygen limitation the redox potential of the culture i n -creased dramatically with l i t t l e corresponding increase in the oxygen lev e l . Considering these observations, the u t i l i z a t i o n of the redox potential of a culture as the sole indicator of the oxygen level seems undesirable, at least with aerobic or facultatively anaerobic organisms. The physiologically and biochemically important concentrations of oxygen for cultures of E. c o l i as indicated by the influence of oxygen limitation on growth appears to be within the sensitivity range of the oxygen electrode and thus preclude the necessity of the sensitivity of the redox electrode. However, the work of Wimpenny and colleagues (Wimpenny, 1969; Wimpenny and Necklen, 1971) on the relationship between cytochrome synthesis and the redox po-tential of the culture demonstrates that redox potential measurements definitely have a place in the investigation of microbial processes. 177 4 PART II: IRON LIMITATION AND THE RESPIRATION AND ENERGY-COUPLING OP E. COLI 4.1 Results 4.1.1 The influence of iron limitation on the respiration and energy-coupling of E. c o l i During a more detailed investigation of the growth of E. c o l i on succinate as carbon source, i t was observed that the oxygen level of a cul-ture of E. c o l i , grown in medium C, reached a plateau value well above zero oxygen saturation (Figure 4.1). The plateau was demonstrated to be real, and not an artifact of the electrode, by ceasing aeration and establishing the residual current of the electrode (the electrode current in the absence of oxygen) by flushing with nitrogen. During this period a l l growth ceased but recommenced immediately once aeration was restarted. The oxygen level returned slowly to the level of the plateau with reaeration of the culture. Further investigation of this phenomenon (Figure 4.2) established that the plateau in the level of oxygen saturation of the culture medium continued with l i t t l e change u n t i l a l l of the succinate had been u t i l i z e d at which point the oxygen level returned to close to saturation. The onset of the plateau in oxygen level coincided with an i n i t i a l change from an expon-ential to a linear growth rate which subsequently became a progressively decreasing rate. Associated with the plateau in oxygen level and the de-crease in growth rate were a decrease in the efficiency of conversion of succinate to c e l l mass, and a decrease in the level of cytochrome bj per unit of wet c e l l mass. Similar patterns of response were obtained with E. c o l i B and E. c o l i B-SG1. During the plateau in oxygen level the c e l l mass increased 2 to 3 fold while the rate of oxygen u t i l i z a t i o n by the entire culture remained 178 Pig. 4.1 Plateau in the oxygen level of a culture of E. c o l i growing on 0.6$ succinate. A: aeration ceased; B: N2 flushing commenced; C: aeration recommenced; Absorbance at 420 nm (methods: sec. 2.2.6.1). 179 Fig. 4.2 Growth and oxygen level (A), efficiency (B) and cytochrome b levels (c) of culture of E. c o l i growing on 0.6$ succinate. Absorbance at 420 nm; units: cytochrome b-|» nmoles/g cells (wet weight); (methods: sec. 2.2.6.1, 2.2.6.2 and 2.2.6.7). 181 relatively constant. This would indicate that the rate of oxygen uptake per unit of c e l l mass decreased during the period of the plateau. This suggested that the respiration of the E. c o l i was limited by an inadequate level of some component. As a decrease in the level of cytochrome bj had been observed (Figure 4.2), iron appeared to be a l i k e l y candidate. As shown in Figures 4.3 and 4.4, and table 4.1, the addition of f e r r i c citrate to a f i n a l concentration of 6 uM resulted, after a 15 min lag, in a rapid drop in the level of oxygen saturation and an increase in the growth rate from 1.2 absorbance units/hr to 2.95 absorbance units/hr. How-ever, the increased growth rate, subsequent to the addition of f e r r i c citrate (7.5 hr), is less than the exponential growth rate prior to the onset of iron limitation (5.5 hr) (Figure 4.4). Measurement of the levels of nonheme iron and cytochrome b^ prior to, and following the addition of f e r r i c citrate indicated that during the two hours preceding the addition of f e r r i c citrate the nonheme iron levels decreased by 70$ while there was a corresponding de-crease in cytochrome b-j of 20$. Consequently the ratio of nonheme iron the cytochrome bj f e l l from 19.7:1 to 7.5:1 (Table 4.1). Subsequent to the addi-tion of f e r r i c citrate, and prior to any apparent changes in the growth rate or the rate of oxygen consumption (i.e., within the 0.25 hr following the addition of f e r r i c citrate to the culture), the nonheme iron levels increased by greater than 290$. During the same period cytochrome b^ levels increased by only 8$. Although both the rate and the amount of increase in the levels of nonheme iron were much greater, the increase in the level of cytochrome b^ was considerable, being an increase of approximately 160$, in the 3 hr follow-ing the addition of f e r r i c citrate. With such marked changes in the levels of known respiratory chain 182 Fig. 4.3 The response of growth and oxygen level (A), and nonheme iron and cytochrome levels (B) to the addition of f e r r i c citrate (Fe) to a culture of E. c o l i growing on 0.6% succinate. Absorb-ance at 420 nm; units: cytochrome b^, nmoles/g cells (wet weight); nonheme iron, natoms/g cells (wet weight); (methods: sec. 2.2.6.1, 2.2.6.5 and 2.2.6.7). 184 0.1 6 12 Hours Fig. 4.4 A semi-log plot of the growth data from Fig. 4.3. 185 Table 4.1 The level of iron-containing respiratory chain components in c e l l extracts of iron-limited, succinate-grown, E. c o l i prior to, and following the addition of f e r r i c citrate (final cone, 6 p_M), Time 1 (hr) Nonheme T 2 Iron <& Cytochrome b i ^ Ratio 4 A420 - 2 . 0 227 329 11.5 125 19.7 3.68 - 1 . 0 73 106 10.4 113 7.0 4.83 0 69 100 9.2 100 7.5 5.98 100 0.25 272 394 9.9 108 27.5 6.27 . 105 0.5 406 588 10.7 116 37.9 6.73 113 1 . 0 516 748 13.1 142 39.4 8.07 135 2 . 0 688 997 15.2 165 45.3 1 1 . 2 0 187 3.0 686 994 23.8 259 28 .8 13.36 223 4.0 705 1022 —— —— — 13.44 225 1 0 corresponds to the time < of addition of f e r r i c citrate; negative time indicates time prior to the addition of f e r r i c citrate. 2 Values expressed as natoms per g cells (wet weight). ^ Values expressed as nmoles per g cells (wet weight). 4 Values expressed as natoms per nmoles. 186 components i t was of considerable interest to investigate the influence of the addition of fe r r i c citrate to an iron-limited culture on the level of respiratory chain associated enzymes. The results presented in Figure 4 . 5 and table 4 . 2 indicate the observed changes in the level of cytochrome b^ and in the a c t i v i t i e s of NADH oxidase, succinate oxidase and succinate dehy-drogenase. The rate of increase in the enzyme a c t i v i t i e s and cytochrome b_^  levels were comparable (Table 4 . 