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Studies on the aerobic and anaerobic cytochromes of Escherichia coli Hackett, Neil Robert 1982

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c. STUDIES ON THE AEROBIC AND ANAEROBIC CYTOCHROMES OF ESCHERICHIA COLI by NEIL ROBERT HACKETT B . S c , The U n i v e r s i t y of Edinburgh, 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department of Biochemistry We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1982 0 N e i l Robert Hackett, 1982 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 for 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 or her representatives. I t 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 Biochemistry  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T.1Y3 Date 29th Sept 1982 i i ABSTRACT Escherichia c o l i can produce several r e s p i r a t o r y chains which transfer electrons to oxygen, n i t r a t e or other acceptors. Cytochromes are amongst the electron c a r r i e r s of these chains, and can be studied by a number of spectroscopic techniques. Of p a r t i c u l a r interest i s the formate-nitrate reductase r e s p i r a t o r y pathway which i s composed of two complexes, formate dehydrogenase and nitrate reductase each with a cytochrome associated. The cytochromes of E_^  c o l i a f t e r growth under a v a r i e t y of conditions have been studied by spectrophotometry redox t i t r a t i o n and other spectroscopic methods. These have 'shown t h a t , contrary to some previous r e p o r t s , t h e r e are at l e a s t s i x cytochromes produced irrespective of the culture c o n d i t i o n s used. This emphasizes the need for simplified systems and two of these have been investigated. By f r a c t i o n a t i o n of cytochromes p r i o r to s p e c t r o s c o p i c a n a l y s i s a cytochrome b of redox p o t e n t i a l OmV was i d e n t i f i e d in c e l l s grown 556 a e r o b i c a l l y . In a d d i t i o n the a s s o c i a t i o n of two cytochromes of potential +20mV and +120mV with n i t r a t e reductase was demonstrated. Secondly the formate-nitrate reductase pathway has been investigated by spectroscopic s t u d i e s of c h i mutants which are defective in t h i s a c t i v i t y . Three phenotypes of cytochrome production were observed. Mutants at l o c i associated with the production of the cofactor for both nitra t e reductase and formate dehydrogenase were shown to produce the same cytochromes as the wi l d - t y p e . Mutants mapping at the chlC locus and defective i n nitrate reductase but not formate dehydrogenase were found to lac k only the two cytochromes associated with nitrate i i i reductase. They had high l e y e l s of a cytochrome of redox potential -100mV which was shown to be associated with formate dehydrogenase. A thi r d class of pleiotropic regulatory mutants was i d e n t i f i e d which was not r e l a t e d with a s p e c i f i c genotype. These produced none of the a n a e r o b i c r e s p i r a t o r y pathways but overproduced the a e r o b i c r e s p i r a t o r y pathway l e a d i n g to cytochrome d. The second aerobic respiratory pathway leading to cytochrome o was most evident i n double mutants l a c k i n g both of the cytochromes associated with n i t r a t e reductase and that associated with formate dehydrogenase. On the basis of these results a model f o r the arrangement of the cytochromes in the r e s p i r a t o r y chains of E. c o l i i s proposed. i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i i LIST OF FIGURES .• ix ABBREVIATIONS x i i ACKNOWLEDGEMENTS , x i i i INTRODUCTION 1 PART I. The cytochromes of aerobic respiratory pathways 5 The existence of two oxidases..... 6 Spectroscopic studies showing the d i v e r s i t y of b-type cytochromes and their arrangement into electron transport chains 7 The properties of purified respiratory complexes containing cytochromes 13 PART I I . Anaerobic respiratory pathways 15 TMAO reduction 15 Nitrate reduction 16 PART I I I . Mutations of the formate-nitrate reductase pathway... 20 Regulation of the production of nit r a t e reductase.... 24 Objectives of this thesis 28 MATERIALS AND METHODS. 30 Chemicals 30 Strains of E s c h e r i c h i a c o l i 30 Growth of c e l l s 33 Medium 33 Preparation of membranes 34 Low temperature difference spectra 35 V Carbon monoxide difference spectra 3 6 Spectrophotometry redox t i t r a t i o n 37 Analysis of t i t r a t i o n data * 39 Kinetic measurements 40 Fractionation of aerobic cytochromes by gel f i l t r a t i o n 40 Fractionation of anaerobic cytochromes 41 SDS polyacrylamide gel electrophoresis ,42 Assay of n i t r a t e , fumarate and TMAO reductase a c t i v i t i e s 43 Assay of oxidase a c t i v i t i e s . . . . . 43 Assay of ATPase a c t i v i t i e s 44 Assay of formate dehydrogenase a c t i v i t y 44 The assay of nitrat e reductase a c t i v i t y by measuring n i t r i t e production 45 Measurement of the capacity of a culture to reduce n i t r i t e 45 i Measurement of the formate hydrogenlyase a c t i v i t y of growing c e l l s using Durham tubes 46 Protein estimation 46 P1 transduction . 46 RESULTS 48 PART I. THE SIMULTANEOUS PRESENCE OF SEVERAL RESPIRATORY CHAINS... 48 The cytochromes of E_^  c o l i studied by difference spectroscopy.... 48 The dependence of respiratory a c t i v i t y on growth conditions 54 The analysis of cytochromes by spectrophotometric redox t i t r a t i o n 57 Studies of cytochrome o and cytochrome d content using carbon monoxide difference spectroscopy 72 v i D i f f e r e n t i a l r e d u c t i o n o f h i g h - p o t e n t i a l cytochrome u s i n g a s c o r b a t e and PMS 76 P o t e n t i o m e t r i c s t u d i e s o f f r a c t i o n a t e d c y t o c h r o m e s . . . 78 K i n e t i c s t u d i e s o f cytochrome c o n t e n t 85 PART I I . THE CYTOCHROMES OF MUTANTS DEFICIENT IN THE FORMATE-NITRATE REDUCTASE PATHWAY 95 The cytochrome c o n t e n t and r e s p i r a t o r y a c t i v i t y o f c h i mutants 96 S p e c t r o p h o t o m e t r y r e d o x t i t r a t i o n s o f c h i mutants 106 The cytochrome o and c y t o c h r o m e d c o n t e n t o f c h i mutants 112 S e p a r a t i o n o f t h e c y t o c h r o m e s o f c h i mutants i n t o h i g h - and l o w - p o t e n t i a l s p e c i e s u s i n g a s c o r b a t e and PMS 117 E l e c t r o p h o r e t i c a n a l y s i s o f mutants f o r n i t r a t e r e d u c t a s e and formate dehydrogenase s u b u n i t s 120 K i n e t i c s o f c y t o c h r o m e r e d u c t i o n i n c h i mutants.... 123 F r a c t i o n a t i o n o f a n a e r o b i c c y t o c h r o m e s from c h i mutants 125 Double c h i mutants ^ 133 C o n s t r u c t i o n o f c h i I d o u b l e m u t a n t s 137 The cytochrome c o n t e n t o f c h l l d o u b l e mutants 138 DISCUSSION 144 S p e c t r o p h o t o m e t r y redox t i t r a t i o n . . . 1 144 The c o n s t i t u t i v e p r o d u c t i o n o f a l l r e s p i r a t o r y c h a i n s 147 Scheme f o r t h e arrangement o f cy t o c h r o m e s i n the membranes o f E ^ c o l i ; 150 Cytochromes o f t h e TMAO r e d u c t a s e pathway 152 Cytochromes o f t h e f o r m a t e - n i t r a t e r e d u c t a s e pathway 154 The cytochrome o pathway 158 The cytochrome d pathway 161 v i i K i n e t i c d e m o n s t r a t i o n o f t h e i n t e r a c t i o n o f the cytochromes o f d i f f e r e n t r e s p i r a t o r y c h a i n s 163 The r e t e n t i o n o f an i n a c t i v e f o r m a t e - n i t r a t e r e d u c t a s e pathway i n many c h i m u t a n t s 165 The membrane assembly o f r e s p i r a t o r y complexes 171 The r e g u l a t i o n o f cytochrome p r o d u c t i o n i n c l a s s I I I mutants 173 BIBLIOGRAPHY 176 Appendix I 183 Appendix I I . . . 184 Appendix I I I 185 v i i i L I S T OF TABLES Ta b l e Page 1 S t r a i n s o f E s c h e r i c h i a c o l i Used i n t h i s Study 31 2 The Dependence o f Cytochrome Content and R e s p i r a t o r y A c t i v i t y o f W i l d - T y p e E s c h e r i c h i a c o l i on C u l t u r e C o n d i t i o n s 55 3 Comparison o f R e s o l v e d Redox T i t r a t i o n Data w i t h P u b l i s h e d R e s u l t s 67 4 R e p r o d u c i b i l i t y o f T i t r a t i o n Data 69 5 pH-Dependence o f M i d - P o i n t P o t e n t i a l o f Cytochromes o f C e l l s Grown A n a e r o b i c a l l y w i t h N i t r a t e 73 6 Cytochrome C o n t e n t and R e s p i r a t o r y A c t i v i t y o f c h i Mutants 99 7 C l a s s i f i c a t i o n o f c h i M u t a n t s by G e n e t i c L o c a t i o n and P h e n o t y p i c C l a s s 101 8 Temperature Dependence o f R e s p i r a t o r y A c t i v i t i e s and Cytochrome C o n t e n t o f TS9A and PK27 105 9 F i t s o f T i t r a t i o n Data f o r C e l l s Grown A n a e r o b i c a l l y w i t h N i t r a t e 109 10 Cytochrome o Content and R e d u c i b i l i t y by A s c o r b a t e and PMS f o r c h i Mutants 116 11 Summary o f R e s p i r a t o r y C h a i n C o n t e n t o f c h i and f d h Mutants 144a ix L I S T OF F I G U R E S F i g u r e . Page 1 The m a j o r r e s p i r a t o r y p a t h w a y s o f E^ c o l i •. ^ 2 Proposed arrangement o f components i n t h e a e r o b i c r e s p i r a t o r y c h a i n s 12 3 Proposed arrangement o f r e s p i r a t o r y c a r r i e r s i n c e l l s grown a n a e r o b i c a l l y w i t h n i t r a t e . 18 4 The cytochromes o f w i l d - t y p e c e l l s grown a e r o b i c a l l y 49 5 D i f f e r e n c e s p e c t r a o f t h e membranes o f E ^ c o l i grown a n a e r o b i c a l l y w i t h v a r i o u s ' t e r m i n a l e l e c t r o n a c c e p t o r s . . . 51 6 S p e c t r o p h o t o m e t r i c redox t i t r a t i o n o f cytochrome c 59 7 Redox t i t r a t i o n s o f the b - t y p e c y t o c h r o m e s o f w i l d - t y p e E. c o l i grown u n d e r v a r i o u s c o n d i t i o n s 60 8 Redox t i t r a t i o n s o f t h e b - t y p e c y t o c h r o m e s o f the w i l d -t y p e s t r a i n s PK27 and H f r H grown a n a e r o b i c a l l y w i t h n i t r a t e 62 9 Membrane cytochromes o f two p a r t i a l l y d i p l o i d s t r a i n s grown a n a e r o b i c a l l y w i t h n i t r a t e 64. 10 Comparison o f t h r e e - and four-component b e s t f i t s o f redox t i t r a t i o n d a t a 66 ' 11 E l e c t r o d i c t i t r a t i o n o f - s o l u b i l i s e d c y tochromes b and d.. 70 12 E f f e c t o f growth c o n d i t i o n s on t h e c a r b o n monoxide d i f f e r e n c e s p e c t r u m o f w i l d - t y p e c e l l s 75 13 R e d u c t i o n o f b-type cytochromes by a s c o r b a t e and PMS 77 14 F r a c t i o n a t i o n by g e l f i l t r a t i o n o f cytochromes from c e l l s grown a e r o b i c a l l y 80 15 D i f f e r e n c e s p e c t r a and redox t i t r a t i o n s o f b - t y p e cytochromes f r a c t i o n a t e d from c e l l s grown a e r o b i c a l l y . . . . . 81 16 F r a c t i o n a t i o n o f s o l u b i l i s e d a n a e r o b i c cytochromes by i o n - e x c h a n g e chromatography 83 17 Redox t i t r a t i o n and d i f f e r e n c e s p e c t r u m o f cytochrome a s s o c i a t e d w i t h a p a r t i a l l y p u r i f i e d n i t r a t e r e d u c t a s e . . . 84 X I 18 ' Kinetics of cytochrome reduction by formate and reoxidation by nitrate or TMAO ; 86 19 Effect of QNO on cytochrome reduction by formate and reoxidation by nitrate 89 20 Trapping of intermediates i n the cycle of cytochrome • reduction and reoxidation 90 21 Kinetics of cytochrome reduction and reoxidation in c e l l s grown anaerobically with TMAO... 92 1 22 D i f f e r e n t i a l cytochrome reduction by substrate in membranes of c e l l s grown anaerobically with TMAO 94 23 Difference spectra of membranes of c h i and fdh mutants 97 24 The derepression of cytochromes b562 and c552 i n respiratory mutants 98 25 Difference spectra of the, membranes of TS9A after growth at the permissive and non-permissive temperatures 107 26 Redox t i t r a t i o n s of the b-type cytochromes of chi mutants 108 27 Tit r a t i o n of the membranes of an fdhA mutant compared with that of the wild-type 111 28 Titration of Qthe t o t a ^ b-type cytochrome of TS9A after growth at 29 C or 42 C 113 29 Carbon monoxide difference spectra of the membranes of chi and fdh mutants 114 30 D i f f e r e n t i a l cytochrome reduction by ascorbate and PMS in chi and fdh mutants 118 31 SDS-polyacrylamide gels of membrane and soluble f r a c t i o n s of c h i , fdh and w i l d - t y p e E. c o l i 121 32 Kinetics of cytochrome reduction i n chi mutants 124 33 Elution p r o f i l e s from DEAE Bio-Gel A of cytochromes sol u b i l i s e d from respiratory mutants 127 34 Titration of cytochromes associated with a p a r t i a l l y p urified preparation of formate dehydrogenase 128 35 . SDS-polyacrylamide gel electrophoresis of nitrate reductase and formate dehydrogenase complexes fractionated from chi mutants • 131 xi 36 D i f f e r e n c e s p e c t r a o f c y t o c h r o m e s f r o m c h i mutants f r a c t i o n a t e d on DEAE BIO-Gel A 132 37 D i f f e r e n c e s p e c t r u m o f a c h l E , c h l l d o u b l e mutant and i t s i s o g e n i c c h l E p a r e n t 135 38 Redox t i t r a t i o n o f t h e b - t y p e c y t o c h r o m e s o f a c h l E , c h l l d o u b l e m u t a n t and i t s c h l E p a r e n t 136 39 D i f f e r e n c e s p e c t r a o f r e s p i r a t o r y c h a i n d o u b l e mutants... 139 40 Redox t i t r a t i o n s o f the b - t y p e c y t o c h r o m e s o f double c h i mutants ... 140 41 Carbon monoxide d i f f e r e n c e s p e c t r a o f r e s p i r a t o r y c h a i n d o u b l e mutants 142 42 R e d u c t i o n o f cytochrome by a s c o r b a t e and PMS i n the membranes o f c h i d o u b l e m u t a n t s 143 43 Proposed arrangement f o r t h e c y t o c h r o m e s i n t h e a e r o b i c and a n a e r o b i c r e s p i r a t o r y c h a i n s o f E. c o l i 151 x i i ABBREVIATIONS adenosine-5'-triphosphate nicotinamide adenine dinucleotide (reduced form) trimethylamine-N-oxide N trimethylamine mid-point redox potential redox p o t e n t i a l r e l a t i v e to the standard hydrogen electrode 2,6-dichlorophenol indophenol carbonyl cyanide m-chlorophenylhydrazone dicyclohexyl carbodiimide 2-heptyl-4-hydroxyquinoline-N-oxide d i t h i o t h r e i t o l ' sodium dodecyl sulphate formate dehydrogenase nitrate reductase cytochrome associated with n i t r a t e reductase cytochrome associated with formate dehydrogenase phenazine methosulphate x i i i 'ACKNOWLEDGEMENTS I am very grateful for the p a t i e n t s u p e r v i s i o n of Dr. P h i l i p D. Bragg throughout my graduate s t u d i e s . I appreciate the great interest and enthusiasm which he always communicated. In addition I wish to thank the many people i n the Biochemistry Department who have helped my research and career i n various ways. In particular I would mention Grant Mauk and Lome Reid for their help with various aspects of the redox t i t r a t i o n experiments. This work would not have been p o s s i b l e without many g i f t s of s t r a i n s from a number of researchers who are l i s t e d i n Table 1 . I espec i a l l y thank Marc Chippaux and V a l l e y Stewart f o r t h e i r s t r a i n s and discussion of results. I also wish to acknowledge the f i n a n c i a l support the Canadian Commonwealth Scholarship Committee. INTRODUCTION ATP and an energised membrane are both necessary for the growth of E s c h e r i c h i a c o l i . They a r e i n t e r c o n v e r t e d by the proton translocating ATPase. This enzyme allows the organism two d i s t i n c t modes of growth. I t can use some compounds, notably glucose, for fermentation. These substrates are used for ATP synthesis by substrate le v e l phosphorylation and the ATP i s hydrolysed by the ATPase causing energisation of the membrane. A l t e r n a t i v e l y i t can derive energy from oxidative phosphorylation. In -this process the membrane i s energised by a number of o x i d a t i o n - r e d u c t i o n r e a c t i o n s known as r e s p i r a t i o n . R e s p i r a t i o n i s e f f e c t e d by membrane bound enzyme complexes which usually contain several redox centres, each of which i s associated with a prosthetic group. Electrons are passed s e q u e n t i a l l y through these centres from the substrate to the ter m i n a l e l e c t r o n acceptor. I t i s b e l i e v e d t h a t t h e s e e l e c t r o n t r a n s f e r r e a c t i o n s d r i v e the translocation of protons across the membrane. The proton gradient can then, be used to drive ATP synthesis by the ATPase running in reverse. (1,2). Respiratory enzymes have been most i n t e n s i v e l y studied in the mammalian mitochondrion. The p r i n c i p l e methods of investigation have been a number.of spectroscopic techniques. These are applicable due to the s t r i k i n g d i f f e r e n c e s between the s p e c t r a of the reduced and o x i d i s e d forms of r e s p i r a t o r y enzymes. The u n d e r s t a n d i n g of mitochondrial e l e c t r o n transport has been helped by the a b i l i t y to s p l i t the e l e c t r o n t r a n s p o r t c h a i n i n t o four complexes and the a v a i l a b i l i t y of several i n h i b i t o r s of e l e c t r o n transport. In spite of 2 this the complete d e f i n i t i o n of the minimum components necessary for electron transport has not been achieved. This i s attributed to the extreme complexity o f the system and the d i f f i c u l t y of fractionating i n t r i n s i c membrane p r o t e i n s . In many mitochondria there i s only one major respiratory pathway which reduces oxygen using NADH. By contrast E. c o l i can synthesize s e v e r a l r e s p i r a t o r y chains and under most growth conditions more than one of these i s produced. The simultaneous presence of two or more r e s p i r a t o r y chains g r e a t l y complicates the analysis of any one of them. This complexity has offset the advantages that E_^  c o l i o f f e r s because of i t s s u s c e p t i b i l i t y to genetic manipulation (2-4). The overall arrangement of the r e s p i r a t o r y chains of mitochondria and E_^  c o l i i s s i m i l a r . Substrates are o x i d i s e d by dehydrogenases which are usually flavoproteins with iron-sulphur centres associated. These transfer electrons to quinones which subsequently transfer them to cytochromes. In both systems cytochromes, or complexes containing cytochromes, are the t e r m i n a l e l e c t r o n c a r r i e r s of the e l e c t r o n transport chains (2-4). The known r e s p i r a t o r y chains of E_^  c o l i are shown i n Figure 1. There are several substrates that reduce quinones through specific dehydrogenases, for example NADH, formate and ^-glycerophosphate. The reduced quinones (quinols) can be reoxidised by several pathways. There are two quinol oxidase pathways terminating i n oxygen which i s the preferred terminal e l e c t r o n a c c e p t o r . Under anaerobic c o n d i t i o n s n i t r a t e , fumarate and trimethylamine-N-oxide (TMAO) can be used instead (3-7). I t has been suggested that there i s just one pool of quinones in the/membrane which may be reduced by a l l substrates and oxidised by a l l terminal electron acceptors. For example, i t i s found that a large number of s u b s t r a t e s can reduce TMAO ( 7 ) . T h i s model i s not consistent with the m u l t i p l e i n h i b i t i o n s i t e s of the quinone anolog 2-heptyl-4-hydroxyquinoline-N-oxide (QNO) or with the involvement of two species of quinone, ubiquinone-8 and menaquinone, i n electron transport (8,9). There also appear to be preferred pairs of electron donor and acceptor. Thus, a formate-nitrate reductase pathway of high s p e c i f i c a c t i v i t y can be induced w h i l s t NADH i s the preferred electron donor to oxygen (3-5). Figure 1 attempts to summarise a l l of these data. In addition to the r e s p i r a t o r y chains i l l u s t r a t e d , other redox a c t i v i t i e s are found i n c e l l s grown anaerobically." None of these have been shown to be proton t r a n s l o c a t i n g . In the case of the NADH-nitrite reductase system, for example, the soluble nature of the a c t i v i t y and the absence of any effect of n i t r i t e on anaerobic growth-yields argues against this being a true respiratory system (10,11). The present study was i n i t i a t e d to i n v e s t i g a t e the components required for e l e c t r o n transport i n c o l i . I t was proposed to exploit the spectroscopic techniques developed for the study of mitochondria and r e c e n t developments i n the methodology of fractionating i n t r i n s i c membrane p r o t e i n s . There follows a description of previous studies of t h i s type which, for reasons to be presented have not provided a consensus on the composition of either of the two oxidase pathways. However two models have been proposed which are relevant to the r e s u l t s of the present study. Recent reports on the p r o p e r t i e s of pur i f i ed - cytochromes and t h e i r , r o l e i n e l e c t r o n transport w i l l be reviewed. The most studied enzymes are the formate 4 h y d F D H > i Q N A D H . D H S D H D - L D H G P D H Q M K * N R > n i t r a t e c y t d c y t o o x y g e n T R — — • T M A O F R > f u m a r a t e 4a \ FIGURE 1. The major r e s p i r a t o r y pathways of E_£ c o l i . The tr a n s f e r of electrons from a number of donors to a number of terminal electron acceptors through what may be a s i n g l e pool of quinone i s represented. The p r e f e r r e d p a i r s of donor and a c c e p t o r are i n d i c a t e d . The a b b r e v i a t i o n s a r e : FDH, formate' dehydrogenase; D-LDH l a c t a t e dehydrogenase; SDH, s u c c i n a t e d e h y d r o g e n a s e ; NADH.DH, NADH dehydrognase; GPDH, OC-glycerolphosphate dehydrogenase; Q, ubiquinone; c y t , cytochrome; NR, n i t r a t e r e d u c t a s e ; TR, TMAO reductase; FR, fumarate reductase; hyd, hydrogenlyase; MK, menaquinone. 