2 ) . However, the level of succinate oxidase ac t i v i t y increased significantly faster than did the other three a c t i v i t i e s . The rate of increase in the levels of NADH oxidase, succinate oxidase, suc-cinate dehydrogenase or cytochrome b^ (Figure 4 . 5 ) did not compare with the rapidity of the increase i n nonheme iron levels (Figure 4 . 3 ) . To investigate the possi b i l i t y that the observed changes in oxygen le v e l , growth rate, cytochrome b^, nonheme iron, NADH oxidase, succinate oxidase and succinate dehydrogenase levels following the addition of f e r r i c citrate might be due to the citrate ion rather than the f e r r i c ion, sodium citrate was added to a culture of E. c o l i during the plateau in oxygen level (Figure 4 . 6 ) . The addition of sodium citrate instead of f e r r i c citrate to a f i n a l concentration of 6 uM did not result in marked changes in growth rate, the level of oxygen saturation, or cytochrome b_^  levels. A comparison of the results obtained when no addition was made during the plateau in the oxygen saturation of the culture (Figure 4 . 2 ) and those obtained following the addition of sodium citrate (Figure 4 . 6 ) indicates that they are nearly identical, and di s t i n c t l y different from the results obtained following the addition of f e r r i c citrate during the plateau in oxygen saturation (Figure 4 . 3 ) . Consequently, i t can be concluded that the active component of the fe r r i c citrate i s the fe r r i c ion. 187 Fig. 4.5 The response of growth and oxygen level (A) and, enzyme levels (B) to the addition of f e r r i c citrate to a culture of E. c o l i growing on 0.6$ succinate. Absorbance at 420 nm; units: specific activity, umoles/min/g cells (wet weight); succinate oxidase, • ; succinate dehydrogenase,T; NADH oxidase, • ; (methods: sec. 2.2.6.1, 2.2.6.8, 2.2.6.9 and 2.2.6.10). 189 Table 4.2 Enzyme activites in c e l l extracts of iron-limited, succinate-grown E. c o l i following the addition of f e r r i c citrate (final cone. 6 uM). Time (hr) Cytochrome b. NADH 2 Oxidase at 1° Succinate Oxidase 3 to 7 Succinate Dehydrogenase % A 4 2 0 0 9.7 100 1 0 . 2 100 41.4 100 27.2 100 6.88 100 0.25 8 . 8 91 1 0 . 8 106 46.5 112 25.5 94 7.00 102 0.5 9.4 97 1 1 . 8 116 65.5 158 31.9 117 7.46 108 1 . 0 1 1 . 2 116 15.9 156 70.7 171 55.0 159 8.70 126 2 . 0 15.6 161 16.1 158 8 7 . 8 212 49.7 185 11.60 169 4.0 17.5 180 16.7 164 8 4 . 2 205 57.8 212 15.08 190 Values expressed as nmoles per g c e l l s (wet weight). 2 Values expressed as umoles substrate oxidized per min per g c e l l s (wet weight). Temperature 22°C. 3 Values expressed as umoles substrate oxidized per min per g cells (wet weight). Temperature 57°C. 190 Fig. 4.6 The response of growth and oxygen level (A), efficiency (B) and cytochrome b-j levels (C) to the addition of sodium citrate (SC) to a culture of E. c o l i growing on 0.6$ succinate. Absorbance at 420 nm; units: cytochrome b.., nmoles/g cells (wet weight); (methods: sec. 2.2.6.1, 2.2.6.2 and 2.2.6.7). Hours Fig. 4.6 192 However, i t was observed that the addition of f e r r i c citrate during the plateau in oxygen saturation of a nitrogen-limited culture of E. c o l i * failed to cause a decrease in the level of oxygen saturation (Figure 4.7). Subsequently addition of ammonium sulfate did result in a stimulation of res-priation after a 15 min lag, suggesting that protein synthesis might be re-quired for respiration to be stimulated by the addition of iron. Pertinent to this p o s s i b i l i t y was the observation that i f E. c o l i were grown in the presence of trace elements (Holms et a l . , 1970) other than iron (Appendix C), the addition of f e r r i c citrate during the plateau in oxygen saturation resulted in an immediate and rapid decline in the level of oxygen saturation (Figure 4.8). That i s , growth of E. c o l i in the presence of trace elements other than iron abolished the 15 min lag in the response of oxygen saturation to the added f e r r i c citrate. The response of growth to the added f e r r i c citrate appeared to demonstrate the usual lag prior to attaining the new growth rate (Figure 4.8). Having investigated an "iron-limited" culture (Figure 4.2), and the response of an "iron-limited" culture to the addition of f e r r i c citrate (Figures 4.3 and 4.5, Tables 4.1 and 4.2), i t was of considerable interest to investigate the growth, oxygen u t i l i z a t i o n , redox potential, cytochrome and nonheme iron levels, and the efficiency of conversion of carbon source to c e l l mass of an "iron-sufficient" culture of E. c o l i NRC 482. The results of this investigation are presented in Figures 4.9 to 4.12 inclusive. The data presented were obtained from the same experiment. Growth and oxygen u t i l i z a t i o n were rapid with the level of oxygen saturation in the culture approaching, but not reaching zero. Preceding the period of oxygen limitation, growth of the culture demonstrated two 193 Fe AS Hours Fig. 4.7 The response of growth and oxygen level to the addition of f e r r i c citrate (Fe) and ammonium sulfate (AS) to an iron-deficient, nitrogen-limited culture of E. c o l i growing on 0.6% succinate. Absorbance at 420 nm; (methods: sec, 2.2.6.1) 194 Fe 0) o c ro n o w Fig. 4.8 The response of growth and oxygen level to the addition of f e r r i c citrate (Fe) to a culture of E. c o l i growing on 0.6$ succinate in a medium containing the trace metals Ca + +, Zn + +, Co + +, Mn + + and Cu + +. Absorbance at 420 nm, (methods: sec. 2.2.6.1). 195 Fig. 4.9 The growth and oxygen level (A) and cytochrome a 2, cytochrome and nonheme iron levels (B) of an iron-sufficient culture of E. c o l i growing on 0.6$ succinate. Absorbance at 420 nm; units: cytochromes a? and b-j, nmoles/g cells (wet weight); nonheme iron, natoms/g cells (wet weight); (methods: sec. 2.2.6.1, 2.2,6.5 and 2.2.6.7). 197 Fig. 4.10 The growth and oxygen level (A) and efficiency (B) of an iron-sufficient culture of E. c o l i growing on 0.6$ succinate. Absorbance at 420 nm; (methods: sec. 2.2.6.1 and 2.2.6.2). 190 i i- 1 J 1 1 i I 0 6 12 Hours Fig. 4.10 Fig. 4.11 The growth, oxygen level and redox potential of an iron-sufficient culture of E. c o l i growing on 0.6$ succinate. Absorbance at 420 nm; units: redox potential, mV x 10" ; (methods: sec. 2.2.6.1). 200 Hours Fig. 4.12 A semi-log plot of the growth data from Fig. 4.9. 