5 dehydrogenase and nitrate reductase complexes which together form the formate-nitrate reductase pathway. A number of mutants are known which are defective i n th i s system and the characteristics of these are known in some d e t a i l . However they have not previously been investigated by spectroscopic means and t h i s approach has yielded much information on the nature of the mutants as w e l l as on the aerobic and anaerobic respiratory chains. PART I. The Cytochromes of Aerobic Respiratory Pathways E. c o l i has sev e r a l d i f f e r e n t types of cytochrome which are named on the basis of t h e i r haem p r o s t h e t i c group. The reduced minus oxidised difference spectrum of a t y p i c a l cytochrome shows three peaks i n the v i s i b l e region. There i s a large Soret peak at around 410nm, a small ^ -peak at around 530nm and an #-peak at 548nm to 628nm. The exact absorption maxima and r e l a t i v e i n t e n s i t y of the three peaks are dictated by the type of cytochrome concerned. In aerobically grown E.  c o l i ' b-type cytochromes are u s u a l l y the most prominent. Their 0^-band absorption maximum i n the d i f f e r e n c e spectrum l i e s between 548nm and 562nm. Measurement of d i f f e r e n c e s p e c t r a at l i q u i d n i t r o g e n temperature (77K) greatly sharpens the absorption bands and allows the r e s o l u t i o n of at l e a s t three b-type cytochromes. These are named b , b and b on the b a s i s of t h e i r absorption maximum at 556 558 562 77K. The peak observed in the d i f f e r e n c e spectrum of whole c e l l s i s a composite of the absorption spectra of a l l these species. This peak may a l s o have a s h o u l d e r at around 550nm which i s the fiC-band absorption peak of c-type cytochromes. Another minor peak i n the 6 difference spectrum i s seen at 594nm which i s associated with the reduced form of cytochrome a^. Cytochrome d absorbs at 628nm i n the reduced form and 650nm i n the o x i d i s e d form. This leads to a complex difference spectrum i n t h i s region ( 3 t 4 ) . The amount of these cytochromes i s d i c t a t e d by the growth c o n d i t i o n s . Thus, cytochromes b , b , a and d are abundant 556 558 1• in c e l l s grown under conditions of poor aeration. When c e l l s are grown under conditions of vigourous ae r a t i o n the t o t a l cytochrome level i s much lower and cytochromes b , and b tend to be the major 556 562 species (12). The Existence of Two Oxidases. E. c o l i has two oxygen r e a c t i v e 1 c y t o c h r o m e s , a b-type cytochrome c a l l e d cytochrome o and cytochrome d. Both of these bind carbon monoxide and so aerobic r e s p i r a t i o n i s completely inhibited by t h i s compound. However i n h i b i t i o n can be r e l i e v e d by a photodissociation process. The p h o t o d i s s o c i a t i o n action spectrum may show one or two peaks depending on the growth conditions of the c e l l s being s t u d i e d . C e l l s of the e x p o n e n t i a l phase have only one peak corresponding to the absorption maximum of the cytochrome o-carbon monoxide complex. However c e l l s grown to the stationary phase have a second peak corresponding to the absorption maximum of the cytochrome d-carbon monoxide complex (13). A refinement of the p h o t o d i s s o c i a t i o n technique has suggested that cytochrome a^ may be a t h i r d carbon monoxide binding oxidase (14). This idea does not agree with stopped-flow k i n e t i c studies which 7 suggest that cytochrome i s o x i d i s e d by oxygen too slowly to support the known oxidase c a p a c i t y -of E_^_ c o l i membranes (15). Moreover cytochrome a^ always appears to be a s s o c i a t e d w i t h cytochromes d and b both i n the membrane (12) and i n p u r i f i e d 558 preparations (16,17). These three cytochromes form a quinol oxidase complex with a low K for oxygen which i s induced under conditions m of poor aeration, for example at the s t a t i o n a r y phase of growth. The cytochrome d or 'low a e r a t i o n ' oxidase pathway i s also induced by aerobic growth i n the presence of c y a n i d e (18) or with l i m i t i n g concentrations of sulphate (19)- Cytochrome o always appears to be associated with xcytochromes b a n d b,__„ which are most evident 555 562 , in c e l l s of the exponential phase of growth, p a r t i c u l a r l y when these are grown on a r i c h medium (20). The pathway to cytochrome o w i l l be c a l l e d the 'high a e r a t i o n ' oxidase pathway since i t has a high K m for oxygen. The two peaks i n the photodissociation action spectrum of s t a t i o n a r y phase c e l l s show t h a t both oxidases can be present simultaneously (13). Spectroscopic Studies Showing the D i v e r s i t y of b-type Cytochromes and  their Arrangement i n t o Electron Transport Chains Difference spectra suggest there to be several b-type cytochromes i n the membrane, the amount of each being d i c t a t e d by the growth conditions. A variety of spectroscopic techniques have been employed to resolve these species f u l l y and arrange them into a sequential chain of e l e c t r o n t r a n s f e r . However i t should be sa i d that none of these studies have been u n e q u i v o c a l , a l t h o u g h two working models are 8 presented; This i s a t t r i b u t e d to the f a i l u r e to recognise that two oxidase pathways are being monitored simultaneously, which makes i n t e r p r e t a t i o n of the data extremely d i f f i c u l t y Some workers have overinterpreted t h e i r data, not reco g n i s i n g that only a variety of approaches can f u l l y d i f f e r e n t i a t e a number of cytochromes with rather similar properties. Fourth-order f i n i t e d i f f e r e n c e a n a l y s i s i s a mathematical procedure which can resolve i n d i v i d u a l components from the composite spectrum of a mixture of absorbing species (21). However i t has recently been shown to have a low t h e o r e t i c a l l i m i t i n i t s capacity to resolve multiple components from complex absorption spectra (22). When applied to c o l i t h i s method showed the presence of at least three b-type and two c-type cytochromes. The same species at different r e l a t i v e concentrations were reported to be present after growth under several different conditions (23). A re-examination of t h i s procedure has confirmed the presence of m u l t i p l e cytochromes, but the existence of the same species under a l l growth c o n d i t i o n s has been questioned (24). An alternative way of resolving cytochromes from complex mixtures i s through kin e t i c measurements. A dual-wavelength stopped-flow study has shown the b-cytochromes of c o l i grown under a v a r i e t y of conditions to be k i n e t i c a l l y heterogeneous (15). This approach suggested the presence of two oxidases k i n e t i c a l l y competent to support the observed rates of respiration. An independent study on the kinetics of cytochrome reduction over a much longer time-scale on c e l l s grown anaerobically with n i t r a t e a l s o suggested the presence of multiple b-type cytochromes ( 8 ) . However i t i s very d i f f i c u l t to i n t e r p r e t 9 kinetic data i n terms of the numbers of components involved and their possible arrangement into respiratory chains. Spectrophotometric redox t i t r a t i o n provides another method of resolving s p e c t r a l l y i n d i s t i n c t cytochromes (25). The value of t h i s r approach i s that i t resolves cytochromes of i d e n t i c a l absorption p r o p e r t i e s on the b a s i s of a d i s t i n c t parameter, t h e i r redox p o t e n t i a l . The redox p o t e n t i a l i s an important value to know i n arranging cytochromes into h y p o t h e t i c a l schemes of sequential electron transfer on the basis that electrons are usually.passed from components of low potential to components of higher p o t e n t i a l . Whilst t h i s method i s t h e o r e t i c a l l y capable of resolving multiple species of cytochrome i t has some p r a c t i c a l d i f f i c u l t i e s . For example, three published a p p l i c a t i o n s to the cytochromes of c o l i have given quite disparate r e s u l t s on the number and p r o p e r t i e s of the cytochromes present (26,27,28). Pudek and Bragg (26) reported the existence of just two cytochromes i n c e l l s grown aerobically of redox potential +l65mV and +35mV. The r e l a t i v e amount of these two and their exact redox potential was reported to depend on the growth conditions used. These cytochromes presumably correspond to the species of potential +186mv and +57mV. reported by Hendler and Shrager (27) , who in a d d i t i o n postulate a species of p o t e n t i a l -105mV. These values d i f f e r markedly from those of +260mV, +80mV and -50mv reported by Reid and Ingledew (28) f o r c e l l s grown a e r o b i c a l l y . For c e l l s grown a n a e r o b i c a l l y with n i t r a t e these authors reported cytochromes of potential +260mV, +140mV and +10mV. There has been very l i t t l e discussion of the relationship between cytochromes i d e n t i f i e d by t i t r a t i o n and those i d e n t i f i e d by other 10 means. Pudek a n d B r a g g ( 2 6 ) s u g g e s t e d t h a t t h e h i g h p o t e n t i a l c ytochrome t h e y f o u n d was c y t o c h r o m e b w h i l s t t h e l o w p o t e n t i a l 558 s p e c i e s was b . R e i d a n d I n g l e d e w ( 2 8 ) s u g g e s t e d t h a t t h e i r 5 5 6 s p e c i e s o f p o t e n t i a l +80mV a b s o r b e d a t 556nm and was c y t o c h r o m e o. They found t h a t t h e i r h i g h p o t e n t i a l c y t o c h r o m e had a s p l i t -peak w i t h maxima a t 556nm and 563nm. T h i s m i g h t be i n t e r p r e t e d t o mean t h a t two h i g h p o t e n t i a l s p e c i e s were p r e s e n t a l t h o u g h t h e y s u g g e s t e d one s p e c i e s w i t h a s p l i t s - p e a k . R e i d e_t a l . ( 2 9 ) h a v e a t t e m p t e d t o o b t a i n p r e p a r a t i o n s o f s i m p l e r cytochrome c o n t e n t t h a n w h o l e membranes t o s t u d y by t i t r a t i o n . T h i s was a c h i e v e d u s i n g a hemA- m u t a n t . No c y t o c h r o m e was produced when t h i s s t r a i n was grown i n t h e a b s e n c e o f a m i n o l e v u l i n i c a c i d . H o w e v e r , a f u n c t i o n a l r e s p i r a t o r y c h a i n was r e c o n s t i t u t e d b y i n c u b a t i o n o f membranes o f t h e m u t a n t w i t h h e m a t i n and ATP. O n l y b - t y p e c y t o c h r o m e s were r e c o n s t i t u t e d u n d e r t h e s e c o n d i t i o n s so c y t o c h r o m e o was t h e s o l e t e r m i n a l o x i d a s e ( 1 2 ) . T i t r a t i o n o f the mem b r a n e s a f t e r r e c o n s t i t u t i o n > s u g g e s t e d t h e p r e s e n c e o f two cy t o c h r o m e s . The m a j o r one had a p o t e n t i a l o f +80mV and bound carbon m o n o x i d e s u g g e s t i n g i t was c y t o c h r o m e o. The c y t o c h r o m e o f h i g h p o t e n t i a l (+250mV) r e s o l v e d b y t i t r a t i o n was f o u n d t o be two s p e c i e s o f s i m i l a r p o t e n t i a l , a c y t o c h r o m e b _ _ _ and t h e c y t o c h r o m e b 55 5 55o a s s o c i a t e d w i t h cytochrome d. To r e s o l v e t h e c y t o c h r o m e s o f t h e c y t o c h r o m e - o p a t h w a y and d e t e r m i n e t h e o r d e r o f e l e c t r o n t r a n s f e r , K i t a and An r a k u (20) have s t u d i e d membranes t r a p p e d i n t h e a e r o b i c s t e a d y s t a t e . To a v o i d c o m p l i c a t i o n s due t o t h e c y t o c h r o m e s o f t h e ' l o w a e r a t i o n ' pathway t h e y used c e l l s grown u n d e r c o n d i t i o n s l e a d i n g t o a v e r y l o w c o n t e n t 11 of cytochrome d. The trapping method involves adding substrate to an oxygen saturated membrane suspension followed by rapid freezing and difference spectroscopy. In an a c t i v e e l e c t r o n transport chain those electron c a r r i e r s c l o s e r to the substrate w i l l be more reduced than those nearer to the t e r m i n a l e l e c t r o n a c c e p t o r . Therefore the difference spectrum of the aerobic steady state shows the preferential reduction of redox components of low p o t e n t i a l . The introduction of a kinet i c block w i l l a l t e r the p r o p o r t i o n a l reduction of cytochrome i n a chain in the aerobic steady s t a t e . E l e c t r o n c a r r i e r s situated before the block w i l l become more reduced w h i l s t those s i t u a t e d after the block w i l l become more oxidised. Using t h i s procedure with a v a r i e t y of substrates and respiratory i n h i b i t o r s K i t a and Anraku were ab l e to propose a sequence for electron transfer through the cytochrome o pathway (20, Figure 2a). This involves the sequential t r a n s f e r of electrons from the substrate dehydrogenases to ubiquinone, cytochrome b cytochrome b and 556 562, f i n a l l y to cytochrome o. These three cytochromes were proposed to be the only ones of t h i s pathway and the major ones of c e l l s from the exponential phase of aerobic growth. A different arrangement of the same cytochromes has been proposed by Downie and Cox (30, Figure 2b). They also used >the technique of trapping the aerobic steady s t a t e . In t h i s case k i n e t i c blocks were produced using a mutant d e f i c i e n t i n the production of ubiquinone. However the membranes of the c e l l s they used c l e a r l y had both the cytochrome d and cytochrome o pathways which greatly complicates the int e r p r e t a t i o n of t h e i r data. They proposed that e l e c t r o n transfer occurs from substrate dehydrogenases through quinone to cytochrome 12 NADH-*Fe.S-> Q cyt T 556 DLDH cyt .cyt '562 NADH—» Fe cyt b562 B cyt 556 T cyt b558 t Fe * Q D L D H cyt d cyt o 2 12a FIGURE 2. Proposed arrangement of components i n the a e r o b i c respiratory chains as published by (A) K i t a and Anraku (20) and (B) Downie and Cox (30). Q, ubiquinone; Fe, Fe.S, iron-sulphur centre; D-LDH, D-lactate dehydrogenase; cyt, cytochrome. 13 1 b , cytochrome b and f i n a l l y cytochrome o. They also proposed 562 556 that cytochrome b , and i n d i r e c t l y cytochrome d, can accept 558 electrons from' a quinone pool s i t u a t e d between cytochromes b , and o. Neither of these two schemes has been c l e a r l y c o r r e l a t e d with data from other sources. I t i s not known how the species found by t i t r a t i o n might correspond to the cytochromes of the postulated schemes. Neither have k i n e t i c measurements or the species -found by fourth-order f i n i t e difference a n a l y s i s been discussed in terms of the models proposed. In view of t h i s the schemes i l l u s t r a t e d in Figure 2 should be considered as working models requiring futher study. The Properties of Purified Respiratory ' Complexes Containing - \ Cytochromes The contradictory i n t e r p r e t a t i o n s on the, presence of particular cytochromes i n membrane preparations and t h e i r r o l e i n respiration shows the need for much simpler systems. Many sol u b i l i s e d respiratory complexes have been reported but few are w e l l c h a r a c t e r i s e d . For ins t a n c e , Reddy and Hendler (31) r e p o r t the s o l u b i l i s a t i o n and f r a c t i o n a t i o n o f t h r e e r e s p i r a t o r y c o m p o n e n t s , s u c c i n a t e dehydrogenase, a cytochrome b^ complex and a cytochrome'oxidase complex. These three components can be mixed with another factor to re c o n s t i t u t e a succinate oxidase a c t i v i t y . However they report no details of the composition of thei r respiratory complexes. S i m i l i a r l y there are some report s on the i s o l a t i o n of s i n g l e cytochromes from E. c o l i a l t h o u g h none of these have a c l e a r 14 functional r o l e . A preparation c a l l e d cytochrome has been purified and i s reported to have a redox p o t e n t i a l of -340mV in the pure form although t h i s i s not i t s p o t e n t i a l i n the membrane (32). Another preparation known as, cytochrome b,_^ „ has a p o t e n t i a l of +110mV (33) • 5o2 This protein has been very i n t e n s i v e l y studied s t r u c t u r a l l y (34). In a d d i t i o n a soluble cytochrome c of redox -potential -200mV and 552 which may have a role i n n i t r i t e reduction (11) has been pur i f i e d from c e l l s grown anaerobically with nitra t e (35). Reid and-Ingledew (16) have neported the p u r i f i c a t i o n of a respiratory oxidase complex. Their preparation appears to be the whole of the 'low a e r a t i o n ' oxidase pathway. I t contains cytochromes d, a , b and b . They suggested t h a t cytochrome b had a 1 555 558 558 redox potential of +250mV while that of b was +140mV. These data 555 were not based on t i t r a t i o n of t h e i r oxidase complex. The carbon monoxide d i f f e r e n c e spectrum of the p r e p a r a t i o n shows i t to be uncontaminated by cytochrome o but very l i t t l e else has been reported about i t . A similar preparation has been reported by M i l l e r and Gennis (17). Their procedure yielded cytochromes a and d with only one associated b-type cytochrome. This species absorbed at 558nm and had a redox potential of +l60mV while cytochrome d had a potential of +230mV. The preparation contained two polypeptides of apparent molecular weight 55.000 and 28,000. Ubiquinol and- TMPD oxidase a c t i v i t i e s were present. The other oxidase, cytochrome o has also been i s o l a t e d as a complex with u b i q u i n o l oxidase a c t i v i t y (20), but t h i s preparation was not pure. It contained equimolar amounts of cytochromes b and 555 b . The cytochrome a b s o r b i n g at 555nm was suggested to be 5 62 ( 15 cytochrome o. The- procedure which yie l d e d t h i s complex also gave a pure preparation of a cytochrome b This protein had a molecular 556 weight of 17,500 and a redox p o t e n t i a l of -45mV (36). The success of K i t a and Anraku (20,36) i n i s o l a t i n g cytochrome b. and t h e i r 556 oxidase complex r e f l e c t s t h e i r choice of s t a r t i n g material. They used aerobic c e l l s grown to the early exponential phase on a minimal medium supplemented with Casamino a c i d s . These c e l l s were shown to be almost devoid of cytochrome d. PART I I . Anaerobic Respiratory Pathways. > There a r e , as shown i n F i g u r e 1, at l e a s t four anaerobic r e s p i r a t o r y pathways i n c o l i . Two of these, the TMAO reductase and~nitrate reductase pathways are of relevance to t h i s thesis. TMAO Reduction. TMAO has been shown to be an electron acceptor for resp i r a t i o n . It . allows E. c o l i to grow a n a e r o b i c a l l y on non-fermentable carbon sources (37) and i t s reduction i s coupled to proton translocation (38) . Anaerobic growth with TMAO leads to the production of cytochromes absorbing at around 550nm which have been shown to be of the c-type (39) . These are presumed to be associated with TMAO reduction since they can be r e o x i d i s e d by TMAO a f t e r reduction by NADH or formate (40) . Reoxidation was inhibited by QNO. These results were interpreted to imply the existence of a r e s p i r a t o r y chain between NADH"and TMAO in which quinones and b- and c-type cytochromes are involved (40). This 16 c o n c l u s i o n was c o n f i r m e d t h r o u g h t h e u s e o f r e s p i r a t o r y mutants ( 4 1 ) . In a d d i t i o n t h e use o f mutants has shown t h a t t h e t e r m i n a l enzyme o f t h e e l e c t r o n t r a n s p o r t c h a i n , TMAO r e d u c t a s e , i s a molybdeno-p r o t e i n ( 4 1 ) . N i t r a t e R e d u c t i o n P r o b a b l y t h e b e s t c h a r a c t e r i s e d r e s p i r a t o r y c h a i n o f E. c o l i i s t h a t between fo r m a t e and n i t r a t e . I t i s q u i t e a s i m p l e r e s p i r a t o r y pathway which i s i n d u c i b l e t o v e r y h i g h l e v e l s . In a d d i t i o n i t has been f r a c t i o n a t e d and r e c o n s t i t u t e d t f r o m p u r i f i e d components. B a s i c a l l y , i t c o n s i s t s o f two c o mplexes, f o r m a t e d e h y d r o g e n a s e and n i t r a t e r e d u c t a s e ( r e v i e w e d i n 3,6). Formate dehydrogenase has been p u r i f i e d t o near homogeneity ( 4 2 ) . The p r e p a r a t i o n c o n t a i n e d a b - t y p e c y t o c h r o m e , b ^ d h , w h i c h was c o m p l e t e l y r e d u c i b l e by f o r m a t e . I n a d d i t i o n i t c o n t a i n e d molybdenum, s e l e n i t e and i r o n - s u l p h u r c e n t r e s b u t no f l a v i n c o m p o n e n t . T h r e e p o l y p e p t i d e s cc, /2and )S o f a p p a r e n t m o l e c u l a r w e i g h t 110,000, 37,000 and 20,000 r e s p e c t i v e l y were p r e s e n t . W h i l s t t h e & - s u b u n i t i s known t o c o n t a i n s e l e n i u m ( 4 3 ) . i t i s n o t known w i t h w h i c h s u b u n i t t h e o t h e r p r o s t h e t i c groups are a s s o c i a t e d . N i t r a t e r e d u c t a s e h a s b e e n p u r i f i e d b y s e v e r a l d i f f e r e n t p r o c e d u r e s ( 4 2 , 4 4 , 4 5 , 4 6 ) . A l l p r e p a r a t i o n s h ave 06 and / 2-subunits o f a p p a r e n t m o l e c u l a r w e i g h t a p p r o x i m a t l y 155,000 and 60,000 r e s p e c t i v e l y . O f t e n m u l t i p l e / 3 - s u b u n i t s a r e f o u n d w h i c h may r e p r e s e n t p r o t e o l y t i c f r a g m e n t s o r m o d i f i e d s u b u n i t s ( 4 7 , 4 8 , 4 9 ) . Some n i t r a t e r e d u c t a s e p r e p a r a t i o n s c o n t a i n i n a d d i t i o n t h e ^ - s u b u n i t o f a p p a r e n t m o l e c u l a r 17 weight 19,000. The presence or absence o f t h i s s u b u n i t depends on a number of f a c t o r s i n c l u d i n g the s o l u b i l i s a t o n procedure used and the d e t a i l s of t h e p r e p a r a t i o n method ( 4 7 , 4 8 ) . The ^ - s u b u n i t i s a i . , nr cytochrome known as b . O t h e r r e d o x c e n t r e s c o n t a i n i n g i r o n , sulphur, molybdenum and a p t e r i n d e r i v a t i v e (50) are associated with the 0C- and ft -sub u n i t s (45,46) E l e c t r o n paramagnetic resonance studies of a p u r i f i e d n i t r a t e reductase (46,51) showed s i g n a l s associated with molybdenum o f redox p o t e n t i a l +180mV and +220mV. The p o t e n t i a l s associated with two other s i g n a l s p r o b a b l y due to iron-sulphur centres were found to be +50mV and +80mV. Formate dehydrogenase, n i t r a t e r e d u c t a s e and ubi q u i n o n e - 6 are s u f f i c e n t f o r e l e c t r o n t r a n s p o r t between formate and n i t r a t e using p u r i f i e d components (52). Therefore i t i s l i k e l y t h a t only s i x types of polypeptide are involved i n t h i s r e s p i r a t o r y c h a i n . This makes i t one of the s i m p l e s t known. A p r e p a r a t i o n o f n i t r a t e r e d u c t a s e l a c k i n g n r c y t o c h r o m e b b u t r e t a i n i n g t h e c a p a c i t y t o r e d u c e n i t r a t e reductase with reduced b e n z y l v i o l o g e n as donor can be produced. This p r e p a r a t i o n was not a c t i v e i n r e c o n s t i t u t i o n which i m p l i e s t h a t nr cytochrome b i s a mandatory e l e c t r o n c a r r y i n g i n t e r m e d i a t e i n the r e s p i r a t o r y c h a i n . In a d d i t i o n i t can be shown i n the r e c o n s t i t u t e d nr system t h a t cytochrome b i s f u l l y r e d u c i b l e by formate and i s re o x i d i s e d by n i t r a t e (52). These r e c o n s t i t u t i o n s t u d i e s l e a d t o the proposed arrangement of e l e c t r o n c a r r i e r s i n the r e s p i r a t o r y c h a i n shown i n Figure 3a (52). T h i s arrangement had p r e v i o u s l y been s u g g e s t e d on the b a s i s of dual-wavelength s t u d i e s by R u i z - H e r r e r a and DeMoss (53). Their studies fdh nr suggested the presence o f two major c y t o c h r o m e s , b and b , i n 18 F o r m a t e - > F D H - > b f d h ^ Q - » b n r - > N R — > N O 3 B N A D H NO; N A D H D H N R c y t F o r m a t e Q \ c y t c y t b f d h b 5 5 8 " F D H c y t o " c y t d 1 18a F IGURE 3. P r o p o s e d a r r a n g e m e n t o f r e s p i r a t o r y c a r r i e r s i n c e l l s grown a n a e r o b i c a l l y w i t h n i t r a t e as p u b l i s h e d by (A) Enoch and L e s t e r ( 52 ) and R u i z - H e r r e r a and D e M o s s ( 5 3 ) and (B) S a n c h e z - C r i s p e n e t  a l . ( 8 ) . FDH, fo rmate d e h y d r o g e n a s e ; NR, n i t r a t e r e d u c t a s e ; NADH-DH, NADH dehyd rogenase . 