201 successive phases of exponential growth, (i) 0 to 3.0 hr, and ( i i ) 3.0 to 6.0 hr (Figure 4.12). During conditions of oxygen limitation the growth of the culture was linear with a rate of increase of 2.83 absorbance units/hr (.Figure 4.9). During the same interval the nonheme iron level dropped by approximately 35$. Cytochrome b^ and ag levels commenced to increase after 6.5 hr of growth. Cytochrome a^ demonstrated an increase in level of approx-imately 200$ between 6.5 and 7.5 hr with no further increase. Cytochrome b-j levels increased linearly between 6.5 and 9.5 hr, increasing by a total of 70$. The redox potential of the culture was 230 mV (Figure 4.11) when the level of the cytochromes began to increase (Figure 4.9). The level of cyto-chrome _2 increased no further after the redox potential had reached 210 mV. The increase in the level of cytochrome b^ was continuing when the redox potential of the culture was 180 mV. The efficiency of conversion of suc-cinate to c e l l mass increased up u n t i l between 5.0 and 6.0 hr. At approx-imately the same time as oxygen became limiting the efficiency began to de-cline (Figure 4.10). For the purpose of comparing the response to iron limitation of cells grown on a fermentable carbon source with the response of cells grown on a nonfermentable carbon source, B. c o l i NRC 482 was grown on glucose as the carbon source under "iron-limited" conditions. As is apparent from Figure 4.13, although nonheme iron and cytochrome b^ levels were low and decreased during growth, the cells were capable of completely depleting the medium of oxygen with no apparent adverse effects on the growth rate (Figure 4.14). The lack of effect of iron limitation on the growth of E. c o l i on glucose raised the possibility that iron was important in the coupling of energy production to growth when the carbon source was nonfermentable. This 202 Fig, 4.13 The growth and oxygen level (A) and cytochrome and nonheme iron content (B) of an iron-limited culture of E. c o l i growing on 0.4$ glucose. Absorbance at 420 nm; units: cytochrome b^ nmoles/g cells (wet weight); nonheme iron, natoms/g cells (wet weight); (methods: sec 2.2.6.2, 2.2.6,5 and 2.2.6.7). 203 12 \-Fig. 4.15 Hours 204 Hours Fig. 4.14 A semi-log plot of the growth data from Pig. 4.13. 205 p o s s i b i l i t y was investigated by (i) calculating the conversion of succinate to c e l l mass prior to, and following the addition of f e r r i c citrate, and ( i i ) determining the respiratory control ratio of the cells under "iron-limited" and "iron-supplemented" conditions as a measure of the degree of coupling of energy production to the respiratory chain. The results (Fig-ure 4.15) demonstrate that the efficiency of the conversion of succinate to c e l l mass decreased u n t i l the plateau in the level of oxygen saturation was attained, at which point the efficiency became relatively constant. Follow-ing the addition of f e r r i c citrate the efficiency increased for a period of 1 hr and then declined. The influence of iron limitation on the respiratory control ratio (RCR) was more dramatic. The RCR decreased progressively during the development of iron limitation. The addition of f e r r i c citrate to the iron-limited culture resulted in a rapid increase in the value of the respiratory control ratio. The rapidity of the increase in the RCR, greater than 280$ in the 15 min immediately following the addition of f e r r i c citrate, was comparable to the rate of increase in the level of nonheme iron which increased by greater than 290$ over the same time interval (Table 4.1). These results supported the concept that iron was involved in the coupling of energy production to the respiratory chain and also provided the information required for obtaining "highly coupled" E. c o l i grown on succinate as the carbon source, 4.2 Discussion 4.2,1 Batch culture versus continuous culture One of the primary considerations when commencing an investigation of this type i s whether to u t i l i z e batch or chemostat culturing techniques. As pointed out by Clegg and Garland (1971) and Light and Garland (1971), 206 Fig. 4.15 The response of growth and oxygen level (A), efficiency (B) and the respiratory control ratio, RCR (c ) to the addition of f e r r i c citrate (Fe) to a culture of E. c o l i growing on 0.6$ succinate. Absorbance at 420 nm; 2,4-dibromophenol concentration,•, 125uM;B, 50 uM; (methods: sec. 2.2.6.1, 2.2.6.2, 2.2.6.3 and 2.2.6.4). 208 the obvious attractions of the batch culture are i t s simplicity and lack of expense while those of the continuous culture are a reliable, convenient and reproducible supply of c e l l s available at a l l times. More important, chem-ostats provide the a b i l i t y to make accurate and repeatable alterations of the growth conditions leading to changes in respiratory or other functions. Pirt (see Demain and Gooney, 1972) has also pointed out that chemostats extend the range of conditions to be examined since they permit substrate limited growth for an indefinite period. Some of the additional beneficial features of the chemostat include the a b i l i t y to vary the growth rate with-out otherwise changing the environment, the maintenance of a constant growth rate while varying other conditions such as temperature, oxygen tension and pH. However, as indicated by Maal/e and Kjeldgaard (1966) the completely undifferentiated behavior of a continuous culture i s also a limitation in that the number of observable characters i s reduced to a minimum. The relationship between two parameters may be more readily apparent as a result of the transition of conditions occurring in a batch culture. Consequently i t i s frequently advantageous to use batch cultures in establishing relation-ships between observed characteristics of the culture, and to u t i l i z e the more stringently controlled conditions of continuous culture to examine the relationship of these characteristics in greater d e t a i l . The reason that batch cultures were chosen was the a b i l i t y to measure the change in cellular parameters as the cells depleted the iron content of the medium and the response of these parameters to the rapid re-establishment of iron sufficient growth conditions. 209 4.2.2 The influence of iron limitation on the respiration and energy-coupling of E. c o l i The interpretation of the data presented in section 4.1.1 relies heavily on the results of investigations on the involvement of nonheme iron in site I oxidative phosphorylation of the yeast Candida u t i l i s as reported by Garland and co-workers (Clegg e_t a l . , 1969; Light and Garland, 1971; Clegg and Garland, 1971; Haddock and Garland, 1971) and Ohnishi and colleagues (Ohnishi et __1. , 1969; Ohnishi e_t a l . , 1971; Ohnishi et a l . , 1972a), and on the vali d i t y of the respiratory control ratio as an i n d i -cator of the extent of coupling of energy conservation to energy generation via the respiratory chain (Hempfling, 1970b). These topics have been di s -cussed in sections 1.6 and 1.4, respectively, and w i l l not be dealt with further at this point. There i s l i t t l e evidence available that iron i s involved in energy-coupling in bacterial systems. Oxidative phosphorylation has been shown to be uncoupled by chelating agents in Azotobacter vinelandii (Jones e_ a l . , 1972), Strepto coccus faecalis (Faust and Vandemark, 197G) B. c o l i {Kashket and Brodie, 1963a) and Mycobacterium phlei (Kurup and Brodie, 19^7). The energy-dependent transhydrogenase of E. c o l i i s also very sensitive to chel-ating agents (Bragg and Hou, unpublished data). The data presented in this thesis supports the hypothesis that nonheme iron i s involved in energy-coupling in E. c o l i . The greater sensitivity to iron deficiency of the growth and res-piration of the culture grown on succinate (Figures 4.2, 4.3, 4.5 and 4.15) compared with that grown on glucose (Figure 4.13) is in accordance with the hypothesis that