19 frozen-thawed c e l l s previously grown anaerobically with n i t r a t e . Both cytochromes had the same a b s o r p t i o n maximum which these workers suggested to be 555nm at 77K, but other groups have shown to be 556nm (15 i28). The l a t t e r value w i l l be used for the purposes of t h i s thesis. Ruiz-Herrera and DeMoss found a high p o t e n t i a l species reducible by ascorbate which was r a p i d l y r e o x l d i s e d by n i t r a t e . This was presumed nr to- be cytochrome b . They also found a cytochrome reducible only by formate which was re-oxidised by n i t r a t e only after some delay, which fdh they, suggested to be cytochrome b . E l e c t r o n t r a n s f e r between these two cytochromes was e n t i r e l y blocked by QNO. These data led them to propose the same model as that derived from r e c o n s t i t u t i o n studies (52,53, Figure 3a). A re-examination of cytochrome reduction by the dual wavelength technique (8) gave rather different results from those of Ruiz-Herrera and Demoss (53). When studied i n d e t a i l the k i n e t i c s of cytochrome r e d u c t i o n and r e o x i d a t i o n i n t h e membranes of c e l l s grown anaerobically with n i t r a t e were found to be quite complex (8). The data were interpreted by the scheme shown i n Figure 3b. The kin e t i c s of reduction were found to i n v o l v e at l e a s t four phases. This was suggested to be due to the simultaneous presence of both aerobic and anaerobic b-type cytochromes i n the membrane . There appeared to be an equilibration of electrons between these pathways which accounted for the complex k i n e t i c behaviour found. The revised scheme retains the electron t r a n s p o r t chain proposed e a r l i e r but merely adds branches indicating e l e c t r o n t r a n s f e r to other r e s p i r a t o r y chains (8). This idea i s fundamentally d i f f e r e n t from that of Ruiz-Herrera and DeMoss (53) who suggested t h a t cytochromes b f d h and, b n r were r a p i d l y 20 auto-oxidised rather than suggesting an i n t e r a c t i o n of aerobic and anaerobic respiratory chains. PART I I I . Mutations i n the Formate-Nitrate Reductase Pathway Our understanding of the formate-nitrate respiratory pathway owes a l o t to the ready a v a i l a b i l i t y of mutants defective in t h i s a c t i v i t y . Because the system i s dispensable under most growth conditions mutants are quite simple to i s o l a t e and handle. They were o r i g i n a l l y isolated on the basis of their resistance to chlorate under anaerobic conditions (54). I t was believed that n i t r a t e reductase reduced c h l o r a t e to c h l o r i t e which was t o x i c to the c e l l . Mutants obtained by t h i s procedure were c a l l e d c h i mutants and were found to map to several l o c i . Most of them l a c k e d both formate hydrogenlyase a c t i v i t y ( r eflecting their lack of formate dehydrogenase) and nitrate reductase, a c t i v i t y . In a d d i t i o n , many c h i mutants have patterns of cytochrome production d i s t i n c t from that of the wild-type (55) but a l l seemed to grow normally under anaerobic c o n d i t i o n s (56). The pleiotropic lack of two respiratory a c t i v i t i e s r e s u l t s from the involvement of the same cofactor i n both enzymes ( 5 7 ) . T h i s i s a p t e r i n d e r i v a t i v e with associated molybdenum and i s c a l l e d Mo-cofactor (50). This involvement was shown by studies of the molybdeno-protein content of wild-type E. c o l i and c h l o r a t e r e s i s t a n t i s o l a t e s . Many of the mutants studied were found to l a c k a l l molybdeno-proteins (57). In addition the phenotype of a chlD mutant and l a t e r a chlG mutant were shown to be p a r t i a l l y r e v e r s e d by growth at a h i g h c o n c e n t r a t i o n of molybdenum (58,59). 21 In a l l , f i v e l o c i appear to be a s s o c i a t e d with Mo-cofactor b i o s y n t h e s i s and i n s e r t i o n . The c h i A l o c u s at 17min, the chlE locus at 18min,and the c h l G l o c u s at Omin s p e c i f y components required for Mo-cofactor b i o s y n t h e s i s . Soluble extracts from mutants at a l l of these l o c i can complement a s o l u b l e e x t r a c t from chlB mutants to r e c o n s t i t u t e n i t r a t e reductase a c t i v i t y (59»60,61). The c h l B l o c u s , which maps t o 86min i s b e l i e v e d to s p e c i f y an a s s o c i a t i o n f a c t o r . T h i s f a c t o r , F . a l l o w s the i n s e r t i o n of A Mo-cofactor i n t o apoproteins to form a c t i v e enzymes (62). In the absence of F the Mo-cofactor i s b e l i e v e d to accumulate i n the A cytoplasm of chlB mutants and can be i n s e r t e d i n t o apoproteins and a c t i v i t y r e c o n s t i t u t e d when the soluble F^ from other c h i mutants i s added (61). F i n a l l y the chlD locus (17min) may specify components required i n molybdenum transport and processing since i t s effects are largely overcome by growth at high concentrations of molybdenum (58). The chlC locus corresponds to the n i t r a t e reductase structural gene and maps to 27min . T h i s was s u g g e s t e d on the b a s i s of immunoprecipitation of what appeared to be fragments of n i t r a t e reductase from the cytoplasm of a chlC mutant (63). The assignment was confirmed by the i s o l a t i o n of mutants temperature sensitive for n i t r a t e reduction (64). S o l u b i l i s e d n i t r a t e reductase from these mutants was found to be more thermolabile than that of the parent. Since t h i s mutation mapped to the c h i C l o c u s t h i s i s very good evidence that t h i s locus includes the n i t r a t e reductase structural gene. Orth et a l . (65) have i s o l a t e d a mutant which w i l l reduce nitrate with benzyl viologen as e l e c t r o n donor but not with any other donor. This mutant retained formate hydrogenlyase a c t i v i t y but had only 50% of the cytochrome content of the parent. I t was suggested to nr s p e c i f i c a l l y lack cytochrome b . Mapping of the gene causing this phenotype (designated c h i I ) showed i t to be very close to chlC. Subsequently c h i C and c h i I were shown to be p a r t of the same operon in which chlC i s t r a n s c r i b e d f i r s t (66). Insertion mutants in the chlC gene have reduced cytochrome l e v e l s due to t h e i r polar effect on c h l l gene expression (59.66). Most c h i l o c i have been shown to contain more than one gene (67). In some cases t h i s leads to several phenotypes being observed from mutation at one locus (59.68). However t h i s can also apparently arise from the tendency of c h i mutants to become double mutants by mutation at the fnr locus to be described l a t e r (59). Although most mutants selected on the basis of chlorate resistance p l e i o t r o p i c a l l y lack both formate dehydrogenase and nitrate reductase, mutants lacking either one of these a c t i v i t i e s can be selected. There are, for example, two procedures f o r s e l e c t i o n of mutants defective in formate dehydrogenase (fdh mutants, 69.70). These mutants map to two l o c i , fdhA at 80min and fdhB at 40min (71). The genetic lesion i n fdhA and fdhB mutants i s unknown but they have been shown to lack immunologically detectable formate dehydrogenase subunits (71). In the c l o s e l y r e l a t e d Salmonella typhimurium a t h i r d class of formate dehydrogenase mutant known as fdn mutants has been obtained which was mapped to 87min ( 7 2 ) . These mutants reta.in formate hydrogenlyase and nitrate reductase a c t i v i t y but cannot reduce nitrate using formate. They appear to lack a component which connects the formate dehydrogenase complex to n i t r a t e reductase but i s not needed 23 f o r e l e c t r o n t r a n s f e r i n t h e h y d r o g e n l y a s e s y s t e m . The f d n mutants had no l o w p o t e n t i a l c y t o c h r o m e s i n c e a l l t h e i r c y t o c h r o m e was r e d u c i b l e by a s c o r b a t e , a h i g h p o t e n t i a l e l e c t r o n d o n o r . On t h e b a s i s o f t h e r e s u l t s o f R u i z - H e r r e r a and DeMoss (53)» t h e m i s s i n g component' f d h was s u g g ested t o be c y t o c h r o m e b (72). A l t e r n a t i v e l y t h e f ormate d e h y d r o g e n a s e r e s p o n s i b l e f o r n i t r a t e r e d u c t i o n may be c o m p l e t e l y d i s t i n c t from t h a t i n v o l v e d i n t h e h y d r o g e n l y a s e system (43). However t h e d a t a on t h e c h i m u t a n t s w h i c h l a c k f o r m a t e d e h y d r o g e n a s e a c t i v i t y w i t h any e l e c t r o n d o n o r means t h a t b o t h o f t h e s e would have t o use M o - c o f a c t o r (57). Mutants s p e c i f i c a l l y d e f e c t i v e i n n i t r a t e r e d u c t a s e b u t r e t a i n i n g f o rmate dehydrogenase can be s e l e c t e d r e a d i l y . M u t a g e n i s e d c e l l s a r e t e s t e d f o r t h e i r i n a b i l i t y t o p r o d u c e n i t r i t e ( d e t e c t e d c o l o r i m e t r i c a l l y ) f r o m f o r m a t e and n i t r a t e (55,71 .73). About h a l f o f the mutants s e l e c t e d b y t h i s m e t h o d l a c k e d n i t r a t e r e d u c t a s e b u t not form a t e d e h y d r o g e n a s e and mapped t o t h e c h l C l o c u s . By c o n t r a s t 98% o f t h e mutants s e l e c t e d on t h e b a s i s o f c h l o r a t e r e s i s t a n c e mapped t o t h e c h i A, c h i B, c_h 1D a n d ch 1E l o c i (73). I n a n o t h e r s t u d y m u t a n t s were s e l e c t e d f o r t h e i r c a p a c i t y t o r e d u c e TMAO i n t h e pre s e n c e o f n i t r a t e on t h e p r e m i s e t h a t a f u n c t i o n a l n i t r a t e r e d u c t a s e i n h i b i t s TMAO r e d u c t i o n (59). M o r e o v e r p l e i o t r o p i c c h i mutants cannot reduce TMAO i n s p i t e o f t h e d e r e p r e s s i o n o f TMAO r e d u c t a s e because t h e y cannot produce e i t h e r M o - c o f a c t o r o r F , b o t h o f w h i c h a r e r e q u i r e d f o r t h e p r o d u c t i o n o f a f u n c t i o n a l TMAO r e d u c t a s e . T h e r e f o r e , t h e o n l y s t r a i n s c a p a b l e o f r e d u c i n g TMAO i n t h e p r e s e n c e o f n i t r a t e l a c k or have a d e f e c t i v e n i t r a t e r e d u c t a s e . A l l o f t h e s e w e r e f o u n d t o map c l o s e t o t h e c h l C l o c u s a n d i n c l u d e d m u t a n t s a t t h e c h l C and 24 c h l l l o c i as w e l l as r e g u l a t o r y mutants. By contrast no mutants selected on the basis of ch l o r a t e r e i s t a n c e mapped to the chlC locus (79). Therefore i t has been proposed by Stewart and MacGregor (59) that there.are two c h l o r a t e r e d u c t a s e s i n E . c o l i . These are n i t r a t e reductase and TMAO reductase, both of which are molybdeno-proteins. Only when the Mo-cofactor cannot be synthesised and inserted i s the le t h a l synthesis of c h l o r i t e prevented. So, mutants l a c k i n g only n i t r a t e reductase, are c h l o r a t e s e n s i t i v e since they r e t a i n TMAO reductase which w i l l perform the l e t h a l s y n t h e s i s . This idea i s somewhat complicated by the degree of ind u c t i o n of the two enzymes. Thus, a c e r t a i n c h i C a l l e l e may or may not c o n f i r m c h l o r a t e resistance depending on the degree of repression of TMAO reductase. This appears to be rather variable between st r a i n s . The Regulation of the Production of Nitrate Reductase It i s clear that the expression of anaerobic respiratory systems i s c l o s e l y r e g u l a t e d , b e i n g r e p r e s s e d by oxygen and induced by substrate. The s p e c i f i c a c t i v i t y of n i t r a t e reductase in c e l l s grown with vigourous a e r a t i o n i s approximatly 3% of that i n c e l l s grown anaerobically. The enzyme i s induced by another f a c t o r of twenty by the introduction of n i t r a t e i n t o the anaerobic growth medium (74). It can be s t i l l f u r t h e r induced by the presence of 0.1mM azide (75). Indeed, one report (45) suggested that n i t r a t e reductase can constitute as much as 15% of the membrane p r o t e i n . In the same way other anaerobic respiratory pathways are repressed by oxygen and induced by their s p e c i f i c substrates. 25 In addition, as mentioned above, a f u n c t i o n a l nitrate reductase represses the production of other anaerobic r e s p i r a t o r y systems. Thus, mutants defective in n i t r a t e reductase are derepressed for other anaerobic r e s p i r a t o r y systems. For i n s t a n c e , chlA and chlB mutants have been shown to be derepressed for n i t r i t e reductase under a l l anaerobic conditions (76). In the wild-type s t r a i n t h i s enzyme i s expressed only at low c o n c e n t r a t i o n s of n i t r a t e or n i t r i t e . Also chlC mutants are derepressed for TMAO reductase (59). This has led to the suggestion (6) that the a c t i v a t i o n of the genes f o r other anaerobic r e s p i r a t o r y systems i s prevented by a f a c t o r which i s missing i n c h i mutants. This could even be a component of n i t r a t e reductase i t s e l f . I t has been found i n A s p e r g i l l u s nidulans that the synthesis of n i t r i t e reductase i s regulated by a component of nitrate reductase (77). Several groups have i s o l a t e d mutants d e f i c i e n t in the regulation of anaerobic respiratory systems, although the functional significance of any of these has yet to be c l e a r l y e s t a b l i s h e d . Many of them map very close to the ch i C l o c u s . Stewart and MacGregor (59) have described mutants i n a gene designated narK which were derepressed for TMAO reductase i n the presence of n i t r a t e . Whilst t h i s i s t y p i c a l of c h i mutants, these s t r a i n s had a f u l l y f u n c t i o n a l n i t r a t e ' reductase. I t was proposed that the mutants lacked a protein which, in the presence of a f u n c t i o n a l n i t r a t e r e d u c t a s e represses other respiratory enzymes. They also reported a mutation i n the narL gene which expressed nitra t e reductase only under anaerobic conditions, but at a low level not futher increased by the presence of n i t r a t e . The narL gene was proposed to s p e c i f y a n i t r a t e - r e s p o n s i v e activator of 26 the expression of the n i t r a t e reductase genes. There are two other r e g u l a t o r y genes mapping i n t h a t a r e a . Mutants at the ana locus prevent anaerobic growth i n the absence of any added terminal electron acceptor (78). Mutants at the adh locus lead to the overproduction of alcohol dehydrogenase and a low l e v e l of n i t r a t e reductase when grown anaerobically with nitrate (79). It i s known that n i t r a t e usually represses alcohol dehydrogenase and induces n i t r a t e reductase. There i s no information published on what r e l a t i o n s h i p , i f any, the four genes described bear to each o t h e r a l t h o u g h some of them may be a l l e l i c . In addition, several workers have isolated mutants p l e i o t r o p i c a l l y lacking a l l known anaerobic r e s p i r a t o r y systems, regardless of whether or not they use molybdenum. These a l l mapped to 29min and were named v a r i o u s l y f n r ( 8 0 ) , n i r A (76) and n i r R ( 8 1 ) . n i r A and fnr mutants do not complement which i m p l i e s that they are a l l e l i c and a mutation i n n i r A has been shown to be r e c e s s i v e (76). The mutants were proposed to l a c k a p o s i t i v e r e g u l a t o r n e c e s s a r y f o r the production of a l l anaerobic r e s p i r a t o r y systems, perhaps by being oxygen s e n s i t i v e . A l t e r n a t i v e l y i t could be s e n s i t i v e to the redox state of the respiratory chain. This idea i s a t t r a c t i v e i n view of the observation that u b i q u i n o n e - d e f i c i e n t mutants are derepressed for n i t r a t e reductase under a e r o b i c c o n d i t i o n s ( 8 2 ) . P o s s i b l y the unusually reduced state of the l o w - p o t e n t i a l c a r r i e r s of the aerobic respiratory chain caused the mutational block to be sensed. A similar state might be expected when oxygen i s l a c k i n g . The nirR gene product may sense t h i s and become m o d i f i e d to become an a c t i v e positive e f f e c t o r allowing the expression of anaerobic respiratory 27 pathways (6). An approach to studying the r e g u l a t i o n of n i t r a t e reductase i s through the c o n s t r u c t i o n o f ch.1 C-l_a__c_Z f u s i o n s t r a i n s . The construction of these s t r a i n s has f a c i l i t a t e d the i d e n t i f i c a t i o n of a large number of mutations which a f f e c t the expression of n i t r a t e reductase. In fusion strains^-galactosidase i s repressed by oxygen and induced by nitrate (83.84,85). The expression of -galactosidase can be determined on i n d i c a t o r p l a t e s a l l o w i n g the ready i s o l a t i o n of r e g u l a t o r y mutants. Moreover i t s a c t i v i t y does not require the p r o d u c t i o n of M o - c o factor or F A and t h e r e f o r e the p o t e n t i a l r e g u l a t o r y e f f e c t s of p l e i o t r o p i c c h i mutants can be studied i n fusion strains (83). In f a c t , the e f f e c t of only two chlE mutations on the expression o f y S - g a l a c t o s i d a s e i n a f u s i o n s t r a i n has been r e p o r t e d ( 6 8 ) . A s m a l l d e l e t i o n , Aj^hJLEJJ^ had no e f f e c t on yB-galactosidase gene expression. However, a l a r g e r deletion ^ chlE68 o r i g i n a l l y isolated as a r e g u l a t o r y mutation, caused a high l e v e l of ^-galactosidase production under anaerobic conditions irrespective of the presence of n i t r a t e . In a d d i t i o n , at l e a s t three phenotypes of expression of ^-galactosidase i n the f u s i o n s t r a i n can be induced by mutation (84). Mutants were found i n which i t was not repressed by oxygen, not induced by n i t r a t e , or both of these. However none of these mutants has been c h a r a c t e r i s e d f u r t h e r . When a n i r R mutation was introduced i n t o a f u s i o n s t r a i n no y8-galactosidase was expressed under any conditions (84,85). Neither could any of the other mutations affecting expression i n a f u s i o n s t r a i n l i f t the repression caused by the n i r R m u t a t i o n . This suggests t h a t the nirR gene product i s absolutely required for the expression of n i t r a t e reductase. 28 Objectives of t h i s Thesis The introduction given above has o u t l i n e d some of the previous work done on E s c h e r i c h i a c o l i r e s p i r a t o r y chains with p a r t i c u l a r reference to the f o r m a t e - n i t r a t e r e d u c t a s e pathway. Whilst many approaches have been taken to d e f i n i n g these enzyme complexes, they are s t i l l poorly understood. The model schemes presented are based upon a limited repertoire of techniques and do not account for the data found by other means, such as redox t i t r a t i o n . In addition, there has i been no standard way of growing c e l l s which has meant that attempting to c o r r e l a t e two sets of data may be f u t i l e because two d i f f e r e n t populations of ^cytochromes may be i n v o l v e d . With t h i s background i t was decided to apply a series of spectroscopic techniques to study the cytochromes- of c e l l s grown under various c o n d i t i o n s and to t r y to correlate results obtained by one technique with those from another. The method of spectrophotometric redox t i t r a t i o n was found to be the most sensitive and unambiguous way of d e f i n i n g the cytochrome content but i n s p i t e of i t s s e n s i t i v i t y i t c o u l d not f u l l y define the cytochrome content of any membrane preparation. This was attributed to the simultaneous presence of at l e a s t s i x b-type cytochromes, a l l of which could be detected only by a range of methods. I t proved necessary to introduce s i m p l i f i c a t i o n s i n o r d e r to i d e n t i f y components of p a r t i c u l a r r e s p i r a t o r y pathways. Three approaches were attempted. Various growth c o n d i t i o n s were e x p l o r e d to f i n d one i n which E.  c o l i produces a very small number of cytochromes. Secondly, some e f f o r t s were made to f r a c t i o n a t e the r e s p i r a t o r y chain before spectroscopic i n v e s t i g a t i o n . F i n a l l y , r e s p i r a t o r y mutants were sought 29 lacking p a r t i c u l a r cytochromes. The only mutants available were in the formate-nitrate pathway so t h i s has been studied in most d e t a i l . However, an i n v e s t i g a t i o n of the p r o p e r t i e s of a number of mutants a l s o gave much i n f o r m a t i o n on the components of the a e r o b i c r e s p i r a t o r y pathways and the r e g u l a t i o n of r e s p i r a t o r y chain s y n t h e s i s . On the basis of the r e s u l t s . a n arrangement of the E. c o l i cytochromes i n the "membrane has been proposed. 30 MATERIALS AND METHODS C h e m i c a l s i V A l l chemicals were obtained from commercial sources and were of reagent grade. Special chemicals were obtained as follows: NADH, PMS, phenazine ethosulphate, QNO, TMAO, DCIP, N-napthyl ethylenediamine, CCCP, horse heart cytochrome c (Type IV) , DCCD and tetracycline from Sigma Chemical Company; 1,2-naphthoquinone and 2-hydroxy-1 ,4-naphthoquinone from Eastman; menadione from N u t r i t i o n a l Biochemicals Corporation; anthraquinone-2-sulphonate from Fisher S c i e n t i f i c Co.; ruthenium hexamine from Alfa D i v i s i o n ; 3,6-diaminodurene from Aldrich; sulphanilamide from Matheson, Coleman and B e l l ; benzyl viologen from ICN Pharmacuticals Inc.; components of media from Difco Laboratories and electrophoresis reagents from Bio-Rad Laboratories. Some batches of potassium n i t r a t e may cause anomalous pigment production and the repression of r e s p i r a t o r y a c t i v i t y i n some s t r a i n s of Esc h e r i c h i a c o l i . Therefore sodium n i t r a t e (Fisher ACS C e r t i f i e d Grade) was used to grow c e l l s for most of the experiments reported i n t h i s thesis. E scherichia c o l i S t r a i n s The E^ c o l i s t r a i n s employed and constructed are l i s t e d in Table 1. Most strains were stored in small v i a l s of Penassay broth-agar o at 4 C and were p e r i o d i c a l l y subcultured a f t e r checking n u t r i t i o n a l markers. For permanent storage an e x p o n e n t i a l l y growing culture in Penassay broth was d i l u t e d with an equal volume of 50% glycerol in a 31 TABLE 1 STRAINS OF ESCHERICHIA COLI USED IN THIS STUDY S t r a i n Genotype O r i g i n W1485E + F ,supE J.R.Guest MR43L F , t h i , l a c , g a l , r e c A R.B.Gennis HfrH Hfr,thi,relA,spoT,supQ ,\ J.A.DeMoss PK27 H f r , t h i n TS9A As PK27 but c h l C 1 1 ' 8 ' ii RK4353 lacU,araD,non,thi,rp sL,gyrA V.Stewart (=MC4100) RK5206 As RK4353 but chlG::Mu ^ c t s II RK5231 II II RK5256 II II RK5201 As RK4353 but chlE::Mu . c t s II RK5218 II II RK5223 ti II RK5274 As RK4353 but narI::TnlO II RK5266 As RK4353 but narK::TnlO II RK5270 As RK4353 but narK::TnlO II RK5269 As RK4353 but narG::TnlO II RK7 (KL96) H f r , g a l , t h i , r p s L C.H.MacGrego: RK7-16 As RK7 but chlA II RK7-36 As RK7 but chlB it RK7-19 As RK7 but chlC II LCB61 lacU,araD,thi,rpsL M. Chippaux (=MC4100) LCB68 As LCB61 but chlE68 II LCB79 As LCB61 but ( c h l l - l a c Z ) II LCB61-22 As LCB61 but n i r R II LCB61-357 As LCB61 but chlE16 it LCB79-357 As LCB79 but chlE16 II 32 TABLE 1 (cont) S t r a i n LCB356 LCB517 LCB320 (=C600) LCB155 LCB160 LCB162 W945T 541 356-15 356-24 CGSC 4444 CGSC 4459 KL718 KL702 KL703 NH10 NH20 NH30 NH40 NH50 Genotype F .leu,thr,arg,his,pro,purE,supE,lacY, malA,xyl,ara,mt1,gal,tonA,rpsL As LCB356 but fdhA .-F~, t h i , l e u , t h r ,,supE, l a c Y , tonA, rpsL As LCB320 but a n a , n i r C , c h l I As LCB320 but chl l : : M u O r i g i n M. Chippaux c t s As LCB320 but chlC::Mu ^ c t s F~, t h i , t r p , l a c , m a l , g a l , x y l , a r a , m t l , s d h As LCB356 As 541 but chlA As 541 but chlB t h i , leuB,sue,bioA,£alT,rp_sL,chlC F + , g a l , s u p E , c h l E F'(F1 5 2 ) , p y r D , t r p , t y r A , r e c A , t h i , g a l K , x y l ^ t l ^ a l A ^ p s L ^ i s F ' ( F 1 5 2 ) , t h i , p y r D , h i s , t r p , r e c A , m t l , x y l , ma!A,galK,rpsL F' ( F 1 2 6 ) , p y r D , t r p , h i s , r e c A , t h i , g a l K , xyl,mtl,malA,rpsL As LCB68 but narl::TnlO As LCB517 but narl:;TnlO As HfrH but narl::TnlO As CGSC 4459 but narl::TnlO As RK7-16 but narI::TnlO W.Kay E.Azoulay B.Bachmann K.B.Low By PI trans-d u c t i o n of parent to r t e t u s i n g RK5274 33 o v i a l and s t o r e d at -70 C. S t r a i n s w i t h Tn10 i n s e r t i o n s were maintained and grown i n the presence of 20>ug/ml tetracycline. Strains with F 1 f a c t o r s were s t o r e d on mimimal medium with appropriate supplements that prevented growth of" c e l l s losing the episome. Growth of C e l l s To' grow a batch of c e l l s a 10ml volume of Penassay broth was innoculated with viable c e l l s from a v i a l and was grown overnight. A l l of t h i s was then used to i n n o c u l a t e l a r g e r volumes of up to 61. Aerobic growth was performed i n 500ml of medium i n a 21 conical flask shaken at 250 rpm in a New Brunswick Rotary Incubator. For anaerobic growth , n o n - s t i r r e d s t a n d i n g c u l t u r e s i n 21, 41 or 61 f l a s k s completely f i l l e d with medium were used. A l l c e l l s were grown at 37°C except s t r a i n s h a r b o u r i n g M u c t s which were grown at 29°C — o , o and the s t r a i n TS9A which was grown at 29 C or 42 C as s p e c i f i e d i n p a r t i c u l a r experiments. Growth was monitored by measuring the opti c a l density of the cultures at 660nm. M£d_i__u m The minimal medium consisted of the mixture of salts described by Davis and Mingioli (86). This was supplemented with 1 ^ g/ml thiamine, 1uM each of ammonium molybdate and selenous acid (87) and 12juM f e r r i c c i t r a t e . For anaerobic growth sodium bicarbonate was added to the above medium from a s t e r i l e stock s o l u t i o n after autoclaving. Glucose was the usual carbon source. This was autoclaved separately and added 34 to a concentration of 0.4% for aerobic growth or 1% for anaerobic growth. 0.5% peptone was i n c l u d e d i n the anaerobic growth medium except where s p e c i f i e d otherwise. Terminal e l e c t r o n acceptors were s t e r i l i s e d separately except f o r n i t r a t e which was s t e r i l i s e d with the s a l t s . They were added to a f i n a l concentration of 0.5% except for n i t r i t e which was used at 0.25%. When carbon sources other than glucose were used for a e r o b i c growth, these too were autoclaved separately. When necessary, e s s e n t i a l n u t r i e n t s or tetracycline were added from s t e r i l e stock solutions to a l e v e l of -20 pg/ml. i Preparation of Membranes Cells were harvested by c e n t r i f u g a t i o n at 5000rpm for 20min in a JA10 rotor of a Beckman J-21 c e n t r i f u g e . The c e l l s were resuspended in ,25mM T r i s - H C l , pH 8.0 c o n t a i n i n g 0.9% NaCl and resedimented by c e n t r i f u g a t i o n at 10,000rpm i n a JA20 r o t o r for 15min. They were resuspended to a concentration of approximatly 1g wet weight per 5ml in 50mM T r i s - H ^ O ^ , pH 7.8 c o n t a i n i n g 10mM M g C l ^ The c e l l s were disrupted i n a French press at 20,000psi. Whole c e l l s were removed by centrifugation in a JA20 rotor as described above. The resulting crude extract was separated into a membrane f r a c t i o n and a soluble fraction by u l t r a c e n t r i f u g a t i o n f o r 2h e i t h e r at 40,000rpm i n a Beckman 42.1 rotor or at 45,000rpm i n a Beckman Ti60 rotor. The membrane p e l l e t was then resuspended i n the T r i s - s u i phate-magnesium b u f f e r and resedimented as before. P e l l e t s were stored at 4°C and were used within 48h of preparation. This type of membrane preparation was used i n nearly a l l of the experiments described i n t h i s thesis, they,were 35 resuspended i n an appropriate buffer immediately before use. Low Temperature Difference Spectra t Spectra were taken at the temperature of l i q u i d nitrogen (77K) in a Perkin-Elmer 356 spectrophotometer operated i n i t s s p l i t beam mode using i t s cryogenic unit. 1ml of a membrane suspension was diluted in an equal volume of^ 2. OM sucrose and the sample d i v i v e d i n t o two portions. These two samples were reduced and oxidised as appropriate before being transferred to the cuvette compartments of the cryogenic c e l l holder. The whole c e l l holder was then immersed in l i q u i d nitrogen for about two minutes. When reductants other than d i t h i o n i t e were used, freezing was delayed by f i v e minutes a f t e r a d d i t i o n of the sample to the cuvette to allow the de p l e t i o n of any oxygen introduced i n the t r a n s f e r p r o c e s s . The f r o z e n samples were"then d e v i t r i f i e d by illumination from a distance of 5cm with a 150W incandescent bulb for three minutes. This step improves the s e n s i t i v i t y and i s necessary for obtaining reproducible spectra. The samples were then refrozen i n the cryogenic unit and scanned. T y p i c a l l y , a rapid scan of the wavelength region 700nm to 500nm was performed at 60nm/min with a s l i t width of 3nm and the pen response switch at the 'medium' p o s i t i o n . This was then followed by a slow scan at 10nm/min of the <£-peak region of the c- and b-type cytochromes with a 1nm s l i t width and 'slow' response s e t t i n g . This allowed an accurate determination of the absorption maximum of a particular sample. The height of the ££-peak of the b-cytochrome was measured above a baseline drawn between the two adjacent troughs of the spectrum. It 36 ( was found that the procedure described for measuring spectra at 77K increased the absorbance for a particular sample by a factor of 23 over that found at room temperature. E x t i n c t i o n c o e f f i c e n t s f o r ty p i c a l -1 -1 b-type cytochromes at room temperature are about 17 mM cm (32,35,42). Therefore an e x t i n c t i o n c o e f f i c e n t of 390 mM~1 cm"1 has been taken f o r t o t a l b-cytochrome measured at 77K. Whilst this value may be in error i t was found u s e f u l i n comparing the cytochrome content of various p r e p a r a t i o n s . An e x t i n c t i o n c o e f f i c e n t of 8.5 mM ^ cm ^ at 628nm using 650nm as reference wavelength was taken at room temperature for cytochrome d (88). A l l of the spectra reported i n t h i s t h e s i s (except the carbon monoxide difference spectra) were measured at 77K. Therefore c- and b-type cytochromes have been named with a s u b s c r i p t denoting their absorption maximum at t h i s temperature. However, some cytochromes have already, been named by other conventions and where possible the previous names of these species have been r e t a i n e d . The r e l i a b i l i t y of the absorption maxima quoted i n t h i s t h e s i s was ensured by confirming the wavelength c a l i b r a t i o n of the Perkin-Elmer 356 using the spectrum of holmium oxide as a standard. Carbon Monoxide Difference Spectra In this thesis the phrase 'carbon monoxide difference spectrum' refers to the di t h i o n i t e reduced plus carbon monoxide minus dit h i o n i t e reduced difference spectrum. These were recorded at room temperature. A membrane suspension i n each of two cuvettes was reduced by the add i t i o n of a few grains of d i t h i o n i t e . One was used as reference 37 w h i l s t the other was bubbled w i t h carbon monoxide for 2min. The difference spectrum was then scanned at 60nm/min with a 3nm s l i t width at 'medium' pen response. No change was seen i n the spectrum after longer periods of bubbling with carbon monoxide. No ef f o r t was made to keep samples i n the dark d u r i n g t h i s p r o c e d u r e . An e x t i n c t i o n c o e f f i c e n t of 170 mM ^ cm ^ between the peak at 417nm arid the trough at 432nm was taken for cytochrome o (89). Spectrophotometric Redox T i t r a t i o n Potenti-ometric t i t r a t i o n s were performed by the method of Dutton (25). A sample of membranes was suspended i n 15ml of degassed 0.1M potassium phosphate b u f f e r of pH 7.0 to a pr o t e i n concentration of approximately 10mg/ml. 12.5ml of t h i s was placed i n a cuvette of the design shown by Dutton (25) but with j u s t one side-arm. The vessel was sealed with a serum stopper i n the side-arm and a rubber stopper in the top containing ports f o r the e l e c t r o d e and for gassing. The electrode was a Fisher combination platinum electrode (13-639-82) which had an i n t e r n a l reference e l e c t r o d e . This was standardised p e r i o d i c a l l y against a s e r i e s of cross-checked standard calomel electrodes. The atmosphere i n the cuvette was replaced by nitrogen (Linde U.S.P) and the c u v e t t e was i n t e r m i t t e n t l y flushed with nitrogen throughout the t i t r a t i o n . The redox mediators used were: sodium anthraquinone-2-sulphonate, phenazine methosulphate (PMS), phenazine e t h o s u l p h a t e , 2 , 6 - d i c h l o r o p h e n o l indophenol (DCIP), menadione, 1,2-naphthoquinone and 2-hydroxy-1, 4-naphthoquinone . These were added from concentrated stock s o l u t i o n s to f i n a l concentrations 38 of 1 CyuM to IOOJUM. To reduce a sample, small a d d i t i o n s of stock s o l u t i o n s of NADH or d i t h i o n i t e were added. To o x i d i s e the sample atmospheric oxygen was introduced or a small volume of a PMS stock solution was added. Reductant stock solutions (approximately 25mM) were always made up in 0.1M T r i s - H C l , pH 8.0. The use of ferricyanide was avoided (27). The remaining 2.5ml' of membrane suspension were oxidised by the addition of f e r r i c y a n i d e and used as reference. The material being t i t r a t e d was scanned against t h i s at a s e r i e s of measured potentials. The wavelength .range covered was 590nm to 530nm scanned at a speed of 60nm/min with a 3nm s l i t w idth. The p o t e n t i a l range of +250mV to -200mV was covered at in t e r v a l s of approximately 15mV. However, i t was sometimes d i f f i c u l t to obtain redox e q u i l i b r a t i o n over the whole range. This was due i n p a r t to the inadequacy of the s t i r r i n g apparatus, which was operated at a l l times except when spectral scans were being performed. Therefore some spectra have been collected when there was a discernable d r i f t i n the redox p o t e n t i a l . The absence of hysteresis between the o x i d a t i v e and reductive t i t r a t i o n s suggested that the components not e q u i l i b r a t i n g r a p i d l y with the electrode were not e q u i l i b r a t i n g with the cytochromes' e i t h e r . Large changes i n the redox p o t e n t i a l upon the a d d i t i o n of r e d u c t a n t or oxidant were combatted by the a d d i t i o n of more mediator of an a p p r o p r i a t e po t e n t i a l . At the end of a l l t i t r a t i o n s , the redox potental was lowered to about -300mV and the difference spectrum scanned. This ensured that no cytochrome of very low p o t e n t i a l had been overlooked and that cytochromes had not been degraded d u r i n g the lengthy t i t r a t i o n procedure. 39 Analysis of T i t r a t i o n Data The amount of cytochrome reduced at a g i v e n p o t e n t i a l was estimated by measuring the height of the -peak above a l i n e drawn tangentally to the troughs on e i t h e r side of the peak in the spectrum. This method i s equivalent to the "three point f i t " of Hendler and Shrager (27). The data were then f u r t h e r analysed using two computer programs. The f i r s t (BMDP:3R) was a non-linear least-squares program (90) supported by the U.B.C. computing centre. This,program optimises the parameters i n a f u n c t i o n s p e c i f i e d by the user to give the best f i t of the experimental data. The f u n c t i o n and i t s derivatives with respect to each of the parameters are s p e c i f i e d i n a separate FORTRAN subroutine. The general form of the function i s : PJJJ 1_ PJ3) -y = 1 + exp((x-P(2))/25.6) + 1 + exp((x-P(4))/25.6) where y i s the -peak height, x i s the solution redox potential, P(D and P(2) are-the -peak height and mid-point p o t e n t i a l of the f i r s t components, P(3) and P(4) are the same parameters for the second component. Four subroutines were w r i t t e n describing the equation with 1, 2, 3 or 4 components each t r a n s f e r r i n g one electron. Therefore, any set of data could be f i t t e d by any number of cytochromes up to four. The subroutine (F4) used i n four component f i t s i s given i n appendix I. The program BMDP:3R was run i n batch mode using a control program i 40 RUNPRO which i s l i s t e d i n Appendix- I I . F i t s were evaluated both from the output of BMDP:3R and by plotting them. The p l o t t i n g was performed by a BASIC program called FAKDAT which was adapted by Lome Reid to plot the results of t i t r a t i o n experiments. This program uses as input the experimental data and the f i t calculated by BMDP:3R. The output i s a p l o t of the f i t with the experimental points i n d i c a t e d . A sample conversation showing the running of th i s program i s given i n Appendix I I I . K i n e t i c Measurements the kinetics of cytochrome reduction and oxidation were measured in a Perkin-Elmer 356 spectrophotometer i n the dual-wavelength mode. 1ml of a concentrated stock suspension of membranes was diluted to a volume of 2ml or 3ml im m e d i a t e l y b e f o r e b e g i n n i n g a k i n e t i c measurement. Small volumes of c o n c e n t r a t e d s t o c k s o l u t i o n s of reductants or oxidants were added using a plumper. Control experiments in which an equal volume of buffer was added were also performed. These allowed compensation for s h i f t s i n the baseline due to d i l u t i o n and allowed the e f f e c t s of oxygen to be d i s t i n g u i s h e d from those of other oxidants. When NADH was used as reductant appropriate controls were necessary to compensate f o r the absorbance of the substrate i t s e l f . Fractionation of Aerobic Cytochromes by Gel F i l t r a t i o n The procedure used was based on that of K i t a et a l . (36). 41 Membranes (1000mg p r o t e i n ) were prepa r e d as described above and suspended i n 25ml of 3mM Na^EDTA, pH 7-2. To t h i s was slowly added 18.75ml of 10% (w/v) Sarkosyl (3% f i n a l concentration), 6.25ml of 20% (w/v) cholate (2% f i n a l c o n c e n t r a t i o n ) and 12.5ml of saturated ammonium sulphate, pH 7.0 (20% f i n a l c o ncentration). The mixture was o s t i r r e d at 4 C for 60min and then c e n t r i f u g e d for 2h at 50,000rpm i n a Beckman Ti60 rotor. The supernatant was d i a l y s e d overnight against 0.05% Sarkosyl (w/v) in 10mM T r i s - H C l , pH 8.0 and then concentrated to a volume of 10ml by u l t r a f i l t r a t i o n i n an Amincon model 402 u l t r a f i l t r a t i o n apparatus with a PM10 membrane. The concentrated material was applied to a column (120 x 2.5cm) of Sephacryl S-300 equilibrated in the d i a l y s i s buffer to which 0.6M NaCl had been added. The column was'run o v e r n i g h t i n the same b u f f e r . The e l u t i o n of cytochromes was determined by measuring the absorbance at 410nm or 412nm of the column f r a c t i o n s . Pooled f r a c t i o n s were concentrated by u l t r a f i l t r a t i o n as above. Subsequently, 1.0M potassium phosphate, buffer, pH 7.0 was added to give a f i n a l concentration of 0.1M before spectral analysis was carried out. Fractionation of Anaerobic Cytochromes The cytochromes of c e l l s grown a n a e r o b i c a l l y with n i t r a t e were f r a c t i o n a t e d by a procedure based on that of Clegg (44). Formate dehydrogenase has been reported to be oxygen sensitive (42) and so a l l buffers were thoroughly degassed before use. C e l l membranes (1000mg protein) were suspended i n 10mM potassium phosphate buffer, pH 7.0, containing 0.2mM DTT to a p r o t e i n c o n c e n t r a t i o n of approximately 42 15mg/ml. To this suspension 20% (v/v) T r i t o n X-100 was slowly added to a f i n a l concentration of 2%. The mixture was s t i r r e d 1 at room temperature for 30min and then c e n t r i f u g e d at 50,000rpm i n a Beckman Ti60 r o t o r . The supernatant was loaded onto a 35 x 1.5cm column of DEAE Bio-Gel A. (DEAE Sephacel has also been used but i t gives an in f e r i o r separation). A f t e r a p p l i c a t i o n , the column was washed with 200ml of the e q u i l i b r a t i o n buffer (10mM potassium phosphate pH 7.0, 0.2mM DTT, 0.156 T r i t o n X-100). Cytochromes were then eluted by a linear gradient of 0 to 0.3M NaCl i n the e q u i l i b r a t i o n buffer (400ml t o t a l volume). 7ml f r a c t i o n s were c o l l e c t e d and t h e i r cytochrome c o n t e n t was e s t i m a t e d from t h e i r a b s orbance at 410nm. When appropriate, fractions were also assayed for formate dehydrogenase and n i t r a t e r e d u c t a s e a c t i v i t i e s . P o o l e d column f r a c t i o n s were concentrated by u l t r a f i l t r a t i o n as described i n the previous section. 1.0M potassium phosphate, pH 7.0 was added to 0.1M f i n a l concentration before spectral analysis was performed. SDS-Polyacrylamide Gel Electrophoresis SDS polyacrylamide gels were run using the discontinuous buffer system of Laemmli (91). E i t h e r l i n e a r or exponential acrylamide gradients of 5% to 22.5% (w/v) were used with 3% (w/v) acrylamide stacking g e l s . Slab gels were run i n a Bio-Rad 220 apparatus using spacers of 0.75mm thi c k n e s s . They were run at 25mA per gel u n t i l the t r a c k i n g dye l e f t the g e l . Gels were stained by the procedure of Fairbanks et a l . (92) and then d e s t a i n e d and f i x e d i n 10% (v/v) ace t i c a c i d . A l l samples were prepared by b o i l i n g for 5min i n the 43 sample buffer of Laemmli (91). S a l t s or detergents were not removed before electrophoresis. Assay of Nitrate, Fumarate and TMAO Reductase A c t i v i t i e s These enzymes were assayed spectrophotometrical-ly using benzyl viologen as electron donor by the method of Jones and Garland (93)r The cuvettes (Hellma, 110-OS, K—181, 10mm) had round t e f l o n stoppers through which a small hole was bored. A small volume of enzyme was placed in the cuvette which was then f i l l e d up to the neck with 0.3mM benzyl viologen i n degassed 50mM T r i s - H C l , pH 8.0. A few small glass beads were added and the cuvette was stoppered taking care to exclude bubbles. Small amounts of fresh 10mM d i t h i o n i t e i n 5mM NaOH were added u n t i l the absorbance at 600nm reached a steady value of approximatly 1.8. Mixing was achieved by i n v e r s i o n of the cuvette. To begin an assay, 25pl of 1.0M fumarate, n i t r a t e or TMAO was added using a Hamilton microsyringe and the o x i d a t i o n of the benzyl viologen radical was monitored at 600nm. The s p e c i f i c a c t i v i t y was calculated using an -1 -1 e x t i n c t i o n c o e f f i c e n t of 7.4 mM cm and a cuvette capacity of 3.7ml. Assay of Oxidase A c t i v i t i e s Oxidase a c t i v i t i e s were measured u s i n g a Y e l l o w S p r i n g s Instruments model 55 oxygen monitor coupled to an Aminco s t r i p chart recorder. The recorder was c a l i b r a t e d so that a f u l l scale deflection represented the oxygen concentration of oxygen saturated buffer. This 44 value was taken to be 0.26umol/ml. A concentrated membrane suspension I (approximately 15mg/ml) was d i l u t e d i n 50mM T r i s - H C l , pH 8.0,^to a volume of 4ml i n the electrode v e s s e l . 0.1ml of substrate was then added from a concentrated stock s o l u t i o n using a Hamilton microsyringe to give a f i n a l concentration of 1mM for NADH and 10mM for formate. Assay of ATPase A c t i v i t y ATPase was assayed by the method described by Davies and Bragg o (94). The assay was performed at 37 C with 5mM ATP, 2.5mM CaCl 2 m Tris-HCl buffer, pH 8.5. The r e a c t i o n was i n i t i a t e d by the addition of a s m a l l volume of enzyme and stopped by the a d d i t i o n of t r i c h l o r o a c e t i c a c i d to a f i n a l c o n c e n t r a t i o n of 5%. After centrifugation to remove denatured' protein the supernatant was assayed for phosphate by the method described by Ames (95). Assay of Formate Dehydrogenase A c t i v i t y Formate d e h y d r o g e n a s e was a s s a y e d by e s t i m a t i n g t h e formate-dependent reduction of DCIP by the method of Ruiz-Herrera et  a l . (55). Whilst the assay was not done under s t r i c t l y anaerobic conditions a l l solutions were degassed before use. The buffer used, in which a l l stock solutions were made up, was 50mM potassium phosphate, pH 7.0. Each assay c o n s i s t e d of 0.1ml of 9.0mM DCIP, 0.1ml of 13.1mM PMS, and buffer and enzyme to a f i n a l volume of 1.4ml. The absorbance of t h i s mixture was monitored at 600nm u n t i l i t reached a steady value of approximatly 1.6. The assay was then i n i t i a t e d by the addition of 45 0.1ml of 34mM formate. The reduction of DCIP was followed at 600nm and the s p e c i f i c a c t i v i t y was calc u l a t e d using an extinction coefficent of 20 . 6 mM~1 cm" 1 . The Assay of. N i t r a t e Reductase A c t i v i t y by Measuring N i t r i t e  Production N i t r i t e was determined by the procedure of Showe and DeMoss (74). The n i t r i t e e stimation reagent was made by mixing two volumes of 4% (w/v) sulphanilamide in 3M HCl with one volume of 0.08% N-napthyl r ethylenediamine i n e t h a n o l . 2.5ml of t h i s was added to 1.0ml of sample. The absorbance at.540nm was measured a f t e r 30min. An absorbance of 1.0 corresponds to 68.3nmol of n i t r i t e i n the sample. N i t r a t e reductase was sometimes e s t i m a t e d from the rate of accumulation of n i t r i t e from n i t r a t e . This assay was done i n a volume of 1ml in a small (13 x 100mm) t e s t tube. The enzyme was preincubated in 0.95ml 50mM Tris-HCl, pH 8.0 with reductant (either 5mM formate or 5mM ascorbate with 50uM PMS) to allow oxygen d e p l e t i o n . To i n i t i a t e the r e a c t i o n 0.05ml of 50mM potassium•nitrate was added and 0.1ml samples were withdrawn at i n t e r v a l s . These were added to 0.9ml of 3M HCl and n i t r i t e was estimated as described above. Measurement of the Capacity of a Culture to Reduce N i t r i t e The a b i l i t y to reduce n i t r i t e 'was t e s t e d by growing c e l l s overnight i n a r i c h medium w i t h n i t r i t e and performing a n i t r i t e determination on the spent medium (80). The medium contained per 46 100ml, 0.4g Bactopeptone, 0.2g of yeast e x t r a c t 0.4g of glucose and 1mM n i t r i t e . After overnight growth, 0.1ml of medium was withdrawn and added d i r e c t l y to 0.9ml of 3M HCl. The presence or absence of n i t r i t e in,-this was determined as described above. Measurement of the Formate Hydrogenlyase A c t i v i t y of Growing Cells  Using Durham Tubes Durham tubes were used to i n v e s t i g a t e the formate hydrogenlyase a c t i v i t y of mutants (78). The medium used contained 0.5g Bactopeptone, 0.3g yeast e x t r a c t and 0.5g glucose per 100ml. A f t e r growth for 24 hours the presence or absence of gas in the colle c t o r tube was noted. P r o t e i n E s t i m a t i o n Protein was estimated by the method of Lowry et a l . (96) using bovine serum albumin as a standard. No c o r r e c t i o n was made for the effect of detergents when present. PI T ransduction P1 transduction was c a r r i e d out e s s e n t i a l l y by the procedure of P r i t t a r d (97). A l l s t r a i n s were grown on L-broth (LB) containing per l i t r e , 10g tryptone, 5g yeast e x t r a c t and 10g NaCl. 2mM CaCl,, was added after autoclaving. 