iron limitation affects the respiratory chain and/or coupled energy production since fermentation would provide energy during growth on 210 glucose. Lower NADH oxidase, succinate oxidase, and succinate dehydrogenase a c t i v i t i e s (Figure 4.5, Table 4.2) and lower levels of cytochrome bj (Figure 4.3, Tables 4.1 and 4.2) in iron-deficient as compared to iron-supplemented c e l l s , indicates that a direct effect on the respiratory chain must have occurred as has also been observed in iron-deficient Aerobacter  indologenes (Waring and Werkman, 1944), Corynebacterium diphtheriae (Pappenheimer and Hendee, 1947; Righelato, 1969; Righelato and van Hemert, 1969), Neurospora crassa (Nicholas and Gommissiong, 1957; Padmanaban and Sarna, 19^5), Mycobacterium smegmatis (Winder and O'Hara, 1964), Pseudomonas  fluorescens (Lenhoff e_t a l . , 1956), Spirillum i t e r s o n i i (Clark-Walker et a l . , 1967), Micrococcus denitrificans (imai et a l . , 1968) and Candida u t i l i s (Light and Garland, 1971; Clegg and Garland, 1971; Ohnishi et a l . , 1969). Of the four respiratory chain associated parameters cytochrome b^, NADH oxidase, succinate oxidase and succinate dehydrogenase, the succinate oxi-dase activity increased at a significantly faster rate than did the other three a c t i v i t i e s (Figure 4.5, Table 4.2). Since the level of succinate oxidase increased at a faster rate than that of both cytochrome b-| and suc-cinate dehydrogenase, i t was unlikely that the l a t t e r components were rate limiting. Thus, since the rate of increase in the level of nonheme iron was more rapid than that of any other respiratory chain associated-- parameter measured, i t is probable that the rate of the succinate oxidase was limited by a nonheme iron component. Such a component is known to be present in this system in E. c o l i (Kim and Bragg, 1971a). However, the possib i l i t y that iron may influence the synthesis of a component not measured, cannot be excluded. In addition to the preceding qualification, the reason that there was no 211 change in the rate of oxygen u t i l i z a t i o n during the 15 min immediately following the addition of 6 uM fe r r i c citrate to the iron-limited culture, when the succinate oxidase activity increased by 12$ during this time, re-mains unknown. An effect of iron limitation on respiratory chain-linked energy production in addition to that on the respiratory chain was suggested by the following observations. Luring the development of iron limitation the level of nonheme iron per unit c e l l mass decreased by 70$ to a nonheme iron: heme iron ratio of 7*5:1 (Figure 4.3, Table 4.1) and the respiratory control ratio decreased to close to 1.0 (Figure 4.15). The efficiency of conversion-of succinate to c e l l mass decreased to the point at which the a v a i l a b i l i t y of oxygen became growth limiting and remained more or less constant at this level (Figure 4.15). Subsequent to the addition of fe r r i c citrate the non-heme iron level (Figure 4.3, Table 4.1) and the respiratory control ratio (Figure 4.15) increased dramatically within the f i r s t 15 min. During this interval the increases in the levels of cytochrome b^ (Figure.4.3, Tables 4.1 and 4.2) and the respiratory chain associated enzymes (Figure 4.5, Table 4.2) were minimal with a slight decrease observed in the succinate dehydro-genase levels. There was no observable change in the efficiency (Figure 4.15). Hempfling (1970b) has shown that stimulation of respiration of whole cells of S. c o l i oxidizing glucose, produced by the addition of 2,4-dibromo-phenol, occurs only when a l l three sites of oxidative phosphorylation are coupled. Thus, the similarity of the kinetics of the responses of the level of nonheme iron and the respiratory control ratio, particularly within the f i r s t 15 min of the addition of fe r r i c citrate, compared with the much slower rate of increase in the oxidase a c t i v i t i e s and cytochrome b 1 l e v e l , 212 is compatible with the hypothesis that nonheme iron has a role in energy conservation linked to the respiratory chain of E. c o l i . These experiments do riot indicate i f energy-coupling was effected at one or at a l l three sites in iron-limited cells of E. c o l i . At present there i s no technique which w i l l permit the measurement- of the :individual sites of oxidative phosphoryla-tion in E. c o l i . The rapidity of the increase i n the nonheme iron levels and the RCR values suggests that protein synthesis was not required in the recovery of these parameters. This would agree with the results of Garland's group but not with the recent results reported by Ohnishi et_ a l . , (1972a). If as proposed by Slater (1953) for mitochrondrial systems, the E. c o l i energy coupling mechanism involves the formation of a high energy intermediate, generated either via the electron transport system or by hydrolysis of ATP via ATPase, which i s subsequently u t i l i z e d to drive mem-brane associated cellular processes, then in order for iron limitation to have a minimal influence on the growth of E. c o l i on glucose, the involve-ment of iron in energy coupling would have to be between the respiratory chain and the high energy intermediate. This i s in agreement with the results obtained with Candida u t i l i s in which iron limitation results in the loss of the energy-dependent reversal of electrons from glycerol-1-phosphate to endogenous pyridine nucleotide, a process believed to be driven directly by a high energy intermediate. The fact that the nonheme iron level continued to increase for an additional hour after, and to a f i n a l level 50$ greater than i t s level when the RCR value had reached i t s maximum, was probably the same phenomenon as observed by Clegg and Garland (1971), that i s , that only some 10$ of the 213 total nonheme iron content of the mitochondria of C_. u t i l i s would he required to account for coupling at site I. This fact- coupled with the observation that the cytochrome b_i level of iron-limited E. c o l i , grown on either glucose or succinate, failed to decrease below a level of 7.5 to 8.0 nmoles per gram wet weight of cells while the nonheme iron levels in some of these experiments dropped to levels which were not detectable by the o_-phenan-throline colorimetric assay used, suggests that a large portion of the non-heme iron may function as an iron source for heme synthesis. A second pos-s i b i l i t y that cannot be excluded at this time, is that the high nonheme iron levels may represent iron accumulated within the intact c e l l , in the process of being metabolized, but not associated with any particular system. Returning to a consideration of the efficiency of conversion of suc-cinate to c e l l mass during the development of iron-limited conditions, the decrease in the efficiency was probably due to the decrease in the coupling of energy conservation to the electron transport chain which in turn has been correlated with the decrease in the nonheme iron content of the c e l l . Following the addition of f e r r i c citrate, however, the efficiency