0.5ml of an overnight c u l t u r e of the donor s t r a i n was subcultured i n t o 10ml of LB and grown for 1.25h with shaking. To 1ml of t h i s exponential phase culture was added 0.1ml of a 47 P1 lysate at a m u l t i p l i c i t y of i n f e c t i o n of approximatly 0.01. The o r i g i n a l l y s a t e of P1 was supplied by Pat Dennis (Biochemistry v i r o Department, U.B.C). The mixture was incubated at 37 C for 20min to allow phage adsorption. Then 0.2ml of the mixture was added to 2.5ml of top agar (LB + 0.8% agar) and poured on a fresh LB plate (LB + 1.5% o agar). These were incubated face upwards for 6h at 37 C, or u n t i l a control plate with no added P1 became v i s i b l y turbid. The top agar was then scraped off into a c e n t r i f u g e tube and the surface of the plate was washed twice with 1.5ml of LB i n t o the same tube. 5 drops of chloroform and 1.0M sodium c i t r a t e (to give a f i n a l concentration of 5mM) were added. The tubes were shaken vigourously and l e f t overnight o at 4 C. The c e l l debris was sedimented at the top speed i n an IEC bench top c l i n i c a l c e n t r i f u g e for 10min. The supernatant was stored 7 over chloroform. This procedure gave l y s a t e s with t i t r e s of 10 to 9 10 per ml. They were t i t r a t e d by the procedure described above using s t r a i n W1485E and phage s u i t a b l y d i l u t e d i n LB. The plates were incubated overnight and plaques counted. To transduce a s t r a i n , 0.05ml of an o v e r n i g h t c u l t u r e was subcultured i n t o 10ml of LB and grown for three hours with shaking. The c e l l s were sedimented i n an IEC bench top c l i n i c a l centrifuge run at top speed for 10min. The c e l l s were resuspended i n 1ml of LB and o 50ul of P1 l y s a t e was added. The mixture was incubated at 37 C for 20min and then resedimented as described above. The p e l l e t was washed with 5ml of 0.9% NaCl and f i n a l l y resuspended in a small volume before spreading on selective plates. As a c o n t r o l to give an estimate of the spontaneous mutation frequency, a c u l t u r e of r e c i p i e n t was always carried through the transduction procedure without the addition of P1. 48 RESULTS PART I. THE SIMULTANEOUS PRESENCE OF SEVERAL RESPIRATORY CHAINS The Cytochromes of E . c o l i Studied by Difference Spectroscopy The cytochrome c o n t e n t of w i l d - t y p e E . c o l i i s known to be dependent on the c u l t u r e c o n d i t i o n s ( 13,15,20,28). Many factors such as the terminal e l e c t r o n acceptors present, the phase of growth and the carbon and nitrogen sources-'present were found to a f f e c t the production of cytochrome. Figure 4 presents the d i f f e r e n c e spectra measured at 77K of membrane p r e p a r a t i o n s from E_^  c o l i a f t e r aerobic growth on various media. The cytochrome b and cytochrome d levels are given in Table 2 (p55). At least six different cytochromes were i d e n t i f i e d i n the spectral scans of the 0C- and 2^-band region. The Soret band region was not scanned because of the spectral interference of the oxidant (ferricyanide) at 420nm. The most prominent cytochromes were cytochromes d, a ,-b , and b . In a d d i t i o n cytochromes 1 555 558 b and c could be seen as shoulders on the ^-peak of the major 562 b-type cytochromes. When c e l l s are grown to s t a t i o n a r y phase on a minimal medium the oxygen supply f a l l s (12,13). Figure 4b shows that the membranes from such c e l l s had a prominent amount of the terminal oxidase cytochrome d. This cytochrome always appears to be associated with cytochromes a^ and b,_,_0. These three c o n s t i t u t e the 'low a e r a t i o n ' quinol oxidase 558 \ pathway which i s synthesised i n response to poor aeration (17,18). In the membrane and in some purified preparations they are associated with a cytochrome absorbing at 556nm (12,16). In addition stationary phase 49 Wavelength , nm 49a FIGURE 4. The cytochromes of w i l d - t y p e c e l l s grown a e r o b i c a l l y . Dithionite-reduced minus f e r r i c y a n i d e - o x i d i s e d difference spectra were performed on membrane preparations at 77K. (A), HfrH grown to the stationary phase on a minimal medium with glucose and 0.5% n i t r a t e (<£A=0.06; protein, 3.8mg/ml); (B) , HfrH grown to the stationary phase on a minimal medium with glucose (AA = 0.06; p r o t e i n , 3.2mg/ml); (C), vMR43L grown to the mid-exponential phase on a minimal medium with 0.4% succinate and 0.4% casamino acids (AA=0.06; protein, 5.2mg/ml). , 50 c e l l s appear to synthesise cytochrome c but the absorption at 550nm could be associated with some other cytochrome. Inclusion of n i t r a t e i n the medium decreases the amount of the cytochrome d pathway present at any phase of aerobic growth (Figure 4a and 4b). The l e v e l of cytochrome d was decreased seven-fold by the a d d i t i o n of n i t r a t e w h i l s t the apparent cytochrome b l e v e l was increased from 0.36nmol/mg pr o t e i n to 0.54nmol/mg protein. There was also a qualitative change i n the production of b-type cytochromes. A cytochrome b became r e l a t i v e l y more prominent than cytochrome 556 n r b . This may r e f l e c t the p r o d u c t i o n of cytochrome b which i s 558 \ known to absorb at 556nm (15,53). Cells grown a e r o b i c a l l y on an amino-acid supplemented minimal medium with a non-fermentable carbon source showed yet another pattern of cytochrome p r o d u c t i o n . T h e i r l e v e l of cytochromes d, a^, and b was quite low (Figure 4c); They had a markedly asymmetricoc-peak 558 of b-cytochrome with a maximum at 555nm and a d e f i n i t e shoulder at 562nm. This r e f l e c t e d the presence of the cytochrome o or 'high a e r a t i o n ' q u i n o l o x i d a s e pathway which i s known to c o n s t i t u t e cytochromes b b and o. This i s c h a r a c t e r i s t i c of vigorously 556 562 aerated c e l l s of the e a r l y exponential phase, and of c e l l s grown in supplemented media (20). The l e v e l of cytochromes in these c e l l s was found to be quite low, t y p i c a l l y 0.21nmol/mg protein. The cytochrome content of c e l l s grown anaerobically was also found to be dependent on c u l t u r e c o n d i t i o n s . The d i f f e r e n c e spectra of membranes from wild-type c e l l s grown a n a e r o b i c a l l y with various terminal electron acceptors are shown i n Figure 5 and the cytochrome le v e l s are given i n Table 2. The basic medium was a minimal medium supplemented with peptone and using glucose as carbon source. This / 51a FIGURE 5. Difference s p e c t r a of the membranes of E_^  c o l i grown a n a e r o b i c a l l y w i t h v a r i o u s t e r m i n a l e l e c t r o n a c c e p t o r s . Dithionite-reduced minus f e r r i c y a n i d e - o x i d i s e d difference spectra were recorded at 77K. S t r a i n HfrH was grown on a minimal medium with glucose and peptone to the s t a t i o n a r y phase. The electron acceptors were: (A) 0.5% fumarate G&A=0.03; protein', 8.2mg/ml); (B), 0.5% nitrate (AA=0.1; p r o t e i n , 6.1mg/ml); (C), 0.25% n i t r i t e (AA=0.03; protein, 3.3mg/ml); (D), 0.4% TMAO (4A=0.03; protein, 8.8mg/ml). 52 w i l l support anaerobic growth i n the absence of any added terminal electron acceptor. Cells grown with no terminal e l e c t r o n acceptor, and those grown with fumarate, gave s i m i l a r patterns of cytochrome production (Figure 5a) . They had a conspicuous c o n t e n t of the cytochrome d pathway including cytochrome b The spectrum was quite similar to that of 556 c e l l s grown aerobically to the s t a t i o n a r y phase on a defined medium (Figure 4b). However, the t o t a l l e v e l of cytochrome was 2.5-fold lower suggesting an i n d u c t i v e e f f e c t of oxygen on the production of cytochromes. Fumarate a p p a r e n t l y has no e f f e c t on cytochrome production under these growth conditions. The presence of n i t r a t e as t e r m i n a l e l e c t r o n acceptor had a s t r i k i n g effect on cytochrome production (Figure 5b, 53). Cytochrome d was barely detectable i n the membranes of c e l l s grown anaerobically with nitrate whilst they had larg e amounts b-type cytochromes with fdh an absorption maximum at 555. 5nm. These are ,cytochromes b and nr b . The cytochrome b l e v e l was 0.54nmol/mg p r o t e i n , 2.5 times higher than i n c e l l s grown a n a e r o b i c a l l y with no added terminal electron acceptor. The e f f e c t of n i t r a t e on cytochrome production was similar to that found i n c e l l s grown a e r o b i c a l l y but was even more pronounced. Therefore i t must be independent of the presence of oxygen. This suggested a r e g u l a t o r y f u n c t i o n for n i t r a t e or one of i t s metabolites upon cytochrome production. To investigate t h i s further c e l l s were grown anaerobically with n i t r i t e , the immediate product of ni t r a t e reduction, and their cytochromes studied (Figure 5c). N i t r i t e , l i k e n i t r a t e caused a severe repression of the formation of cytochrome d. It also gave a sharply defined cytochrome b PC-peak but t h i s was 53 c e n t e r e d at a s l i g h t l y lower wavelength (555nm) than i n the nitrate-grown c e l l s . N i t r i t e - g r o w n c e l l s had a n o t i c e a b l y higher content of cytochrome c than d i d n i t r a t e - g r o w n c e l l s . This i s consistent with the r o l e of such cytochromes i n n i t r i t e reduction (11,35). It should be noted that the concentration of n i t r i t e used to grow c e l l s was h a l f the c o n c e n t r a t i o n of n i t r a t e used. Higher concentrations of n i t r i t e were found to i n h i b i t c e l l growth. This may be s i g n i f i c a n t i n studying the r e g u l a t i o n of cytochrome formation since the l e v e l of a l l cytochromes i s markedly a f f e c t e d by the concentration of nitra t e in the growth medium (98). Trimethylamine N-oxide (TMAO) i s another p o t e n t i a l terminal electron acceptor (38,39). I n c l u s i o n of t h i s compound i n the standard growth medium led to the production of a large number of cytochromes (Figure 5d). The major species absorbed at 552nm and 548 nm and have been shown to be of the c-type (39). There was also the production of b-type cytochromes and the shape o f ^ t h e Qi-peak suggested that cytochromes. b , b,.,.,, and b were a l l produced. The presence 555 558 • 562 of cytochromes a^ and d i s c o n s i s t e n t with the presence of cytochrome b . While oxygen and n i t r a t e increased the t o t a l l e v e l of b-type 558 cytochrome TMAO did not have t h i s e f f e c t (Table 2). On the other hand i t s q ualitative effect on cytochrome production was quite s t r i k i n g . The pattern of cytochromes found i n the membranes of c e l l s grown under any of the c o n d i t i o n s studied was very reproducible provided that the growth c o n d i t i o n s were p r e c i s e l y c o n t r o l l e d . However, the values quoted for the amount of cytochrome were^less reproducible due in part to the large v a r i a t i o n i n cytochrome l e v e l with growth phase (30). The procedure used f o r the pre p a r a t i o n of membranes gave a 54 variable content of outer membrane causing futher i r r e p r o d u c i b i l i t y . This source of error could be a l l e v i a t e d by performing spectra on whole c e l l s but with that approach i t may prove d i f f i c u l t to obtain a f u l l y o x i d i s e d sample. I t i s known to' take a lengthy period of starvation to completely deplete a c e l l suspension of a l l endogenous substrate (99). The^apparent cytochrome content i s also dependent on the s t r a i n b e i n g s t u d i e d . For i n s t a n c e , HfrH t y p i c a l l y gave a cytochrome l e v e l of 0.52nmol/mg p r o t e i n when grown anaerobically with n i t r a t e while PK27 gave levels of 0.77nmol/mg protein.' The Dependence of Respiratory A c t i v i t i e s on Growth Conditions The d i f f e r e n c e s p e c t r a suggested the presence of the same respiratory pathways, such as the cytochrome d pathway, under nearly a l l growth conditions. This implied that many respiratory a c t i v i t i e s might also be produced i r r e s p e c t i v e of the growth conditions, since the cytochrome content must r e f l e c t the respiratory pathways present. Table 2 shows that several r e s p i r a t o r y a c t i v i t i e s were present at some le v e l regardless of the c o n d i t i o n s used for growth. The values quoted are the averages of at l e a s t two determinations, but the v a r i a b i l i t y was quite large for the reasons outlined above. The formate and NADH oxidase a c t i v i t i e s of membrane preparations were c o n s i s t e n t l y higher when, they were derived from c e l l s grown aerobically. Cells grown a n a e r o b i c a l l y r e t a i n about one f i f t h of the a c t i v i t y . In the cases of c e l l s grown a n a e r o b i c a l l y with n i t r a t e and c e l l s grown a e r o b i c a l l y without n i t r a t e the oxidase a c t i v i t i e s were shown to be a d d i t i v e s u g g e s t i n g t h a t the formate and NADH TABLE 2 THE DEPENDENCE OF CYTOCHROME CONTENT AND RESPIRATORY ACTIVITY OF WILD-TYPE E. COLI ON CULTURE CONDITIONS Growth Conditions Cytochrome Content Respiratory Activity Carbon Electron Cytochrome^ 2 Cytochrome d NADH3 Formate 3 X T ' . . 4 Nitrate 4 Fumarate 4 TMAO ATPa: Source Acceptor b Cytochrome b oxidase oxidase reductase reductase reductase Glucose °2 0.39 0.30 590 420 80 nd5 nd 180 Glucose °2 + N°3 0.53 0.03 560 610 880 nd nd nd Glucose + None 0.20 0.21 34 107 340 500 nd 200 Peptone II Fumarate 0. 15 0.17 164 58 190 670 60 220 II TMAO 0. 18 0.07 53 140 60 400 1560 nd tt Nitrate 0.59 0.01 209 134 560 10 30 nd Glucose Nitrate 0.75 0.01 50 125 1020 10 nd 150 4 1. nmol/mg protein 2. nmol cytochrome d/nmol cytochrome b 3. ngatoms oxygen/min/mg protein 4. nmol substrate/min/mg protein 5. nd - not determined 56 dehydrogenase a c t i v i t i e s were r a t e - l i m i t i n g rather than the oxidase a c t i v i t y . The retention of- s u b s t a n t i a l oxidase a c t i v i t y by c e l l s grown anaerobically may be p a r t i a l l y due to the presence of traces of oxygen during growth. In a d d i t i o n some oxygen would be introduced during harvesting and washing of c e l l s , although this, process was carried out at low temperature to minimize metabolic a c t i v i t y at t h i s stage. It was noticed that the i n t e r m i t t e n t s t i r r i n g of the standing culture during .growth led to higher oxidase a c t i v i t i e s than i n an unstirred control. Just as c e l l s grown a n a e r o b i c a l l y have a c t i v i t i e s c h a r a c t e r i s t i c , of c e l l s grown aerobically so the opposite i s true. Nitrate reductase i s an a c t i v i t y t y p i c a l of c e l l s grown anaerobically, especially i n the presence of n i t r a t e ( 7 4 ) . I t was a l s o found i n c e l l s grown a e r o b i c a l l y , and i n p a r t i c u l a r when n i t r a t e was included i n the aerobic growth medium and the c e l l s were grown to the stationary phase when the oxygen le v e l in the medium f a l l s (Table 2). In one experiment the s p e c i f i c a c t i v i t y of n i t r a t e reductase was observed to increase from 64 to 520 nmol/min/mg p r o t e i n between the mid-exponential and stationary phases of aerobic growth. The s p e c i f i c a c t i v i t i e s reported here are perhaps greater than those reported elsewhere (74) due to the inclusion of s e l e n i t e and molybdate i n the standard growth medium. These are required components f o r the c o f a c t o r s i n v o l v e d i n the t formate-dependent reduction of n i t r a t e . Nitrate reductase was found i n large amounts i n the membranes of a l l c e l l s grown anaerobically. I t was futher induced by nit r a t e whilst TMAO and fumarate had a re p r e s s i v e e f f e c t . S i m i l a r l y , TMAO reductase was found in a l l c e l l s grown a n a e r o b i c a l l y but was induced by TMAO and 1 57 represed by n i t r a t e . Fumarate reductase seemed to be under a similar type of control. The n i t r a t e reductase a c t i v i t y was a l s o dependent on other factors. Table 2 shows that the e x c l u s i o n of peptone from the standard anaerobic growth medium led to a s u b s t a n t i a l increase in the spe c i f i c a c t i v i t y of t h i s enzyme. In a d d i t i o n the cytochrome b l e v e l was increased. Further experiments were performed in which the ammonia of the medium was omitted or 0.1 mM azide was added (75). Both of these lead to a yet higher n i t r a t e r e d u c t a s e s p e c i f i c a c t i v i t y of 2000 nmol/min/mg protein and a corresponding increase i n the t o t a l l e v e l of b-type cytochrome. C l e a r l y there are s e v e r a l f a c t o r s a f f e c t i n g the synthesis of nitra t e reductase. This might be expected i n view of i t s potential dual role in energy transduction and nitrogen metabolism. F i n a l l y , a limited number of experiments have suggested that the a c t i v i t y of the proton translocating ATPase i s r e l a t i v e l y independent of the growth conditions (Table 2) . The Analysis of Cytochromes by Spectrophotometries Redox Ti t r a t i o n An a l t e r n a t i v e method of attempting to analyse cytochromes i s that of spectrophotometric redox t i t r a t i o n (25). This technique i s capable of d i s t i n g u i s h i n g two cytochromes of i d e n t i c a l absorption properties and was considered appropriate to apply to the membranes of c e l l s grown anaerobically with n i t r a t e . These c e l l s have a sharp and narrow b-cytochrome od-peak'suggestive of a simple p a t t e r n of cytochrome production. In a d d i t i o n published data suggested that such c e l l s contain only two major cytochromes (53). Since previous redox / 58 t i t r a t i o n studies have emphasized the cytochromes of c e l l s grown a e r o b i c a l l y , from both the s t a t i o n a r y and exponential (26,27,28) phases of growth, i t was decided to study these simultaneously. It was hoped that the r e s u l t s from aerobic c e l l s would confirm the v a l i d i t y of the results found for c e l l s grown a n a e r o b i c a l l y with n i t r a t e . In addition the cytochromes of c e l l s grown anaerobically with TMAO have barely been studied (39.40) and so these were also investigated. To v e r i f y the p r o c e d u r e b e i n g used and to c o n f i r m the standardization of the reference electrode, a spectrophotometric redox t i t r a t i o n of cytochrome c was performed with the r e s u l t given in Figure 6. The data obtained are f i t by a t h e o r e t i c a l curve describing the redox behaviour of a s i n g l e component transferring one electon. However there i s some d e v i a t i o n of the data from the t h e o r e t i c a l curve. This i s a t t r i b u t e d to the procedure by which the amount of cytochrome reduced was determined and the lack of complete redox e q u i l i b r a t i o n . This d e v i a t i o n from i d e a l behaviour has als o been observed in redox t i t r a t i o n s of cytochrome c i n an electrodic system (100). The apparent mid-point potential was +76mV. Since the mid-point potential of cytochrome c i s known to be +260mV-(101) th i s means that the electrode used had a p o t e n t i a l of -183mV r e l a t i v e to the standard hydrogen electrode. This agreed with the r e s u l t found by c a l i b r a t i n g the electrode with mixtures of ferrous and f e r r i c EDTA and checking i t against a series of cross-checked saturated calomel electrodes. This corection factor was applied i n a l l subsequent t i t r a t i o n s . ^ Figure 7 shows the results of spectrophotometric redox t i t r a t i o n s of the membranes of c e l l s grown under the conditions described above. Whilst cytochrome c t i t r a t e d from 90% reduced to 90% oxidised over a range of 120mV, membranes t i t r a t e d over the range +250mV to -150mV, 50 0 +200 Measured Redox Potential 5 9 a FIGURE 6. Spectrophotometry redox t i t r a t i o n of cytochrome c. 10mg of horse heart cytochrome c was d i s s o l v e d i n 20ml of 0.1M potassium phosphate b u f f e r , pH 7.0. Phenazine methosulphate (to 50uM f i n a l concentration), phenazine ethosulphate (50^iM) and 2,6-dichlorophenol indophenol (5uM) were added. The cytochrome was t i t r a t e d as described i n ' M a t e r i a l s and Methods' u s i n g d i t h i o n i t e as r e d u c t a n t and f e r r i c y a n i d e as o x i d a n t . The t r i a n g l e s are from the reductive t i t r a t i o n and the c i r c l e s are from the oxidative t i t r a t i o n . The curve i s the best f i t f o r a s i n g l e component of uncorrected mid-point potential +76.5mV with an absorbance peak of 0.243. 60 60a FIGURE 7. Redox t i t r a t i o n s of the b-type cytochromes of wild-type E . c o l i grown under v a r i o u s c o n d i t i o n s . Membranes were used for t i t r a t i o n as described in 'Materials and Methods'. The s o l i d l i n e s are theoretical curves describing the behaviour of four cytochromes giving the best f i t of the experimental data (see Table 3). (a), MR43L grown aerobically to the exponential phase on a minimal medium with 0.4% succinate and 0.4% casamino acids (AA=0.036; protein, I6.4mg/ml); (b), RK7 grown a e r o b i c a l l y on a minimal medium w i t h glucose to the s t a t i o n a r y phase (AA=0.036; p r o t e i n , 13.0mg/ml); ( c ) , HfrH grown anaerobically with glucose, peptone and n i t r a t e (AA = 0.063; protein, 7.4mg/ml) ; -(d) , NH30 grown a n a e r o b i c a l l y with glucose, peptone and 0.4% TMAO (AA=0.018; protein, 10.6mg/ml). 61 Suggesting that there were several species of cytochrome present. This i s true of the membranes of c e l l s grown e i t h e r a e r o b i c a l l y or an a e r o b i c a l l y . However there are c l e a r q u a l i t a t i v e d i f f e r e n c e s in cytochrome content between the two types of c e l l s . A l l t i t r a t i o n data were plotted on e x a c t l y the same axes so that the r e s u l t s could be compared by superimposition. The cytochromes of c e l l s grown a e r o b i c a l l y to the exponential phase on a semi-defined medium were shown to be quite d i f f e r e n t from those of c e l l s grown to s t a t i o n a r y phase on a f u l l y defined medium (Figures 7a and 7b). The e x p o n e n t i a l phase c e l l s have almost no cytochrome of p o t e n t i a l l e s s than OmV w h i l s t having a very large content of cytochrome with a potential approximately 50mV. This type of cytochrome i s much less evident i n c e l l s from stationary phase. Cells grown, under both conditions have an appreciable content of cytochrome with potential approximately +200mV. C e l l s grown a e r o b i c a l l y have r a t h e r l e s s cytochrome of low p o t e n t i a l ( < OmV) than do those grown a n a e r o b i c a l l y with either nitra t e or TMAO (Figure 7) . C e l l s grown a n a e r o b i c a l l y on TMAO have conspicuous amounts of a very low p o t e n t i a l cytochrome (E^ = -180mV) which i s not found i n the membranes of c e l l s grown under any other conditions (Figure 7d). There are notable differences between strains. As mentioned above HfrH and PK27 have quite d i f f e r e n t t o t a l amounts of cytochrome after anaerobic growth with n i t r a t e . Figure 8 shows that PK27 has more cytochrome, of high potential ( > OmV) than HfrH grown under the same conditions. In addition the s p e c i f i c a c t i v i t y of nitrate reductase i s t h r e e - f o l d h i g h e r i n PK27. T h i s emphasizes the n e c e s s i t y for 62 ) 62a FIGURE 8. Redox t i t r a t i o n c urves of the b-type cytochromes of membrane preparations of the wild-type s t r a i n s PK27 and HfrH grown a n a e r o b i c a l l y with n i t r a t e . For PK27 A A was 0.060 at a p r o t e i n concentration of 17.2mg/ml. For HfrH A A was 0.032 at a p r o t e i n concentration of 13.0mg/ml. 63 performing a l l analyses on the same s t r a i n or at l e a s t on sets of mutants which are otherwise isogenic. A dramatic example of the s t r a i n dependence of cytochrome production i s shown in Figure 9 . The two s t r a i n s analysed are p a r t i a l d i p l o i d s . KL718 contains an F 1 f a c t o r (F152) which i s reported to include the structural genes f o r the aerobic cytochromes (102). KL702 contains an episome (F12) c o v e r i n g the t r p operon and n i t r a t e reductase structural gene. A f t e r anaerobic growth with nitrate KL718 was found to have a prominent c o n t e n t of cytochrome b,_r . a 5 0 2 cytochrome characteristic of aerobic c e l l s from exponential phase. In a d d i t i o n KL718 shows the p r o d u c t i o n of a pigment with a broad absorption band around 670nm. This i s c h a r a c t e r i s t i c of some strains when grown anaerobically on certain batches of potassium n i t r a t e . KL702 however has a spectrum t y p i c a l of the wild-type grown anaerobically with n i t r a t e . The t i t r a t i o n of KL718 shows more cytochrome of potential approximately +100mV than was found for KL702 whilst i t has less cytochrome of po t e n t i a l approximately -100mV (Figure 9). In fact the t i t r a t i o n data for KL718 resemble the data obtained from the wild-type a f t e r aerobic growth r a t h e r than anaerobic growth with n i t r a t e . Presumably t h i s r e f l e c t s a gene dosage effect of the aerobic cytochrome genes which cannot b e . f u l l y compensated by the usual regulatory mechanisms. The redox t i t r a t i o n data for the cytochromes of a l l membrane preparations suggested the presence of s e v e r a l cytochromes, and the mid-point potential of these was not immediately obvious from the data. To resolve the t i t r a t i o n data i n t o i n d i v i d u a l components a non-linear least squares program was used as described in 'Materials 64 6 4 a FIGURE 9. Membrane cytochromes of two p a r t i a l d i p l o i d strains grown a n a e r o b i c a l l y with n i t r a t e . The upper h a l f of the f i g u r e shows di t h i o n i t e - r e d u c e d minus f e r r i c y a n i d e - o x i d i s e d d i f f e r e n c e spectra recorded at 77K. For KL718AA was 0.015 at a protein concentration of 9.3mg/ml. For KL702 Ak was 0.015 at a p r o t e i n concentration of 6.6mg/ml. The lower s e c t i o n of the f i g u r e shows redox t i t r a t i o n s of the b-type cytochromes of the same pre p a r a t i o n s . For KL718AA was 0.019 at 10.0mg p r o t e i n / m l and f o r KL702 AA was 0.012 at 9»9mg protein/ml. \ • \ ) 65 and Methods'. The program was tested using i d e a l data generated by using the Nernst equation and found to a c c u r a t e l y f i t the correct parameters. F i t s were found to be quite sensitive to erroneous points. These could r e s u l t , for i n s t a n c e , from not leaving sufficent time for equilibration before performing a s p e c t r a l scan a f t e r a l t e r i n g the redox potent i a l . F i t s were performed only when a l l the data obtained c l e a r l y f e l l on a smooth c u r v e . Moreover t h i s procedure cannot r e l i a b l y i d e n t i f y minor components c o n s t i t u t i n g l e s s than 5% of the t o t a l amount of cytochrome nor d i s t i n g u i s h two cytochromes of mid-point potentials within 50mV of each other. Examples of three- and four-component f i t s are shown i n Figure 10. The data comes from t i t r a t i o n s of the membranes of c e l l s grown a e r o b i c a l l y to the s t a t i o n a r y phase or grown a n a e r o b i c a l l y with n i t r a t e . In both cases the four-component analysis gives a better f i t of the e x p e r i m e n t a l d a t a . This i s p a r t i c u l a r l y evident i n the h i g h - p o t e n t i a l p o r t i o n of the c u r v e s . T h i s r e s u l t cannot be i n t e r p r e t e d to mean that the c e l l s grown under e i t h e r c o n d i t i o n possess only four cytochromes. Rather i t indicates the minimum number of cytochromes l i k e l y to be present. The mid-point potentials and r e l a t i v e amounts of the cytochromes indicated by the f i t t i n g procedure f o r the data i n Figures 7 and 10 are given in Table 3 where they are compared with previously published r e s u l t s . The components resolved by the three component f i t for the data from aerobically grown exponential phase c e l l s are very similar to those reported by Hendler and Shrager (27). This suggests that their data, obtained by a quite d i f f e r e n t technique from that used here are the same, although the i n t e r p r e t a t i o n that this implies the 66 66a FIGURE 10. Comparison of thr e e - and four-component best f i t s of redox t i t r a t i o n data. Curves 1 and 2 are f i t s of the data shown i n Figure 7b (AA=0.020) and ^ curves 3 and 4 are f i t s of the data_ shown i n Figure 7c (AA=0.037). Curves 1 and 3 are three-component f i t s whilst curves 2 and 4 are four-component f i t s . The redox p o t e n t i a l of the components are indicated at the relevant p o s i t i o n s . The calculated f i t s are given in Table 3. , ^ V TABLE 3 COMPARISON OF RESOLVED REDOX TITRATION DATA WITH RESULTS PREVIOUSLY PUBLISHED Growth Conditions Cytochromes Resolved Authors (Reference) E l e c t r o n Phase of''' 9 Acceptor Growth E (%) E (%) E (%) E (%) m m m m Pudek and Bragg (26) Oxygen S 196 (60) 34 (40) -Reid and Ingledew (28) Oxygen E 260 80 -50 II N i t r a t e E 250 140 10 Hendler and Shrager (27) Oxygen E 186 (24) 57 (60) -105 (16) Present Study Oxygen S 182 (43) 45 (35) -122 (22) II N i t r a t e S or E 166 (26) 16 (40) -104 (34) . Oxygen S 190 (39) 74 (27) -2 (16) -136 (19) N i t r a t e S or E 197 (15) 99 (19) -3 (36) -113 (30) Oxygen -E 195 (32) 69 (60) -100 (8) TMAO S 195 (16) 70 (35) -54 (28) -182 (21) 1. S - s t a t i o n a r y , E - exponential., 2. E (%). Mid-point p o t e n t i a l ( i n mV) and r e l a t i v e amount of resolved species. 68 presence of only three cytochromes i s questioned. However the present data d i f f e r somewhat from those of Pudek and Bragg (26) and quite considerably from those of Reid and Ingledew (28). The t i t r a t i o n results were very reproducible as shown in Table 4. Tit r a t i o n experiments were performed over a time period of more than one year on three separate -membrane preparations of HfrH after aerobic growth to the ~ stationary phase on a minimal medium. The three component f i t s for these agree quite c l o s e l y . In addition t r i p l i c a t e t i t r a t i o n s were performed on the same membrane preparation which was derived from RK7 after aerobic growth as described for HfrH. The f i t s for these three t i t r a t i o n s agree extremely closely. To give an idea of the error i n the values quoted i n t h i s thesis for mid-point potentials, the mean and standard deviation of the parameters has been calculated. The t i t r a t i o n s are c l e a r l y quite reproducible but t h i s was true only i f growth conditions were standardised and only for a given s t r a i n . There are some d i f f i c u l t i e s associated with the chemical t i t r a t i o n procedure u t i l i z e d i n t h i s study (100). Therefore the results found were confirmed using a t h i n l a y e r e l e c t r o d i c t i t r a t i o n system (103). Figure 11 shows the r e s u l t of one such t i t r a t i o n . In t h i s case only s i x s p e c t r a l scans were performed at w i d e l y spaced i n t e r v a l s of po t e n t i a l . A l l of the" b-type cytochrome t i t r a t e d between -100mV and +250mV. Cytochrome d by c o n t r a s t t i t r a t e d i n the range +150mV to +350mV in agreement with previous r e s u l t s (26,28). In this particular t i t r a t i o n the Soret band could a l s o be studied since the mediators used did not absorb in the v i s i b l e region. The peak at 428nm seemed to be associated with the reduced form of the b-type cytochromes since i t was completely absent when the redox p o t e n t i a l was greater than +250mV TABLE 4 THE REPRODUCIBILITY OF TITRATION DATA 1 Experiment S t r a i n E (mV) m Components Resolved E (mV) % m E (mV) % m A B C DI D2 D3 HfrH RK7 180 169 173 184 178 173 48 44 5,1 42 43 44 48 39 42 51 47 48 28 30 31 35 36 36 •127 •128 •130 •119 •118 •119 23 26 18 23 21 20 Mean (Standard Deviation) 176 (5.0) 45 (3.1) 46 (4.1) 32 (3.1) -124 (4.9) 22 (2. 1. The s t r a i n s i n d i c a t e d were grown a e r o b i c a l l y on a minimal medium to the s t a t i o n a r y phase.Membranes were prepared and t i t r a t e d as de s c r i b e d i n ' M a t e r i a l s and Methods'. 4 0 0 5 0 0 6 0 0 7 0 0 Wavelength , nm 70a FIGURE 11. Spectroeletrochemical t i t r a t i o n of solub i l i s e d cytochromes d and b. S t r a i n LCB68 was grown a n a e r o b i c a l l y with n i t r a t e and membranes were prepared. These were s o l u b i l i s e d and subjected to gel f i l t r a t i o n by the procedure of K i t a et a l • (36). To the pooled, concentrated fractions containing cytochrome, ruthenium hexammine and diaminodurene were added to f i n a l concentrations of 50uM each. The material was then subjected to spectrophotometric redox t i t r a t i o n with an o p t i c a l l y transparent t h i n - l a y e r e l e c t r o d e . The system used (103) had a 0.2cm l i g h t path and used a gold f o i l working e l e c t r o d e , a platinum counter electrode and a Bioanalytical Systems RE-2 saturated calomel reference e l e c t r o d e . A Princeton Applied Research Model 173 potentiostat was used to impose the desired p o t e n t i a l on the sample. Temperature was monitored w i t h a subminiature copper-constantan thermocouple and a Fluke Model 2175A d i g i t a l thermometer. At least 20min was l e f t between a l t e r i n g the imposed p o t e n t i a l and performing spectral scans. The of- and J% -band region (AA = 0.006) and S o r e t band region (AA=0.03) were scanned at a s e r i e s of solution potentials: (a), +600mV; (b), +350mV; ( c ) , +250mV; ( d ) , +100mV; ( e ) , -100mV; ( f ) , -350mV. 71 (Figure 11 a, b, and c) . The peak at 414nm appeared to be associated with the oxidised forms of both cytochromes b and d since i t t i t r a t e d over the whole range covered by these two cytochromes, that i s -100mV to +250mV. The same preparation was subjected to a chemical t i t r a t i o n and showed no b-type cytochrome t i t r a t i n g outside the range -100mV to +250mV. Therefore the e l e c t r o d i c t i t r a t i o n method was taken to confirm the v a l i d i t y of the chemical method. Neither method showed the presence of a b-type cytochrome of p o t e n t i a l +250mV that has been reported by Reid and Ingledew (28). I t i s a p r e d i c t i o n of the chemiosmotic hypothesis that the energi s a t i o n of the membrane should change the apparent mid-point potential of those cytochromes involved i n energy transduction (104). Membranes of c e l l s grown a e r o b i c a l l y were t i t r a t e d in the presence of either 5QpM CCCP, which would cause membrane de-energisation, or 0.1mM DCCD, which would enhance membrane e n e r g i s a t i o n . In each case the t i t r a t i o n data were i d e n t i c a l to those obtained i n a control, t i t r a t i o n with no additions. Presumably a l l these t i t r a t i o n s r e f l e c t the redox potential of cytochromes i n the de-energised membrane. I t i s d i f f i c u l t to v i s u a l i z e how a t i t r a t i o n of energised E^ c o l i membranes could be achieved since extremely l a r g e amounts of ATP would be required to energise the membrane for the amount of time r e q u i r e d . In addition many of the quinone s p e c i e s used as m e d i a t o r s are known to be li p i d - s o l u b l e and therefore are l i k e l y to have a de-energising effect on the membrane. , An alternative way of de-energising E. c o l i membranes involves making them freely permeable to protons by washing them i n buffer of low ionic strength. When membranes washed in 2mM Tris-HCl buffer of pH 72 8.0, were t i t r a t e d t h e r e was an extreme h y s t e r e s i s between the oxidative and reductive t i t r a t i o n s , and smooth t i t r a t i o n curves could not be obtained. I t may be s i g n i f i c a n t that Ried and Ingledew (28) used such a washing step before some of t h e i r t i t r a t i o n s i n order to remove a cytochrome of low p o t e n t i a l . For membranes prepared as described i n th i s study there was no cytochrome removed by washing at low ionic'strength. The chemiosmotic hypothesis a l s o p r e d i c t s that a cytochrome involved i n energy transduction should have a pH dependent mid-point potential (104). The mid-point p o t e n t i a l should drop by 60mV per pH u n i t . T h e r e f o r e t i t r a t i o n s of the membranes of c o l i a f t e r anaerobic growth with nitrate were performed at a series of pH values. The r e s u l t s are summarised i n Table 5. The p o t e n t i a l of a l l of the cytochromes i n the membrane f e l l w i t h i n c r e a s i n g pH. However the change was much smaller than 60mV per pH unit . Studies of Cytochrome o and Cytochrome d Content Using Carbon  Monoxide Difference Spectroscopy The t i t r a t i o n data in conjunction with the assays of respiratory a c t i v i t y and difference spectra i n d i c a t e d a considerably more complex pattern of cytochrome production than i s g e n e r a l l y r e a l i s e d . The r e s o l u t i o n of i n d i v i d u a l cytochromes from the mixture c a l l e d for a l t e r n a t i v e approaches a l l o w i n g the i d e n t i f i c a t i o n of i n d i v i d u a l cytochromes. Carbon monoxide d i f f e r e n c e spectroscopy was considered to be a us e f u l appproach. I t w i l l i d e n t i f y the two cytochrome oxidases, TABLE 5 pH DEPENDENCE OF MID-POINT POTENTIALS OF CYTOCHROMES OF CELLS GROWN ANAEROBICALLY WITH NITRATE o f T i t r a t i o n B u f f e r Cytochromes R e s o l v e d E (mV) % o f t o t a l E (mV) % o f t o t a l E (mV) % o f t o t a l m m m 5.6 MES 1 201 28 55 43 -64 29 7.0 P o t a s s i u m 2 6 1 6 ^ _ 1 Q 4 3 4 phosphate 8.4 T r i s - H C l 123 33 -43 40 -171 27 1. MES, 2 - ( N - m o r p h o l i n o ) - e t h a n e s u l p h o n i c a c i d 74 cytochrome o and cytochrome d, since these are the only cytochromes which bind carbon monoxide•( 13, 1 05) . Carbon monoxide d i f f e r e n c e spectra of c e l l membranes made a f t e r growth under the four conditions being emphasized are shown i n F i g u r e 12. The spectrum of aerobic exponential phase c e l l s shows p r i m a r i l y the spectrum of cytochrome o with troughs at 556nm and 428nm and peaks at 566nm, 534nm and 416nm (105, Figure 12b). Aerobic s t a t i o n a r y phase c e l l s show in addition the presence of cytochrome d with a peak at 640nm and troughs at 442nm and 621nm (Figure 12c). The membranes of c e l l s grown anaerobically with n i t r a t e show the presence of only cytochrome o. I t has been mentioned previously that these c e l l s contain very l i t t l e cytochrome d. However the Soret band of the carbon monoxide d i f f e r e n c e spectrum i s not the same as that found for aerobic c e l l s of exponential phase. The peak at 416nm i s considerably more prononounced than the trough at 433nm (Figure 12a). The production of a v a r i a n t of cytochrome o i n c e l l s grown with poor ae r a t i o n has already been reported (105). The l e v e l of carbon monoxide-binding cytochrome in nitrate grown c e l l s i s much lower than i n a e r o b i c a l l y grown c e l l s presumably r e f l e c t i n g the reduced need for an oxidase. Likewise c e l l s grown anaerobically with TMAO have a low l e v e l of carbon monoxide binding cytochrome. In th i s case however the c e l l s evidently contain both cytochrome d and o but the composite Soret band absorption spectrum (Figure 12d) makes i t d i f f i c u l t to estimate the r e l a t i v e amount of each. « ' « z\n 400 500 600 700 W a v e l e n g t h , n m 75a FIGURE 12. E f f e c t of growth c o n d i t i o n s on the carbon monoxide difference spectrum of wild-type c e l l s . Carbon monoxide difference spectra were performed as described i n 'Ma t e r i a l s and Methods' on membrane preparations of HfrH. When necessary the absorbance scale was changed between the #-band region (700nm to 500nm) and the Soret band region (500nm to 400nm). C e l l were grown: ( a ) , a n a e r o b i c a l l y with n i t r a t e (AA=0.03 i n the tf-band and Soret band regions; p r o t e i n concentration, 9.0mg/ml); ( b ) , a e r o b i c a l l y with 0.5% glycerol, 0.5% casamino acids to the exponential phase (AA = 0.03 i n ct-band and Soret band regions; protein, 7.1mg/ml); (c) , aerobically with glucose to the stationary phase (AA=0.03 i n #-band r e g i o n , 0.1 i n Soret band region; p r o t e i n , 10.7mg/ml; ( d ) , a n a e r o b i c a l l y with 0.4% TMAO (AA=0.03 in Ct-band region and 0.1 i n Soret band region; protein, 21.0mg/ml). 76 D i f f e r e n t i a l Reduction of High-Potential Cytochrome Using Ascorbate  and PMS The use of ascorbate as reductant with c a t a l y t i c quantities of phenazine methosulphate (PMS) should allow the separation of a sample into low- and h i g h - p o t e n t i a l species of cytochrome. This i s because ascorbate (E = +50m-V) w i l l not reduce many of the cytochromes m reducible by NADH (E = -320mv). These reductants, i n conjunction m with spectroscopy at 77K have been employed to correlate the results of difference spectroscopy and redox t i t r a t i o n . Cytochrome d cannot be studied by t h i s method s i n c e a r e d u c t i o n product of PMS absorbs strongly in the wavelength range 600nm to 700nm. Even at around 555nm th i s compound leads to a baseline which i s markedly -slanted. However t h i s does not prevent the a c q u i s i t i o n of us e f u l data as shown i n Figure 13. For aerobic c e l l s from the exponential phase of growth cytochromes b . and b . are s u b s t a n t i a l l y reduced by ascorbate and PMS. By 562 558 contrast the cytochromes absorbing at 555nm are reduced to a lesser extent (Figure 13b). Fourty p e r c e n t of the t o t a l b-cytochrome i s reduced by t h i s procedure, but t h i s value does not r e f l e c t a true equilibrium. Thus, the measured steady s t a t e potential imposed by the concentrations of ascorbate and PMS used was +10mV and t h i s was obtained 15 minutes a f t e r adding the. reductants. I f t h i s potential were imposed upon a l l the cytochromes of the membrane then the redox t i t r a t i o n predicts that aproximately 80% of the cytochrome should have been reduced. This underlines the need for several mediators i n a t i t r a t i o n of whole membranes. I t also shows that some high-potential 77 i i —i i 1 ~i 535 555 575 535 555 575 W a v e l e n g t h , n m 77a t FIGURE 13. Reduction of b-type cytochromes by ascorbate and PMS Difference spectra were performed at 77K on membranes derived from HfrH. Samples were reduced by 2.5mM ascorbate and 25uM PMS for 20min before freezing. Such samples were scanned against equivalent samples either reduced by d i t h i o n i t e (d-ap, shown on the right of the figure) or oxidised by f e r r i c y a n i d e (ap-f, shown on the l e f t of the figu r e ) . Cells were grown: (a) aerobically with glucose to the stationary phase (AA=0.03; protein, 5.4mg/ml); (b) , a e r o b i c a l l y with 0.5% glycerol and 0.5$ casamino acids to the e x p o n e n t i a l phase (AA=0.03; p r o t e i n , 3.6mg/ml); ( c ) , a n a e r o b i c a l l y w i t h n i t r a t e (AA = 0.03; p r o t e i n , 4.5mg/rnl); (d), anaerobically with TMAO, (AA=0.03; protein, 6.4mg/ml). 78 cytochromes in the membrane are not reducible by ascorbate and PMS for k i n e t i c reasons and that these are not i n rapid equilibrium with ones which are more readily reducible. A e r o b i c s t a t i o n a r y phase c e l l s a l s o show some degree of preferential reduction by ascorbate and PMS (Figure 13a). Cytochromes b and b , are p r e f e r e n t i a l l y reduced over cytochrome b 558 562 555 By contrast the c-type cytochrome absorbing at 550nm i s not reduced. This pattern of d i f f e r e n t i a l reduction of cytochrome absorbing at higher wavelengths was seen for a l l samples studied. Cells grown anaerobically with n i t r a t e also show a subtle pattern of d i f f e r e n t i a l reduction. Ascorbate and PMS reduce cytochrome with an absorption maximum at 556nm whilst that not reduced has a maximum at 555nm (Figure 13c). T h i s d i f f e r e n t i a l r e d u c t i o n has been noted previously (8) . The pattern of reduction i n c e l l s grown on TMAO i s very d i s t i n c t i v e . The b-type cytochromes absorbing at 558nm and 555nm are reduced by ascorbate and PMS while the major species, the c-type cytochromes remain oxidised. Potentiometric Studies of Fractionated Cytochromes Some attempts have been made to f r a c t i o n a t e the cytochromes of both aerobically and a n a e r o b i c a l l y grown c o l i . There are several published methods for o b t a i n i n g r e s p i r a t o r y complexes and individual cytochromes (17,32,33.36), and i t was aimed to follow these procedures and to study the products by the means previously described. Many of the procedures attempted f a i l e d due to the d i f f i c u l t y of-, precisely reproducing the e x p e r i m e n t a l c o n d i t i o n s of o t h e r s . However two 79 p u r i f i e d species of cytochrome have been obtained and studied by redox t i t r a t i o n . , The procedure of K i t a et a l . (36) using c e l l s grown aerobically to the stationary phase gave the column p r o f i l e shown in Figure 14. The results of difference spectroscopy and redox t i t r a t i o n of the pooled column f r a c t i o n s are shown i n F i g u r e 15. The s e p a r a t i o n of cytochromes i n t o d i s c r e t e species was poor but there were d i s t i n c t q u a l i t a t i v e differences between the 'cytochromes of the various pooled frac t i o n s . The most c l e a r d i f f e r e n c e was i n the cytochrome d content which was much h i g h e r i n samples B and C than i n sample A and especially sample D. The d i s t r i b u t i o n of cytochrome i s consistent with the r e s u l t s found by K i t a e_t a l . a f t e r a l l o w i n g f o r extensive contamination of a l l f r a c t i o n s by the high l e v e l of cytochrome d^in the s t a r t i n g m a t e r i a l . Thus, sample A had the highest content of cytochrome b . a l t h o u g h c y t o c h r o m e s d and b were a l s o 5 6 2 5 5 8 prominent. Samples B and C were dominated by cytochromes b_,_,.. b 556 558 and d. Sample D however had a quite low content of cytochrome d and had an absorption maximum at 557nm. The peak was presumably broadened due to the presence of small amounts of cytochromes b and b 556 558 (Figure 15). The t i t r a t i o n data f o r sample D suggested a single major component of mid-point p o t e n t i a l OmV. The contamination by the cytochrome d complex would explain the deviation of the t i t r a t i o n data from the theoretical curve. T i t r a t i o n of sample B showed primarily the presence of cytochrome of high p o t e n t i a l (E <0mV) whereas sample C m t i t r a t e d as a mixture of those species found i n the samples B and D (Figure 15). This feature of sample C might also be inferred from the column p r o f i l e . 