increased slowly, rather than demonstrating the rapid recovery shown by the nonheme iron levels and the degree of coupling of energy conservation to the electron transport system as indicated by the RCR values. The reasons for this remains unclear but may be due to the u t i l i z a t i o n of the increased energy available from the more "highly coupled" respiratory chain to drive processes such as the transport, the metabolism and/or the redistribution of the iron, or the synthesis of a minor ce l l u l a r component(s) required before a detectable i n -crease in c e l l mass could occur. The subsequent decline in the efficiency of conversion of succinate to c e l l mass (commencing at 9.0 hr) seems somewhat anomalous. Since these cells were iron-supplemented, the decrease in the "efficiency" was unexpect-ed as the cells were expected to be well coupled. One would expect the ef-ficiency of conversion of a nonfermentable carbon source to c e l l mass to be dependent upon the degree of coupling energy production to the respiratory chain. A possible explanation was suggested by the observation that the decline in the "efficiency" coincides with the onset of oxygen limited con-ditions, as indicated by the level of oxygen saturation and the growth of the culture. The relationship of these parameters as well as the redox potential of the culture is even more apparent in the iron sufficient culture (Figures 4.9 to 4.12). The conditions of oxygen limitation appeared to influence both the "efficiency" and the cytochrome levels in iron-supplemented (Figure 4.3 and 4.15) and iron-sufficient cells (Figure 4.9 and 4.10). The decrease in growth yield at low oxygen tension has previously been reported with chemo-stat cultures of Aerobacter aerogenes (Harrison and P i r t , 1967; Harrison and Loveless, 1971) and E. c o l i (Dr. D. G. Kilburn,. personal communication; Harrison and Loveless, 1971) and has been shown to occur concurrently with an increased rate of respiration. Harrison and Pirt (19^ 7) have likened the effect of low oxygen tension on the growth yield and the respiration rate to that of an uncoupler of oxidative phosphorylation. Thus, the observed decrease in the efficiency of conversion of succinate to c e l l mass (Figure 4.10 and 4.15) is probably due to the "uncoupling" action of low oxygen tension. Although apparent in both the iron-supplemented and the iron-suf-f i c i e n t cultures the influence of oxygen limitation was most apparent in l a t t e r (Figure 4.9). Within half an hour of the onset of oxygen-limited growth the cytochrome a^ and cytochrome b^ levels began to increase markedly. Similar observation of increased levels of cytochromes at low oxygen tensions have been reported for Pseudomonas fluorescens (Lenhoff e_ a l . , 1956), Spirillum it e r s o n i i (Clark-Walker e_ a l . , 1967), Azotobacter vinelandii (Nishizawa et a l . , 1971), and Aerobacter aerogenes (Harrison, 1972). It is generally proposed that the reason for the increased level of cytochromes would be to make the small amounts of oxygen present at low oxygen tensions more readily available to the reducing systems of the c e l l s . The induction of a cytochrome oxidase, such as cytochrome a^ at low oxygen tensions i s generally assumed to be due to the possession of a lower for oxygen. Thus the reduced efficiency of conversion of succinate to c e l l mass could occur either by an "uncoupling" mechanism as implied by Harrison and co-workers or via the u t i l i z a t i o n of an alternative respiratory chain sequence related to the induction of cytochromes b_^  and a^. With the equipment u t i l i z e d i t was not possible to determine the in s i t u respiration rates. Thus i t i s not known whether there was a cor-responding increase in respiration with the decrease in "efficiency". However, since there was no apparent accumulation of intermediates of suc-cinate oxidation, as indicated by a single phase of oxygen u t i l i z a t i o n , an increase in respiration rate probably did occur. Therefore, the decrease in efficiency of the iron-supplemented and iron-sufficient cultures under conditions of oxygen limitation was probably due to the "uncoupling" action of low oxygen tension. It i s impossible to exclude the poss i b i l i t y that the efficiency of conversion of succinate to c e l l mass may have been i n -fluenced by iron at a level other than the generation and coupling of energy 216 conservation. While there was a rapid recovery of nonheme iron levels and energy coupling (as indicated by the respiratory control ratio) within the f i r s t 15 min following the additon of f e r r i c citrate, there was no detectable increase in either the rate of oxygen u t i l i z a t i o n or the rate of growth (Figure 4.3). The 15 to 30 min lag prior to the increase in rate of oxygen u t i l i z a t i o n suggested that the synthesis of some component, perhaps a protein, was required in order for respiration and/or growth to be stimulated by iron, Support for the concept of a requirement for protein synthesis was obtained when i t was discovered that the addition of fe r r i c citrate late in the growth of a nitrogen-limited culture (Figure 4.7) did not stimulate respir-ation or growth. The subsequent addition of (NH^^SO^ stimulated both growth and respiration following the usual 15 min lag period. However, i t was sub-sequently discovered that i f the iron-deficient medium was supplemented with mixture of trace metals other than iron (Ca + +, Zn + +, Cu + +, Mn + + and Co + +), the results obtained were strikingly different (Figure 4.8). There was an i n i t i a l plateau in the oxygen level at approximately 40$ of saturation. This was followed by a second plateau at an oxygen saturation of 25$. Addi-tion of fe r r i c citrate to the culture during the second plateau resulted in an immediate decrease in the level of oxygen saturation of the medium. There was no 15 min lag prior to the increase in the rate of oxygen consumption. However growth appeared to demonstrate the usual lag prior to attaining the new growth rate. The increase in the rate of oxygen u t i l i z a t i o n was far too rapid to have involved de novo protein synthesis. A possible explanation of the two plateau phases and the rapid response in oxygen u t i l i z a t i o n to the addition of f e r r i c ion may involve a functional replacement of the fe r r i c 217 ion under conditions of iron limitation, by one of the trace metals. Therefore, the f i r s t plateau probably represents a decreased respiration rate due to iron limitation at a particular site in the respiratory chain, as observed previously (Figures 4.3, 4.5 and 4.15). The second phase of oxygen consumption could be due to the substitution for iron of one of the other trace metals at the site(s) at which iron is limiting, resulting in the synthesis of a metal compound with a lower functional a c t i v i t y than the corresponding iron compound. The development of the plateau would then be due to the depletion of this trace metal from the medium. When f e r r i c citrate was added to the medium there may have been a rapid