80 A B C D » 1 ; 1 1 1 I 1 1 "I 20 30 40 50 Fraction Number 80a FIGURE 1_4. F r a c t i o n a t i o n by g e l f i l t r a t i o n of cytochromes sol u b i l i s e d from a e r o b i c a l l y grown,cells. S t r a i n ML308-225 from the s t a t i o n a r y phase of a e r o b i c growth was used to prepare an inner membrane f r a c t i o n as d e s c r i b e d by K i t a e_t a l _ A ( 3 6 ) . The cytochromes were then s o l u b i l i s e d and separated by gel f i l t r a t i o n as described in 'Materials and Methods'. The absorbance of the fractions was measured at 280nm (A ) to detect the e l u t i o n of a l l proteins 280 ' and at 412nm (^^^^ ^° detect the e l u t i o n of cytochrome. The column fractions were pooled into four fractions as indicated. 535 555 575 +200 W a v e l e n g t h , n m E h ( m V ) 81a FIGURE 15. D i f f e r e n c e s p e c t r a and redox t i t r a t i o n s of b-type cytochromes f r a c t i o n a t e d from a e r o b i c a l l y grown c e l l s . The samples correspond to the pooled column f r a c t i o n s shown i n Figure 14. The fractions were pooled, concentrated and their pH adjusted as described i n ' M a t e r i a l s , and Methods'. The l e f t s i d e of F i g u r e 15 shows di t h i o n i t e - r e d u c e d minus f e r r i c y a n i d e - o x i d i s e d d i f f e r e n c e spectra measured at 77K for thefts-peak region of b-cytochromes. For (A ) , A A r 0.1 at 2.0mg protein/ml; (B), AA=0.1 at 0.8mg protein/ml; (D),AA=0.1 at 1.0mg protein/ml. Sample C had a s i m i l a r spectrum to sample B. The right side of the figure shows the r e s u l t s of spectrophotometric redox t i t r a t i o n s of the same pooled f r a c t i o n s . (B): (AA=0.02; p r o t e i n , 1.5mg/ml) The curve drawn i s a three component f i t with cytochromes of potentials +134mV, +22mV and -123mV c o n s t i t u t i n g 44%, 27% and 29% of the t o t a l r e s p e c t i v l y ; (C): (AA = 0.035; p r o t e i n = 2.1mg/ml) The curve i s a three component f i t f o r cytochromes of p o t e n t i a l s +197mV (co n s t i t u t i n g 7% of the t o t a l ) , +117mV (38%) and -25mV (55%); (D): cA.A=0.031; protein, 2.0mg/ml) The curve i s the f i t for one cytochrome of potential -1mV. 82 / In spite of the poor resolution these data support the conclusion of K i t a e_t a l . (36) t h a t t h e r e i s a b-type cytochrome of low potential i n c e l l s grown a e r o b i c a l l y . The data presented above however suggest that i t has a p o t e n t i a l of OmV and an absorption maximum of 557nm i n c o n t r a s t to the v a l u e s of -45mV and 556nm r e p o r t e d previously. The i d e n t i c a l p u r i f i c a t i o n properties suggest that the two cytochromes with apparently d i f f e r e n t p r o p e r t i e s are i n fact one and the same. It has now been i s o l a t e d from c e l l s i n both the exponential and s t a t i o n a r y phases of a e r o b i c growth. Application of the f r a c t i o n a t i o n procedure of Clegg (44) to the membranes of c e l l s grown a n a e r o b i c a l l y with n i t r a t e gave the result shown i n Figure 16. There were two p o s i t i o n s at which cytochrome eluted from the column. The f i r s t corresponded to the elution position of formate dehydrogenase and was l a b e l l e d peak A. The second corresponded to the e l u t i o n p o s i t i o n of n i t r a t e reductase and was labelled peak B. The cytochrome e l u t i n g with n i t r a t e reductase was further investigated by d i f f e r e n c e spectroscopy and redox t i t r a t i o n (Figure 17). The spectrum showed a quite symmetrical#-peak centred at 556nm. However the t i t r a t i o n c l e a r l y showed that.two species of cytochrome having very similar absorption maxima were present. Several experiments of t h i s type gave the same stoic h i o m e t r y f o r the two cytochromes found by t i t r a t i o n . The computed mid-point potentials were +20mV and +120mV r e s p e c t i v e l y and they contributed e q u a l l y to the absorption difference. Subfractions of the peak of n i t r a t e reductase a c t i v i t y were t i t r a t e d and a l l of these gave the same r e s u l t . The peak of cytochrome e l u t i o n always corresponded to the peak of elution of ni t r a t e reductase. Therefore, both of the,species of cytochrome were £8 83a FIGURE 16. F r a c t i o n a t i o n of s o l u b i l i s e d anaerobic cytochromes by ion-exchange chromatography. S t r a i n PK27 was grown anaerobically with nitrat e and membranes were prepared. These were solubilised with Triton X-100 and fr a c t i o n a t e d on DEAE Bio-Gel A as described in 'Materials and Methods'. A l i n e a r g r a d i e n t (0 to 0.3M) of NaCl was applied between f r a c t i o n s 30 and 80. F r a c t i o n s were assayed for n i t r a t e reductase a c t i v i t y (1 graph u n i t = 1/imol/min/ml) and formate dehydrogenase a c t i v i t y (1 graph u n i t = 1nmol/min/ml). Cytochrome content was estimated from the absorbance at 410nm (A, 410 84 E h (mv) 5 5 6 n m I 5 0 0 6 0 0 7 0 0 W a v e l e n g t h , n m 84a FIGURE 17« Redox t i t r a t i o n and d i f f e r e n c e spectrum of cytochrome associated with a p a t i a l l y p u r i f i e d n i t r a t e reductase. Cytochromes were fractionated from PK27 as described for Figure 16. Fractions with n i t r a t e reductase a c t i v i t y were pooled, concentrated and buffered as described i n 'Materials and Methods'. The upper h a l f of the figure shows a redox t i t r a t i o n of t h i s mater i a l . AA = 0. 010 at a pr o t e i n concentration of 10.6mg/ml. ( A ) , t h e o r e t i c a l curve for a s i n g l e component t r a n s f e r r i n g one e l e c t r o n ; (B) , t h e o r e t i c a l curve for two components (E , +122mV and +17mV) each c o n t r i b u t i n g e q u a l l y to the m absorbance d i f f e r e n c e . The lower h a l f of the f i g u r e shows the dithionite-reduced minus f e r r i c y a n i d e - o x i d i s e d spectrum measured at 77K for the same preparation (AA=0.03; protein, 5.3mg/ml). I 85 considered to be genuinely associated with n i t r a t e reductase rather than merely copurifying f o r t u i t o u s l y . Their p o t e n t i a l i s suitable to pass electrons to the higher p o t e n t i a l molybdenum-associated redox centres of nitrate reductase i t s e l f (51). Kinetic Studies of Cytochrome Content The o r i g i n a l s u g g e s t i o n t h a t the membranes of c e l l s grown anaerobically with nitrate possess only two major cytochromes was based on the r e s u l t s of dual wavelength s t u d i e s (53). These data have subsequently been re-examined and a more complex explanation of the k i n e t i c behaviour has. been proposed ( 8 ) . In view of the apparently large number of cytochromes revealed by t i t r a t i o n i t was decided to investigate the cytochromes of c e l l s grown anaerobically with either n i t r a t e or TMAO by dual wavelength spectroscopy. This approach alone gave complex kin e t i c patterns which proved impossible to interpret. By trapping k i n e t i c intermediates some idea of the correspondence of ki n e t i c phases and p a r t i c u l a r cytochromes was obtained, but only i n the case of TMAO grown c e l l s . Dual wavelength traces showing the reduction of cytochrome by formate and i t s reoxidation are shown i n Figure 18. The membranes used i n Figure18(2) were d e r i v e d from c e l l s grown a n a e r o b i c a l l y with n i t r a t e . The kinetics of reduction are quite complex i n accordance with those reported previously ( 8 ) . There i s a phase immediately following formate addition i n which formate i s reducing oxygen. In t h i s phase approximately 15% of the t o t a l b-type cytochrome i s reduced. Within two minutes oxygen i s depleted and some 65% of the cytochrome i s then 86a FIGURE 18. K i n e t i c s of cytochrome r e d u c t i o n by formate and reoxidation by nitrate or TMAO. The redox state of the cytochromes was monitored at 428nm using 453nm as a reference wavelength. Membranes were prepared from HfrH and were suspended i n 50mm potassium phosphate b u f f e r , pH 7.0 c o n t a i n i n g 5mM MgCl^ i n a 3ml assay volume. ( 1 ) , c e l l s were grown anaerobically with TMAO (AA=0.03; protein, 2.8mg/ml); (2), c e l l s were grown a n a e r o b i c a l l y with n i t r a t e (AA=0.1; protein, 4.8mg/ml). At the times i n d i c a t e d 1Oumol formate ( F ) , a trace of oxygen ( 0 ) , 25/Jmol of TMAO (T) or 25/Jmol of n i t r a t e (N) were i n t r o d u c e d . Oxygen was i n t r o d u c e d by s t i r r i n g the suspension vigorously with" a plumper. The di s c o n t i n u i t i e s in the progress curve are indicated as D1, D2 and D3. 87 rapidly reduced. The time of oxygen d e p l e t i o n i s marked D1 i n Figure 18(2). The p r o g r e s s curve shows a d i s c o n t i n u i t y i n t h i s r a p i d reduction phase (D2, Figure 18(2)). A much more pronounced change in rate (D3) occurs approximately 1.5 minutes after oxygen depletion and the remaining 15% of "the cytochrome i s reduced rather slowly over a period of some 15 minutes. The progress curve of t h i s phase i s concave i n shape suggesting that the reduction of more than one species i s involved. The cytochrome which i s slowly reduced by formate i s r a p i d l y r e o x i d i s e d by oxygen. T h i s i s shown by the f a c t t h a t the slow reduction phase i s repeated after the introduction of traces of oxygen by s t i r r i n g . The a d d i t i o n of n i t r a t e l e a d s to the immediate reoxidation of 60% of the t o t a l cytochrome. In fact rather more than t h i s i s b r i e f l y reoxidised due to the introduction of small amounts of oxygen with the n i t r a t e . However a steady state i s quickly obtained in which formate i s reducing n i t r a t e . This l a s t s u n t i l formate i s depleted when more cytochrome i s o x i d i s e d leaving approximatly 10% of that cytochrome o r i g i n a l l y reduced not reoxidised by n i t r a t e . An extensive s e r i e s of i n v e s t i g a t i o n s i n t o the k i n e t i c s of cytochrome reduction and r e o x i d a t i o n i n the membranes of c e l l s grown anaerobically with n i t r a t e has e s t a b l i s h e d the following points: The reduction k i n e t i c s followed i n the #-band region (540nm-559nm) are the same as those observed in the Soret band region (453nm-428nm). The kin e t i c s of cytochrome reduction by NADH and formate are extremely s i m i l a r , both of these reductants reduce approximatly 90% of that cytochrome r e d u c i b l e by d i t h i o n i t e . The' extent of r e o x i d a t i o n by ni t r a t e i s the same for both reductants, and the sp e c i f i c a c t i v i t y for 88 n i t r a t e r e d u c t i o n i s t h e same f o r b o t h s u b s t r a t e s when measured on t h e b a s i s o f t h e d u r a t i o n o f t h e n i t r a t e r e d u c i n g s t e a d y s t a t e . The phase f o l l o w i n g n i t r a t e a d d i t i o n was shown t o be a t r u e s t e a d y s t a t e d u r i n g w h i c h n i t r a t e was b e i n g r e d u c e d b y s u b s t r a t e . I t s d u r a t i o n was p r o p o r t i o n a l t o t h e amount o f s u b s t r a t e o r i g i n a l l y added. A l s o t h e s t e a d y s t a t e l e v e l o f c y t o c h r o m e r e d u c t i o n c o u l d be c h a n g e d by t h e a d d i t i o n o f d i f f e r e n t c o n c e n t r a t i o n s o f n i t r a t e or by t h e a d d i t i o n o f a z i d e w h i c h i s a p o t e n t c o m p e t i t i v e i n h i b i t o r o f n i t r a t e r e d u c t a s e ( 5 3 ) - In a d d i t i o n some e l e c t r o n d o n o r s o f h i g h p o t e n t i a l were shown t o r e d u c e much o f t h e c y t o c h r o m e r e o x i d i s e d b y n i t r a t e . T h e s e were l a c t a t e and a s c o r b a t e w i t h e i t h e r PMS or i t s impermeant anologue p h e n a z i n e m e t h o s u l p h o n a t e . The complex e f f e c t s o f QNO on t h e r e d u c t i o n and r e o x i d a t i o n o f c y t o c h r o m e s a r e shown i n F i g u r e 19 • I t p r o l o n g e d t h e a e r o b i c s t e a d y s t a t e and c o n s i d e r a b l y slowed t h e r e d u c t i o n o f cytochrome upon oxygen d e p l e t i o n . A l s o t h e f i n a l e x t e n t o f c y t o c h r o m e r e d u c t i o n by formate was r e d u c e d . Upon n i t r a t e a d d i t i o n t h e r e was s t i l l a r a p i d r e o x i d a t i o n o f cytochrome i n t h e p r e s e n c e o f QNO b u t t h i s was r a t h e r l e s s t h a n i n i t s a b s e n c e . The t i m e t a k e n f o r f o r m a t e d e p l e t i o n was g r e a t l y i n c r e a s e d but t h e e x t e n t o f r e o x i d a t i o n upon f o r m a t e d e p l e t i o n was the same as i n t h e c o n t r o l . A t t e m p t s were made t o i s o l a t e s p e c t r a l i n t e r m e d i a t e s i n o r d e r t o u n d e r s t a n d t h e n a t u r e o f t h e c o m p o n e n t s u n d e r g o i n g r e d u c t i o n and o x i d a t i o n i n t h e d u a l - w a v e l e n g t h e x p e r i m e n t s . F i g u r e 20 shows th e r e s u l t s o f such e x p e r i m e n t s . The c y t o c h r o m e r a p i d l y r e d u c e d by formate i s d i s t i n g u i s h a b l e o n l y i n amount f r o m t h a t r educed a f t e r 45 m i n u t e s . B o t h s p e c t r a show an a b s o r p t i o n maximum a t 556nm. L i k e w i s e i t i s i d e n t i c a l t o t h a t r a p i d l y r e o x i d i s e d by n i t r a t e and t h a t r e m a i n i n g 89 89a FIGURE 19. E f f e c t of QNO on cytochrome reduction by formate and reoxidation by n i t r a t e . The redox s t a t e of cytochromes was measured at 427nm using 409nm as a reference wavelength. The sample was a membrane suspension from HfrH grown a n a e r o b i c a l l y w i t h n i t r a t e i n 0.1M potassium phosphate b u f f e r pH 7.0, at a p r o t e i n concentration of 2.5mg/ml with a 3ml assay volume. AA r e p r e s e n t s an absorbance difference of 0.003. 8.5/Jmol of formate (F) or 59/imol of nitrate (N) were added at the times indic a t e d . Curve A was recorded i n the absence of QNO and curve B at a QNO concentration of 37/uM. 90 I 1 ' 1 530 555 580 Wavelength ,nm 90a FIGURE 20. Trapping of i n t e r m e d i a t e s i n the c y c l e of cytochrome reduction and r e o x i d a t i o n . Membranes were prepared from HfrH after anaerobic growth with n i t r a t e . They were suspended in 0.1M potassium phosphate b u f f e r , pH 7.0 c o n t a i n i n g 1 .OH s u c r o s e to a p r o t e i n concentration of 5.0mg/ml. The sample was d i v i d e d . One p o r t i o n , o x i d i s e d with H^O^, w a s used as a r e f e r e n c e . This was scanned against the second sample, which was reduced by the a d d i t i o n of formate to 3mM and immediatly frozen i n l i q u i d nitrogen (curve A). The reduced sample was then thawed and l e f t f o r 15min before refreezing and scanning against the reference (curve B) . A f t e r rethawing, 10mM nitrate was added and after one minute the sample was again frozen and scanned agairist the reference (curve C) . F i n a l l y , i t was thawed and l e f t f o r 30min b e f o r e r e f r e e z i n g and r e s c a n n i n g (curve D) . AA represents an absorbance difference of 0.003. The spectra were scanned at 77K. 91 reduced, both i n the steady s t a t e and a f t e r substrate d e p l e t i o n (Figure 20c and 20d). The ki n e t i c s of cytochrome reduction from TMAO grown c e l l s bear some resemblance to those observed i n n i t r a t e grown c e l l s (Figures 18(1) and 21). There i s a steady state of oxygen reduction immediately after the addition of formate. In t h i s case only 10% of the cytochrome i s reduced. Upon oxygen de p l e t i o n 45% of the cytochrome i s rapidly reduced followed by the remainder over a period of about 20 minutes. Again most of the cytochrome slowly reduced by formate i s reoxidised r a p i d l y by oxygen. Upon the a d d i t i o n of TMAO some 36% of the cytochrome o r i g i n a l l y reduced i s r a p i d l y reoxidised. Following t h i s no large change occurs in the l e v e l of cytochrome reduction over a period of at le a s t t h i r t y minutes. This concurs with the slow k i n e t i c s of cytochrome reoxidation by TMAO reported previously (40). The ki n e t i c s of cytochrome reduction and r e o x i d a t i o n have also been studied for the flC-band region of these c e l l s . The data are shown in" Figure 21. I t i s necessary to use the #-band region when using ascorbate and PMS due to the larg e spectroscopic interference i n the Soret band region by PMS. Formate reduced only about 70% of that cytochrome reduced by d i t h i o n i t e and with the k i n e t i c s discussed previously. Ascorbate and PMS reduced only 45% of the t o t a l reduced by di t h i o n i t e . However, regardless of the reductant used the amount of cytochrome reoxidised r a p i d l y upon TMAO addition i s approximately the .same. This suggests t h a t the TMA/TMAO redox couple has a high potential since the cytochrome which i t rapidly reoxidised was reduced by ascorbate and PMS. In the case of TMAO grown c e l l s rapid freezing and spectroscopy of 92 f 92a FIGURE 21. Kinetics of cytochrome reduction and reoxidation i n c e l l s grown' anaerobically with TMAO. Membranes from HfrH were suspended in 50mM potassium phosphate b u f f e r , pH 7.0 containing 5mM MgCl^ to a protein concentration of 5.3mg/ml i n a 2ml assay volume.A A represents an absorbance difference of 0.003 at 555nm using 575ntn as a reference wavelength. 5/jmol of formate ( f ) , and 25/Jmol of TMAO (t) were added at the times i n d i c a t e d f o r curve a. A d d i t i o n of a few g r a i n s of dith i o n i t e instead of TMAO gave the broken trace marked +d. In curve b 5/Jmol of ascorbate and 50nmol of PMS were added at the time indicated (ap) followed by 25/Jmol of TMAO ( t ) . 93 Samples i n the process of r e d u c t i o n and ox i d a t i o n did allow the correlation of kine t i c phases with p a r t i c u l a r cytochromes (Figure 22). This i s a t t r i b u t e d to the g r e a t e r s p e c t r o s c o p i c d i v e r s i t y of cytochromes than i s found i n n i t r a t e grown c e l l s . Figure 22 shows that the cytochrome not reducible by formate (or NADH) was primarily of the c-type with maxima at 552nm and 548nm. However some of t h i s cytochrome was reduced by NADH (Figure 22b). Two species, cytochromes c and 552 b which were not reducible by ascorbate and PMS were reducible by 557 NADH. The cytochrome r a p i d l y r e o x i d i s e d by TMAO consisted / of several species (Figure 22d) . I t c l e a r l y included some of the cytochrome c but also a, b-type cytochrome with an absorption maximum at 555nm. The cytochrome reduced by formate but not ra p i d l y reoxidised by TMAO had a broad absorption band centred at 557nm and probably included b i 555 and b s p e c i e s ( F i g u r e 2 2 c ) . In c o n j u n c t i o n w i t h the data 558 presented e a r l i e r these r e s u l t s allowed a proposal for the arrangement of cytochromes in the membranes of c e l l s grown anaerobically with TMAO to be made. This w i l l be discussed l a t e r . 94 Wavelength , nm 94a FIGURE 22. D i f f e r e n t i a l cytochrome r e d u c t i o n by s u b s t r a t e i n membranes from c e l l s of HfrH grown anaerobically with TMAO. Difference spectra were measured at 77K. ( a ) , dithionite-reduced minus formate-reduced (AA = 0.03; p r o t e i n 9.3mg/ml); (b) , NADH-reduced minus ascorbate and PMS-reduced (AA = 0.03; p r o t e i n , 9.3mg/ml); (c), formate-' reduced, TMAO-reoxidised minus f e r r i c y a n i d e - o x i d i s e d (AA=0.03; p r o t e i n , 5.3mg/ml); (d) , formate-reduced minus formate-reduced, TMAO-reoxidised (AA = 0 . 03 5 p r o t e i n , 5.3mg/ml). The s u b s t r a t e concentrations used were 2.5mM for formate, NADH and ascorbate, 50uM for PMS and 10mM for TMAO. Reduced samples were l e f t 30min before freezing. Reoxidised samples were l e f t 5min after the addition of TMAO before freezing. 95 PART I I . THE CYTOCHROMES OF MUTANTS DEFICIENT IN THE FORMATE- NITRATE REDUCTASE PATHWAY The procedures o u t l i n e d above had o n l y l i m i t e d success i n resolving the complex mixture of cytochromes in the membranes of c e l l s grown under any of the co n d i t i o n s discussed. Therefore a l t e r n a t i v e approaches were sought. I t was b e l i e v e d that mutants of E_^  c o l i s p e c i f i c a l l y lacking one or a few cytochromes would greatly simplify study of the r e s p i r a t o r y chains. This could (be e s p e c i a l l y useful i n view of the simultaneous presence of at least two respiratory pathways under any of the growth conditions investigated. No mutants were available which were s p e c i f i c a l l y affected i n any aerobic cytochrome'. No d e t a i l e d p r o t o c o l for their i s o l a t i o n has been p u b l i s h e d a l t h o u g h i t i s known t h a t o x i d a s e n e g a t i v e mutants i d e n t i f i e d by ov e r l a y i n g a pla t e of p r e v i o u s l y permeabilised c e l l s with reduced N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) w i l l i n c l u d e mutants i n the cytochrome d pathway (106). A number of experiments have been performed i n an attempt to i s o l a t e a mutant defective in the aerobic cytochromes. The f o l l o w i n g approaches were attempted: (a), Mutants d e f i c e n t i n i n d i c a t o r reduction were isolated using either t r i p h e n y l - t e t r a z o l i u m c h l o r i d e i n plat e s or TMPD as an overlay, (b.) , Mutants r e s i s t a n t to 2mM azide (107), aminoglycoside a n t i b i o t i c s (108), or 0.1% c h l o r o l a c t a t e were obtained (109). (c). purE and l i p P1 t r a n s d u c t a n t s were i s o l a t e d from auxotrophs using l y s a t e s made on E_^  c o l i p r e v i o u s l y mutagenised by e i t h e r random Tn5 i n s e r t i o n s or c h e m i c a l l y (102,110,111). The candidate cytochrome mutants were then screened by measuring r e s p i r a t o r y 96 a c t i v i t y and by d i f f e r e n c e s p e c t r o s c o p y . None of the procedures outlined yielded a clear example of a cytochrome mutant. The Cytochrome Content and Respiratory A c t i v i t y of chi Mutants There are a large number of mutants a v a i l a b l e in the respiratory pathway between formate and n i t r a t e and some of these were obtained (Table 1). Their cytochromes were analysed after anaerobic growth on a minimal medium with glucose, peptone and n i t r a t e upon which a l l these s t r a i n s , can r e a d i l y grow. They w i l l c o l l e c t i v e l y be c a l l e d c h i mutants for the purposes of t h i s t h e s i s a l t h o u g h they are not necessarily resistant to c h l o r a t e (59). In a d d i t i o n to chi mutants a single fdhA mutant has also been s t u d i e d . I t was hoped that some of these mutants would lack one or more cytochromes. This would provide a simpler p r e p a r a t i o n than the membrane of the w i l d - t y p e grown anaerobically with n i t r a t e to analyse spectroscopically, and i t might f a c i l i t a t e d e f i n i t i o n of the cytochromes of the f o r m a t e - n i t r a t e respiratory pathway. / D i f f e r e n c e s p e c t r a measured at 77K f o r the membranes of representative c h i mutants a f t e r anaerobic growth with n i t r a t e are shown in Figures 23 and 24. The cytochrome b and cytochrome d levels of these mutants are given in Table 6. Several different phenotypes of cytochrome production were evident. A c l a s s of c h i mutants including RK7-16 and CGSC 4459 have a cytochrome content c l o s e l y resembling that of the w i l d - t y p e grown under the same c o n d i t i o n s . Their cytochrome, d l e v e l i s e x t r e m e l y low (Table 6 ) . The -peak of cytochrome closely resembles that of the wild-type but i s discernibly 97 AA I i i - i 1 r-5 4 0 5 5 5 5 7 0 5 1 0 6 0 0 6 9 0 W a v e l e n g t h , n m 97a FIGURE 23. D i f f e r e n c e s p e c t r a o f membranes of c h i and fdh mutants. The c e l l s were grown a n a e r o b i c a l l y w i t h n i t r a t e . Dithionite-reduced minus ferricyanide-oxidised difference spectra were taken at 77K. The s t r a i n s used were: ( A ) , LCB68 (AA=0.1; p r o t e i n , 8.2mg/ml); (B), LCB517 (&A=0.03 (whole spectrum) or 0.1 (detail of <£-band re g i o n ) ; p r o t e i n , 3.2mg/ml); (C), RK7-16, (AA = 0.03; protein, 2.7mg/ml); (D), CGSC 4459 (AA=0.03; protein, 1.9mg/ml). 