exchange of f e r r i c ion with the less functional cation resulting in an immediate increase in the rate of oxygen u t i l i z a t i o n . When beginning the discussion of the influence of iron on the respir-ation of E. c o l i i t was indicated that interpretation of the results relied heavily upon the report by Hempfling (1970b) that the stimulation of res-piration of whole cells of E. c o l i oxidizing glucose produced by the addition of 2,4-dibromophenol occurred only when a l l three sites of oxidative phos-phorylation were functional. Thus, the respiratory control ratio (RCR) defined as the rate of respiration in the presence of DBP relative to the rate of respiration in the absence of this compound, can be u t i l i z e d as an indication of the degree of coupling of at least one site of energy conser-vation. Gutnick, (personal communication) has subsequently confirmed Hempfling's results. However, during i n i t i a l attempts to u t i l i z e this technique a number of anomalous observations were made. When glucose-dependent respiration rates, in the presence and absence of dibromophenol were determined at 37°C, 218 instead of room temperature as reported by Hempfing (1970b), although a transient stimulation of respiration was observed in a few instances, the majority of the samples showed no stimulation of respiration. Only a pro-gressive inhibition of respiration was observed. A possible explanation for the decrease in the stimulation of respiration and the increase in the inhibition observed at 37°C may be that DBP has a biphasic action on electron transfer, a stimulation and an inhibition, with the l a t t e r possessing the greater temperature coefficient. More d i f f i c u l t to understand was the observation that i f glucose was replaced by succinate, a substrate which must generate the majority of i t s energy via the electron transport chain, and the respiration rates in the presence and absence of DBP were determined, then DBP at concentrations as high as 1 mM had no effect on the respiration rate 37°C. However, when the same measurements were performed at room temperature, the respiration rate was inhibited at DBP concentrations as low as 50 uM. This raises two questions: (i) Why is the succinate-dependent respiration of intact E. c o l i inhibited by DBP at room temperature but not at 37°0, when the reverse i s true for glucose-dependent respiration? ( i i ) Why is the succinate-dependent respiration of E. c o l i not stimulated by DBP when the glucose-dependent res-piration i s stimulated? Cavari et a l . , (19^ 7) have reported similar obser-vations with respect to the influence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) on glucose-, pyruvate-, succinate- and glutamate-dependent respiration of E. c o l i . Preincubation of E. c o l i with CCCP inhibited the succinate-, pyruvate- and glutamate-dependent respiration but stimulated the glucose-dependent respiration. Thus, the results appear to be characteristic of <the interaction of uncouplers with E. c o l i . Cavari 219 et a l . , (1967) also observed that preincubation of E. c o l i with CCCP and a respiratory substrate prevented the inactivation of the respiratory chain. Of interest to this problem was the demonstration by Lo et a l . , (1972b) that the accumulation of succinate by E. c o l i was inhibited by uncoupling agents. This suggested that a possible explanation for the f i r s t question raised could be as follows. At 37°C sufficient succinate may have been accumulated prior to the addition of DBP to protect the electron transport chain from inhibition by DBP and also maintain the respiration rate. Whereas at room temperature the succinate accumulated prior to the addition of DBP may not have been sufficient to protect the electron transport chain and/or to main-, tain the respiration rate. Why the DBP does not stimulate the respiration at 37°C is unclear, unless the "protection" of the respiratory chain via succinate prevents either stimulation or inhibition. Although the preceding i s a possible explanation, considerably more information i s required on the influence of uncouplers such as DBP on the respiration of intact bacteria inorder to c l a r i f y these problems and to validate the emphasis placed on the RCR values. There are a number of questions which have arisen directly or i n -dire c t l y from the research into the influence of iron on the respiration of E. c o l i . Detection of the location of the site responsible for the limi t a -tion of the respiration rate under conditions of iron deficiency should be possible through the determination of low temperature difference spectra of E. c o l i obtained by rapid sampling prior to, and immediately following the addition of f e r r i c citrate to the iron-limited culture. The function of the "excess" nonheme iron i s of considerable interest. Location of the iron in the c e l l membrane, c e l l wall or cytoplasm i s readily 220 determined by c e l l fractionation techniques. Localization of the nonheme iron would provide some indication as to i t s metabolic state and function. The sensitivity of the assay for nonheme iron could be increased by using 59 / \ the radioactive isotope of iron, Fe (Clegg and Garland, 1971J. The pos-s i b i l i t y that a considerable proportion of the nonheme iron functions as a 59 precursor of cytochromes could be examined by adding a quantity of Fe-labelled f e r r i c citrate to the medium such that i t would be taken up into the nonheme iron prior to the induction of significant cytochrome synthesis. The hemes of the cytochromes synthesized subsequently could be examined for 59 the appearance of radioactivity due to Fe. Also of considerable interest would be the determination of the EPR spectra of the E. c o l i membranes during the development of, and recovery from conditions of iron limitation. This would be of particular interest i f determination of the redox potential of the signals were also performed by the technique of Ohnishi et a l . , (1972b). Thus, information on the relative order of the nonheme iron pro-teins involved with the respiratory chain of E. c o l i would be provided. The question of the requirement of protein synthesis following the addition of f e r r i c citrate and prior to the increased rates of oxygen con-sumption and growth could readily be tested via blocking protein synthesis with chloramphenicol. A failure to prevent the usual increase in respira-tion rate would indicate that protein synthesis was not required. Failure to obtain an increase in the rate of respiration in the presence of chlor-amphenicol could not be taken as conclusive evidence for a requirement for protein synthesis. Under these circumstances the rate of incorporation of a radioactivity labelled amino acid into the acid insoluble fraction of the c e l l , following the addition of f e r r i c citrate to the culture, would have 221 to be used in order to provide a conclusive answer. The role of the trace elements in the response of the growth and respiration of the iron-limited culture of E. c o l i i s d i f f i c u l t to investi-gate. The trace metal involved should be identifiable by selective addition of trace metals to the culture medium. This may provide the necessary lead for further investigations. On considering the investigation of the respiration of bacteria and i t s control, i t becomes apparent that there has been no attempt to investi-gate the relationship between the biosynthetic energy demand and the res-piration rate throughout the bacterial c e l l cycle. This would seem to be a logical starting point in any attempt to demonstrate the presence of res-piratory control in the bacterial c e l l . 222 5 PART III: THE TRANSITION FROM AEROBIC TO ANAEROBIC GLUCOSE UTILIZATION 5.1 Results and discussion 5.1.1 The lag in acid production by E. c o l i associated with the transition from aerobic to anaerobic glucose u t i l i z a t i o n During preliminary experiments on the simultaneous measurement of acid production and oxygen consumption (sec.2.2.5.3) by E. c o l i oxidizing glucose, i t was observed that concurrent with the depletion of oxygen from the assay medium, there occurred a 15 to 30 sec lag in acid production. Following the "lag" period, acid production resumed at a rate nearly twice that observed during aerobic glucose u t i l i z a t i o n (Figures 5.1 and 5.2). These observations were not pursued further due to the necessity of com-pleting other research in progress. Subsequently identical information, but of a more extensive nature, was retrieved from the literature (Hempfling et a l . , 1967). These researchers reported that the cessation in acid pro-duction occurred concurrently with: (i) a cessation in the uptake of K + ions, ( i i ) a cessation of glucose u t i l i z a t i o n , ( i i i ) a rapid decrease i n the levels of glucose-6-phosphate and fructose-6-phosphate, (iv) increased levels of dihydroxyacetone phosphate, glyceraldehyde-3-phosphate and fructose-1,6-diphosphate, (v) slight increases in the levels of 3-phosphoglyceric acid and 2-phos-phoglyceric acid, (vi) constant levels of PEP and pyruvate, ( v i i ) a marked decrease in citrate and isocitrate levels, ( v i i i ) a slight increase in the level of malate, (ix) a marked decrease in the level of NAD+, presumably due to the form-223 Fig. 5.1 Oxygen consumption and acid production during aerobic and anaerobic u t i l i z a t i o n of glucose by intact E. c o l i . E. c o l i c e l l s , grown and harvested as described in sec. 2.2.2.1 were resuspended 1:80 (w/v) in 0.85% NaCl and were incubated at 37°C for 10 min. The ce l l s were sedimented by centrifugation for 5 min at approximately 2100 x g_ at 4°C. The supernate was removed and the cel l s were washed once with 3 mM glycylglycine-KOH buffer, pH 7.0. The c e l l pellet was resuspended in the same buffer at a c e l l cons-centration of 1:5 (w/v). The assay system consisted of 2.8 mM glycylglycine-KOH buffer, pH 7.0, 25 mM glucose, and 0.5 mM KC1 in a f i n a l volume of 4.0 ml. Oxygen tension was measured with the Aminco oxygen electrode. C: 40 mg ce l l s ; #: oxygen; A : pH. 225 Fig. 5.2 Oxygen consumption and acid production during aerobic and anaerobic u t i l i z a t i o n of glucose by Tris-EDTA permeabilized E_ c o l i . E. c o l i c e l l s , grown and harvested as described in sec. 2.2.2.1, were suspended 1:80 (w/v) in 0.14 M Tris-HCl, pH 8.0 containing 0.1 mM EDTA and were incubated at 37°C for 10 min. The c e l l s were sedimented by centrifugation for 5 min at approximately 2100 x __ at 4°C. The supernate was removed and the cells were washed once with 3 mM glycylglycine-KOH buffer, pH 7.0. The c e l l pellet was resus-pended in the same buffer at a c e l l concentration of 1:5 (w/v). The assay system consisted of 2.8 mM glycylglycine-KOH buffer, pH 7.0, 25 mM glucose, and 0.5 mM KC1 in a f i n a l volume of 4.0 ml. Oxygen saturation was measured with the Aminco oxygen electrode. C: 40 mg c e l l s ; * : oxygen; A: pH. 227 ation of NADH (Sstabrook et a l , , 1962), and (x) a marked decrease in the level of ATP with a corresponding i n -crease in ADP and a slight decrease in AMP levels. Inorganic phosphate levels remained constant during this period. On the "basis of these and other results Hempfling _et al_., (1967) proposed that the control of glycolysis in E. c o l i was mediated by changes in the relative a c t i v i t i e s of phosphofructokinase and glyceraldehyde-3-phosphate dehydro-genase. At a given time, the identity of the particular enzymatic step determining the flux of glucose depends on the chemical environment ( i . e . , concentration of specific modifiers of enzymatic activity) within the c e l l . However, in attempting to explain their observations during the transition from aerobic to anaerobic glucose oxidation on the basis of this proposal, Hempfling et a l . , (1967) relied greatly upon the information available from the corresponding yeast system. Such an extrapolation i s not valid (sec.1.2). Re-examining the data of Hempfling et a l . , (1967) and Estabrook et a l . , (1962), in terms of present knowledge of the regulation of the amphi-bolic pathways of E. c o l i (sec.1.2), the most dramatic changes observed on the depletion of oxygen from the assay system were: (i) the rapid reduction of NAD+, ( i i ) the marked decrease in the level of ATP, ( i i i ) the accumulation of fructose-1,6-diphosphate, dihydroxyacetone phos-phate and glyceraldehyde-3-phosphate, and (iv) the decrease in the levels of glucose-6-phosphate and fru c t o s e s -phosphate. The high level of NADH possibly explains the accumulation of fructose-1,6-diphosphate, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, due 228 to the fact that the NADH generated at glyceraldehyde-3-phosphate dehydro-genase was not oxidized. The decline in ATP levels may "be due to the absence of oxidative phosphorylation under anaerobic conditions but is more probably due to the cessation of glycolysis resulting from the low levels of NAD+. Decreased levels of ATP would in turn remove the inhibition from phosphofructo-kinase and explain the decrease in glucose-6-phosphate and fructoses-phos-phate, as well as contributing to the build up of fructose-1,6-diphosphate, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. The lack of build up in PEP and pyruvate as well as 2-phosphoglyceric acid and 3-phospho-glyceric acid would also be explained by the inhibition of glyceraldehyde -3-phosphate dehydrogenase as a result of the lack of NAD+. The occurrence of the blockage of glycolysis at glyceraldehyde-3-phosphate dehydrogenase is also supported by the lack of phosphate uptake during the lag in acid pro-duction. Failure of glucose uptake probably resulted from low levels of PEP. Thus in the "lag" a l l evidence supports the interpretation that the primary event i s an inhibition of glycolysis at glyceraldehyde-3-phosphate dehydrogenase resulting from the unavailability of NAD+. The results i n -dicate that NADH i s oxidized following the lag but by what system, and why wasn't there a smooth transition? How is the anaerobic NADH oxidation system initiated? The most l i k e l y candidate to i n i t i a t e the oxidation of NADH would be the level of NADH. The results suggest an important role for the NADH/NAD+ ratio (Sanwal, 1970a), or the NADH level as proposed by Wimpenny and Fi r t h (1972), in the control of metabolism in E. c o l i . The involvement of the NADH/NAD+ ratio in the regulation of the pyruvate dehydro-genase complex has been demonstrated by Shen and Atkinson (1970). 229 Y/hat system i s responsible for the anaerobic oxidation of NADH? Considering, f i r s t of a l l , the acids produced aerobically and anaerobically, the acid accumulating aerobically is probably acetate (Holms and Bennett, 1971). As to the nature of the acid produced anaerobically, examination of Figure 1.5 and table 1.1 indicates that anaerobically there are but three possible reactions for the further metabolism of pyruvate which do not either generate NADH or directly require oxygen. These reactions are (i) the soluble, NAD -requiring, D-lactate dehydrogenase (reaction 38), ( i i ) the D-lactate dehydrogenase of the particulate D-lactate oxidase system (reaction 39), and ( i i i ) the malic enzymes (reaction 20). Of these p o s s i b i l i t i e s the equilibriums of the l a t t e r two favor the formation of pyruvate. Also the NADP +-requiring malic enzyme is a l l o s t e r i c a l l y inhibited by NADH. As there was no increase in the level of pyruvate during the "lag", to shift the equilibrium toward the formation of either D-lactate via the reversal of the particulate D-lactate dehydrogenase, or to malate v i a the reversal of the NAD +-requiring malic enzyme, this would suggest that, under the conditions of the assays, pyruvate i s reduced to lactate via the soluble D-lactate dehy-drogenase, the equilibrium of which favors the formation of D-lactate. There-fore, D-lactate acid would be the acid expected to accumulate anaerobically. Since the soluble D-lactate dehydrogenase of E. c o l i i s an NAD +-requiring enzyme, this would be the most probable system for the anaerobic oxidation of NADH. Tarmy and Kaplan (1968) investigated the kinetics of NADH oxida-tion via the soluble D-lactate dehydrogenase and concluded that they were _3 of the Michaelis-Menten type with a for NADH of 7 x 10 M. However, no attempt was made to evaluate the response of the enzyme to the NADH/NAD + ratio. 230 The reason(s) for the lack of smooth transition from aerobic acid production to anaerobic acid production is unclear. To investigate the poss i b i l i t y that i t might be related to nucleotide levels, particularly pyridine and adenine nucleotides, E. c o l i c ells were permeabilized with Tris-EDTA according to the technique of Leive (1965). Permeabilization of E. c o l i with Tris-EDTA has been demonstrated to cause the release of the nucleotide pool, and the breakdown of ribosomal RNA (Figure 5.3) (Leive and K o l l i n , 1967; Neu et a l . , 1966; 1 9 6 7 ) . The results were inconclusive. The cessation of acid production was of longer duration in the Tris-EDTA treated cells (Figure 5.2) than in those preincubated in 0.85$ NaCl (Figure 5 . 1 ) , but was not of an indefinite duration. This suggested that i f pyridine nucleotides were involved as was presumed, they must not be com-pletely released as a result of the permeabilization procedure. This raises the p o s s i b i l i t y of compartmentation. A possible explanation for the increased duration of the "lag" in the Tris-EDTA treated cells i s that the metabolism of these cells as i n d i -cated by.the rate of oxygen consumption and acid production (Figure 5.2) is considerably slower than observed with the cells preincubated in 0.85$ NaCl (Figure 5 . 1 ) . Consequently, the extended duration of the "lag" may be due to the greater amount of time required to produce the concentration of NADH, or the NADH/NAD + ratio, required to i n i t i a t e the anaerobic oxida-tion of NADH. Definite answers to the questions raised await further re-search. 231 <D O C (0 J Q —. O CO _ Q < 30 Minutes 60 Fig. 5.3 The release of ultraviolet absorbing material from E. c o l i . 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Absorbance Wet Weight of (at 420 nm) (per 100 ml) 3.08 0.20 4.69 0.38 6.22 0.55 7.44 0.68 8.57 0.80 9.64 0.90 10.44 0.95 3.65 0.32 5.27 0.48 6.78 O.65 8.17 0.80 9.38 0.90 10.36 1.00 10.84 1.02 The regression line for the combined data was y = 0.103(x) - 0.081 where x i s the absorbance at 420 nm, and y i s the wet weight of cells per 100 ml expressed i n grams. 251 Absorbance Fig. A The relationship between the absorbance, at 420 nm, of a culture of E. c o l i and the c e l l mass, measured as wet weight of c e l l s . Units: c e l l mass, g/l of culture;#: experiment 1;A: experiment 2. 252 APPENDIX B The influence of buffer concentration and buffer ion on the color development of the Lowry method of protein determination. Standard protein solutions were prepared by dissolving weighed quantities of bovine serum albumin in the buffers indicated. Protein was assayed according to the standard procedure of Lowry et a l . , (1951). Absorbance was determined at 500 nm and/or 750 nm dependent upon the intensity of the color present. The appropriate buffer solution was assayed for color development concurrently with the protein standards prepared in that buffer, and was employed as the reference solution during the determination of the absorbance of the color developed by the protein standards. Buffer Concentration (mM) Protein (ve) A500 A750 D i s t i l l e d water 7.5 0.028 22.0 0.080 37.0 0.134 Glycylglycine- 1 7.5 0.030 K0H,pH7.0 22.0 0.082 37.0 0.137 3 7.5 0.030 22.0 0.083 37.0 0.136 5 7.5 0.030 22.0 0.083 37.0 0.138 10 7.5 0.031 22.0 0.084 37.0 0.140 30 9.0 0.037 26.5 0.097 44.5 0.160 50 9.0 0.034 26.5 0.092 44.5 0.157 100 9.0 0.034 26.5 0.092 44.5 0.153 300 9.0 0.027 26.5 0.066 44.5 0.110 500 9.0 0.013 26.5 0.043 44.5 0.076 Buffer Concentration (mM) HEPES-K0H,pH7.0 3 30 50 300 PIPES-HCl,pH7.0 300 13.5 0.006 0.012 40.0 0.012 0.028 72.0 0.030 0.056 TES-K0H,pH7.0 300 13.5 0.060 40.0 0.144 72.0 0.258 Tris-HCl,pH7.0 300 13.5 0.024 40.0 0.069' 72.0 0.125 Color development of buffer solutions (no protein present) Buffer Cone ent rat ion(mM) A500 —150 Water 0 0 HSPES-K0H,pH7.0 300 .1.101 M0PS-K0H,pH7.0 300 0.057 PIPE3-HCl,pH7.0 300 0.150 TES-K0H,pH7.0 300 0.073 Glycylglycine- 300 0.028 K0H,pH7.0 Tris-HCl,pH7.0 300 0.109 Protein 13.5 40.0 72.0 13.5 40.0 72.0 13.5 40.0 72.0 13.5 40.0 72.0 A500 0.012 0.058 0.112 0.026 0.074 0.111 0.050 0.082 0.115 0.037 0.074 0.080 -750 Conclusions: (i) a l l the buffers investigated gave significant color development in the absence of protein; ( i i ) a l l the buffers, with the exception of TE3-K0H, suppressed or reduced the color development of the protein standards. APi\r:]>:rx c Composition of the trace element solution (Figure 4 . 8 ) . Concentration (M) Compound Stock Solution Medium CaCl2'6H20 • 3.3 x 1 0 ~ 5 3.3 x 1 0 T 6 ZnCl 2 6 . 0 x 10~4 6.o x 10"? C u S 0 4'5H 2 0 6.0 x 1 0 " 4 6.0 x 1 0 " ' MnCl 2«4H 2 0 6 . 0 x 1 0 ~ 4 6.0 x 1 0 ~ 7 CoCl 2.6H 2 0 7 . 0 x 10-4 7 . 0 x 10"? Na2.EDTA«2H20 6.4 x 1 0 ~ 2 6.4 x 1 0 " 5 

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