98 Wavelength ,nm 98a FIGURE 24. The d e r e p r e s s i o n o f cytochromes b r . ^ and c ^ i n 562 552 respiratory mutants. Membranes were prepared from strains (A), HfrH; (B), CGSC 4 4 5 9 ; (C), LCB517; a f t e r anaerobic growth with n i t r a t e . Dithionite-reduced minus f e r r i c y a n i d e - o x i d i s e d difference spectra were measured at 77K and stored in a Bascom-Turner Model 4120 Data-Centre. For each sample two spectra were recorded , averaged and smoothed and then adjusted to the same s i z e . A-B i s the difference between the b-and c-type cytochrome c o n t e n t of HfrH and CGSC 4 4 5 9 . A-C i s the difference between HfrH and L C B 5 1 7 . The v e r t i c a l scale i s arbitrary for a l l spectra. TABLE 6 CYTOCHROME CONTENT AND RESPIRATORY ACTIVITY OF CHL MUTANTS S t r a i n Phenotypic Class 2 Cytochrome b 3 Cytochrome d Cytochrome b NADH4 Oxidase 4 Formate Oxidase Nitrate"* Reductase 6 Formate Hydrogen-l y a s e N i t r : Re dud LCB61 Wild-type 0.60 0.04 40 20 1140 + + HfrH I I 0.64 0.01 59 57 550 + + RK7-16 I 0.80 0.06 225 <2 <10 - + CGSC 4459 I I 0.77 0.02 209 <2 <10 - + LCB79 I I , 0.54 0.08 50 200 <10 + + LCB160 I I 0.44 0.10 143 190 430 + + LCB68 I I I 0.82 0.55 190 <2 <10 - -CGSC 4444 I I 0.41 0.14 209 5 70 - -LCB517 Fdh" 0.46 0.02 64 <2 610 - -1. C e l l s were grown a n a e r o b i c a l l y w i t h n i t r a t e and membranes prepared as described i n ' M a t e r i a l s and Methods' 2. nmol/mg p r o t e i n 3. nmol cytochrome d/nmol cytochrome b 4. ngatoms oxygen/min/mg p r o t e i n 5. nmol nitrate/min/mg p r o t e i n ; Assayed w i t h benzyl v i o l o g e n as e l e c t r o n donor 6. Estimated from gas production i n Durham tubes 7. Estimated from the c a p a c i t y to c l e a r ImM n i t r i t e from a r i c h medium overnight 100 broader. This i s evident from Figure 24 where the spectrum of the oc-peak region of cytochrome b i s shown for CGSC 4459 and HfrH after adjustment to the same h e i g h t . The d i f f e r e n c e between these two s p e c t r a shows t h a t the CJTJLF. mutant has a h i g h e r c o n t e n t of cytochromes c,.^ and b ^  , ^  than the w i l d - t y p e (Figure 24, A-B) . 552 5 o 2 This i s a r e f l e c t i o n of the derepression of other respiratory pathways i n the absence of a f u n c t i o n a l n i t r a t e reductase pathway. Mutants belonging to t h i s phenotypic c l a s s w i l l be ca l l e d class I mutants for the sake of d i s c u s s i o n . The cytochrome l e v e l i n a l l class I mutants was higher than i n t y p i c a l wild-type strains at 0.77nmol/mg protein. A d i s t i n c t phenotype of cytochrome production,,to be found in mutants of class I I , was als o i d e n t i f i e d . Mutants of t h i s class, for example LCB162, have an a b s o r p t i o n maximum for the -peak at a s l i g h t l y lower wavelength than i n the wild-type (Figure 23c). In a d d i t i o n there i s a pronounced s h o u l d e r at 558nm which i s the cytochrome b associated with the r e l a t i v e l y high levels of cytochrome d. These mutants have a low apparent l e v e l of t o t a l b-type cytochrome (0.38nmol/mg p r o t e i n ) s u g g e s t i n g that they la c k a cytochrome (Table 6). A t h i r d phenotype of cytochrome production i s char a c t e r i s t i c of the class I I I mutants. Figure 23a shows that these mutants, such as LCB68 produce very high l e v e l s of cytochromes d and a^ and therefore i t i s not s u r p r i s i n g that cytochromes b and b dominate the 556 558 ot*-pe.ak region for b-type cytochromes. The mutants of t h i s class, but no other phenotypic c l a s s , produced the same cytochromes irrespective of the presence of n i t r a t e i n the growth medium. The l e v e l of cytochrome i n t h i s p a r t i c u l a r mutant i s 0.82mol/mg protein although 101 TABLE 7 CLASSIFICATION OF CHL MUTANTS BY PHENOTYPIC CLASS Phenotypic Class S t r a i n Genotype I RK5200 chlA RK5201 chlE RK5206 chlG RK5208 chlB RK5218 chlE RK5223 chlE RK5231 chlG RK5256 chlG RK5266 narK RK5270 narK BK-7-16 chlA' 356-15 chlA 356-24 chlB CGSC 4459 chlE LCB61-357 chlE II TS9A (42°C) chlC RK5269 narG (=chlC) RK5274 n a r l (=chll) RK7-19 chlC LCB79 c h l l LCB155 c h l l LCB160 c h l l LCB162 chlC LCB79-357 c h l I , c h l E NH30 . c h l l III CGSC 4444 chlC (?) LCB68 chlE NH10 chlE,chlI RK7-36 chlB (?) LCB61-22 nirR (=fnr) -102 t h i s value i s variable for members of phenotypic class I I I (Table 6). The cytochromes of mutants of t h i s class are s t r i k i n g l y different since c e l l p e l l e t s after anaerobic growth with n i t r a t e are noticeably green rather than the pink colour c h a r a c t e r i s t i c of other chi mutants and the wild-type. The d i f f e r e n c e spectrum of the fdhA mutant (LCB517) i s very similar to that of mutants from c l a s s I (Figure 23b). As shown in Figure 24 i t too has a higher content of cytochromes o p r . and b 552 562 than the wild-type. Again t h i s i s thought to r e f l e c t the derepression of other r e s p i r a t o r y components due to a d e f i c i e n c y i n the formate dependent-nitrate reductase pathway. Chi mutants of a phenotype p r o d u c i n g l a r g e amounts of cytochrome b have been r e p o r t e d p r e v i o u s l y ( 5 5 ) . Another 5 5 8 phenotype reported p r e v i o u s l y (55) i n which cytochrome c i s the 5^8 major species has not been i d e n t i f i e d i n t h i s study. This i s probably because the d i f f e r e n c e s p e c t r a r e p o r t e d here were performed on membranes and not whole c e l l s . The cytochrome c observed 5 4 8 previously (55) i s probably the s o l u b l e cytochrome named c which 552 i s associated with n i t r i t e r e d u c t i o n (11,35) which' would not be evident i n the spectra of membranes. This species i s known to be derepressed i n c e r t a i n c h i mutants (76). The r e s p i r a t o r y a c t i v i t i e s and cytochrome l e v e l s of a r e p r e s e n t a t i v e s e r i e s o f chi m u t a n t s , the fdhA mutant and wild-type s t r a i n s are shown i n Table 6. These data show a c l e a r c o r r e l a t i o n between the s p e c t r a l p r o p e r t i e s and the r e s p i r a t o r y a c t i v i t y . Table 7 shows the mutants which have been studied for this t h e s i s c l a s s i f i e d i n terms of t h e i r genotype and phenotype of 103 i cytochrome production which they displayed. Mutants of class I were a l l p l e i o t r o p i c and mapped to the chlA, B, D, E_ and G l o c i (Table 7 ) . They l a c k e d both formate oxidase and n i t r a t e reductase a c t i v i t y . The absence of formate oxidase a c t i v i t y r e f l e c t s a defect in formate dehydrogenase since they did not produce gas in Durham tubes but re t a i n e d an NADH oxidase a c t i v i t y . The deficiency in both enzyme a c t i v i t i e s of the formate-nitrate pathway r e f l e c t s t h e i n a b i l i t y t o s y n t h e s i z e and i n c o r p o r a t e the molybdenum-cofactor (57). These mutants were not affected in n i t r i t e r e d u c t i o n . Their cytochrome l e v e l , as mentioned p r e v i o u s l y , was s l i g h t l y higher than in the wild type but they retained the repression of cytochrome d by nitrate (Table 6). Class II mutants were c h a r a c t e r i s e d by gas production i n Durham tubes (Table 6). These mutants had a formate oxidase a c t i v i t y c o n s i d e r a b l y higher than that of the wi l d - t y p e . The NADH oxidase a c t i v i t y was increased by a smaller f a c t o r . The increase i n formate oxidase, a c t i v i t y presumably r e f l e c t s an i n c r e a s e i n formate dehydrogenase a c t i v i t y since dehydrogenases were shown above to be rate l i m i t i n g i n electron transfer to oxygen. A l l class II mutants retained the c a p a c i t y to reduce n i t r i t e and were somewhat derepressed for cytochrome d but t h e i r t o t a l cytochrome b l e v e l tended to be lower than in the wild-type. They may or may not r e t a i n n i t r a t e reductase a c t i v i t y using reduced benzyl v i o l o g e n as donor depending on the nature of the c h i l e s i o n . Whilst chlC mutants lacked the a c t i v i t y with any e l e c t r o n donor, c h l l mutants retained n i t r a t e reductase a c t i v i t y with benzyl viologen but not with ascorbate and PMS, NADH or formate as donors (65). A l l c l a s s I I mutants mapped to the chlC 104 l o c u s (Table 7 ) , which i n c l u d e s the narG (=chlC) , narH and n a r l ( = c h l l ) genes (59.66). Class I I I mutants were s t r o n g l y p l e i o t r o p i c (Table 6). They did not produce gas in Durham tubes and. had no formate oxidase a c t i v i t y . However they were apparently not d e f i c i e n t i n the aerobic respiratory chains since they had a high NADH oxidase a c t i v i t y . They were deficent in both n i t r a t e and n i t r i t e r e d u c t i o n . Mutants at the chlB, C and E, and nirR (=fnr) l o c i were found i n t h i s c l a s s (Table 7). The fdhA mutant r e t a i n e d n i t r a t e reductase a c t i v i t y with a l l donors except formate (Table 6 ) . It- lacked formate dehydrogenase a c t i v i t y as shown both by Durham tubes and assays of formate oxidase a c t i v i t y . In a d d i t i o n i t was d e f i c i e n t i n n i t r i t e r e d u c t i o n . This observation i s consistent with the observation that formate i s a major electron donor for n i t r i t e reduction (112). The l e v e l of cytochrome in the mutant was a l i t t l e lower than i n the wild-type but the repression of cytochrome d by n i t r a t e was apparently normal. Of p a r t i c u l a r i n t e r e s t are the p r o p e r t i e s of the mutant TS9A which are compared to those of i t s isog e n i c parent PK27 in Table 8. This temperature sensitive mutant i s defective in the n i t r a t e reductase structural gene (64) and so growth at the non-permissive temperature o (42 C) led to a lower s p e c i f i c a c t i v i t y for n i t r a t e reductase. The amount of cytochrome was lower a f t e r growth at the non-permissive temperature (Table 8). These p r o p e r t i e s are the cha r a c t e r i s t i c s of a class I I mutant. However other c h a r a c t e r i s t i c s of c l a s s I I mutants o were p r e s e n t a f t e r growth at e i t h e r the p e r m i s s i v e (29 ) or non-permissive temperatures. That i s , i t had a high formate oxidase a c t i v i t y and a r e l a t i v e l y high content of cytochrome d. The difference TABLE 8 TEMPERATURE DEPENDENCE OF RESPIRATORY ACTIVITY AND CYTOCHROME CONTENT OF PK27 AND TS9A1 Strain Growth Temperature Cytochrome b Cytochrome d~ Cytochrome, b .NADH Oxidase Formate Oxidase Nitrate Reductase PK27 29°C 0.71 0.05 273 69 1480 PK27 42°C 0.67 0.05 210 141 1240 o TS9A o 29 C 0.86 0.20 277 701 1170 TS9A 42°C 0.39 0.18 150 592 150 1. Cells were grown anaerobically with nitrate and membranes prepared as described in 'Materials and Methods' 2. nmol/mg protein 3. nmol cytochrome d/nmol cytochrome b 4. ngatom oxygen/min/mg protein 5. nmol nitrate/min/mg protein 106 s p e c t r a of t h i s s t r a i n a f t e r g r o w t h at t h e p e r m i s s i v e and non-permissive temperatures are shown i n Figure 25- A cytochrome b species was more abundant r e l a t i v e to cytochrome b a f t e r 556 558 growth at the permissive temperature. Cytochrome absorbing at 556nm i s c h a r a c t e r i s t i c of the w i l d - t y p e a f t e r growth a n a e r o b i c a l l y with ni t r a t e and has been shown to be involved in ni t r a t e reduction (15, 53). Spectrophotometric Redox T i t r a t i o n s of chi Mutants The difference spectra and assays of respiratory a c t i v i t y of some of the c h i mutants suggested t h a t s m a l l e r number of cytochromes might be present. Therefore the cytochromes of a number of the chi mutants and the fdhA mutant were studied by spectrophotometric redox t i t r a t i o n . R e p r e s e n t a t i v e data are shown i n Figure 26 and four component f i t s for these t i t r a t i o n s are given i n Table 9'. The t i t r a t i o n data for RK7-16 and related class I mutants closely resembled those described above for the wild-type. There were at least four cytochromes i n the membranes of these c e l l s t i t r a t i n g over a range of +200mV to -150mV. T h i s suggested the r e t e n t i o n of fdh - nr cytochromes b and b by these mutants. There was s l i g h t l y more cytochrome of low p o t e n t i a l ( < -120mV) i n the mutants than i n the w i l d - t y p e , an observation c o n s i s t e n t with the s l i g h t increase i n cytochrome c seen i n the d i f f e r e n c e spectrum and the suggestion 552 that t h i s i s a low potential cytochrome. T i t r a t i o n of a c l a s s I I mutant such as L C B 7 9 gave a quite di f f e r e n t r e s u l t . These mutants were very d e f i c i e n t i n cytochromes of 42° 29° 107a FIGURE 25. Difference spectra of the membranes of TS9A after growth at the permissive and non-permissive temperatures. Dithionite-reduced minus ferricyanide-oxidised d i f f e r e n c e spectra were measured at 77K. o TS9A was grown anaerobically with n i t r a t e at 42 C (AA=0.03; protein, 4.3mg/ml) or 29°C (AA=0.1; pr o t e i n 4.8mg/ml). I 108 108a FIGURE 26. Redox t i t r a t i o n s of the b-cytochromes of c h i mutants. The,strains were grown a n a e r o b i c a l l y with n i t r a t e , membranes were prepared and t i t r a t i o n s performed as described i n 'Materials and Methods'. The s o l i d l i n e s are best f i t s assuming LCB79 and RK7-16 had four components and LCB68 had three (Table 9 ) . The strains used were LCB68 (AA=0.066; p r o t e i n , 13.4mg/ml); RK7-16 (AA=0.053; p r o t e i n , 10.9mg/ml); LCB79 (AA=0.017; protein, 12.2mg/ml). TABLE 9 FITS OF TITRATION DATA FOR THE CYTOCHROMES OF CHL MUTANTS Strain Figure Components Resolved Number E (mV) m % E (mV) m % E (mV) m % E (mV) m t HfrH 8 197 15. 3 99 18.5 -3 35. 6 -113 30. ,6 PK27 8 197 32. 0 97 20.9 9 30. 7 -112 16. 3 KL718 9 218 8. 4 137 49.2 40 28. 5 -84 13. ,9 KL702 9 174 18. 0 100 33.7 5 25. 6 -102 22. ,7 LCB68 26 224 49. 6 HI 41.6 -67 8. 8 LCB79 26 201 8. 4 93 15.6 -13 17. 1 -106 58. ,8 RK7-16 26 188 20. 5 58 31.4 -49 28. 2 -165 19. ,8 LCB517 27 180 24. 6 89 29.3 8 32. 8 -83 13. ,2 TS9A (29°C) 28 207 28. 0 67 26.3 -38 19. 9 -127 25. .8 TS9A (42°C) 28 208 27. 5 130 11.5 17 9. 5 -99 51. .5 LCB61-357 38 185 16. 2 76 36.2 -35 30. 2 -159 16. .8 LCB79-357 38 166 20. 9 1 40.1 -97 39. 0 NH20 40 194 47. 5 80 33.3 -69 19. 2 NH40 40 201 27. 0 96 18.9 -9 24. 6 -99 29. .4 NH50 40 253 19. 6 135 27.2 22 30. 3 -103 22. .9 CGSC 4459 40 158 18. 1 57 32.1 -23 31. 0 -153 18. .7 1. The strains indicated were grown anaerobically with nitrate, membranes prepared and titrated as described in 'Materials and Methods'. The data were analysed as four components except where this gave a f i t no better than the three component analysis. 110 potential 0 to +100mV. This confirmed that they lack cytochrome nr b which was shown above to have a p o t e n t i a l of approximately +60mV. In fact a single cytochrome of p o t e n t i a l approximately -100mV could account for 60% of the cytochrome i n these c e l l s . This cytochrome was found i n both the three and f o u r component f i t s of the data. The appearance of the same cytochrome i n two f i t s of the same data performed using a d i f f e r e n t number of components has previously been suggested to be a c r i t e r i o n upon which the . r e a l i t y of a species could be judged (113). In contrast to class II mutants, c l a s s I I I mutants such as LCB68 had a large content of h i g h - p o t e n t i a l cytochrome. In fact they had almost no cytochrome of redox p o t e n t i a l l e s s than 50mV which implied nr that cytochrome b was absent. The experimental data suggested the presence of two major cytochromes of potentials +220mV and +110mV, and some minor low potential components (Table 9). The fdhA (LCB517) mutant showed a rather d i s t i n c t phenotype in t i t r a t i o n . This i s compared to data obtained from a wild-type s t r a i n i n Figure 27. Whilst the t i t r a t i o n curves may appear to.be somewhat si m i l a r , the mutant c l e a r l y lacked the cytochrome of potential less than -100mV which was quite prominent i n the parent. This absence i s also evident i n the four component f i t s of the data shown in Table 9. fdh Presumably t h i s i s cytochrome b . C l a s s I I mutants have a cytochrome of t h i s p o t e n t i a l and are' suggested to r e t a i n cytochrome fdh b whilst c l a s s I I I mutants have no cytochrome of t h i s potential and are suggested to l a c k i t . The fdhA mutant, l i k e the wild-type, has cytochrome of p o t e n t i a l approximately +50mV and i s therefore nr suggested to r e t a i n cytochrome b I l l 111a FIGURE 27. T i t r a t i o n of, the b-type cytochromes of an fdhA mutant compared to that of the w i l d - t y p e . Membranes were prepared from the fdhA mutant, LCB517, and the wild-type s t r a i n , HfrH, after anaerobic growth w i t h n i t r a t e . These p r e p a r a t i o n s were su b m i t t e d to spectrophotometric redox t i t r a t i o n as described i n 'Materials and Methods'. The s o l i d l i n e s are four component best f i t s (Table 9). For LCB517, AA = 0.032; p r o t e i n , 11.8mg/ml; Hf r H, A A = 0 . 032; p r o t e i n , 7.4mg/ml. 112 Figure 28 shows redox t i t r a t i o n s for the cytochromes of TS9A after growth at the permissive and non-permissive temperatures. Cytochrome of potential around +50mV was much more prominent after growth at the permissive temperature. In i t s lack of cytochrome at around +50mV potential and the conspicuous amounts of cytochrome of potential about -lOOmV, TS9A grown at the non-permissive temperature resembled a class I I mutant. On the other hand, the r e s u l t s of t i t r a t i o n of the membranes of TS9A a f t e r growth at the permissive temperature more closely resembled the data found for the wild-type. The Cytochrome d and Cytochrome o Content of chi Mutants In order to determine whether the cytochromes observed by t i t r a t i o n belonged to the cytochrome d or cytochrome o pathways the c h i and fdhA mutants have a l s o been i n v e s t i g a t e d by carbon monoxide difference spectroscopy. Representative difference spectra are shown i n Figure 29. The spectrum f o r RK7-16 (Figure 29b) shows an apparently normal spectrum f o r cytochrome o with a trough at' 431nm. * and a peak of approximately equal i n t e n s i t y at 416nm. The difference spectrum closely resembled that of the wild-type grown aerobically to the exponential phase. However, the wild-type grown anaerobically with nitrate (Figure 12a) showed the same peak but the trough i n the Soret band region of the spectrum was much l e s s prominent than i n these mutants. Class II mutants, l i k e RK7-19 (Figure 29a) showed a carbon monoxide difference spectrum that more c l o s e l y resembled that of the wild-type, at l e a s t i n the Soret band reg i o n , with a peak at 420nm and a very s l i g h t trough at 433nm. The spectrum i n the CC-band region 1 1 3 113a FIGURE1 28. Tit r a t i o n of t o t a l b-type cytochrome of TS9A after growth o o at 29 C (A) or 42 C ( B ) . C e l l s were grown a n a e r o b i c a l l y with n i t r a t e . Membranes were prepared and t i t r a t e d . For ( A ) , A A was 0.013 at a protein concentration of 6.5mg/ml. For (B) , A A was 0.020 at 7.6 mg protein/ml. The s o l i d l i n e s are the best f i t s assuming four components (Table 9) r— 4 0 0 5 0 0 W a v e l e n g t h , n m —I— 6 0 0 —r 7 0 0 114a FIGURE 29. Carbon monoxide d i f f e r e n c e spectra of the membranes of chi and fdh mutants. C e l l s were grown a n a e r o b i c a l l y with n i t r a t e , ( a ) , LCB517 ( A A = 0.03 i n QC-peak and So r e t band regions; p r o t e i n , 6.3mg/ml); (b), RK7-16 ( A A = 0.03 i n Ct-band and Soret band regions; protein, 6.0mg/ml); ( c ) , RK7-19 ( A A = 0.03 i n Ot-band region and 0.1 in Soret band region; p r o t e i n , 8.9mg/ml); (d) , LCB68 (AA=0.03 i n <*-band region and 0.1 in Soret band region; protein, 9.0mg/ml). 115 c l e a r l y showed the presence of cytochromes d and o which must together contribute to the spectrum i n the Soret band region to give the shape observed. In LCB68, and other c l a s s I I I mutants, cytochrome d was the primary carbon monoxide binding pigment leading to a trough i n the spectrum at 443nm. Superimposed on t h i s was the spectrum of cytochrome o which was c l e a r l y present i n rather smaller amounts (Figure 29d). Using published extinction c o e f f i c e n t s for cytochromes o and b i t was possible to estimate the content of cytochrome o and to determine the c o n t r i b u t i o n made by cytochrome o to the Q£-peak of the b-type cytochromes. The c a l c u l a t e d v a l u e s f o r v a r i o u s mutants and the wild-type are summarized i n TablelO. The values given i n the table for cytochrome o content i n terms of percentage of t o t a l b-type cytochrome cannot be taken too l i t e r a l l y . They are calculated on the basis of extinction coefficents that may be questionable. However they do suggest that mutants of c l a s s e s I and I I have a higher cytochrome o content than the wild-type and that c l a s s I I I mutants have rather l e s s . The c l a s s I I mutants have a higher cytochrome o content than does the w i l d - t y p e . This increase i n the r e l a t i v e amount cannot be w h o l l y accounted f o r by t h e i r reduced l e v e l of t o t a l b-type cytochrome. By cont r a s t the mutants of c l a s s I I I which overproduce cytochrome d had quite low l e v e l s of cytochrome o. The c l a s s I mutants, although they resembled the wild-type i n many respects had a higher content of cytochrome o. Again these data seems to r e f l e c t the derepression of o t h e r r e s p i r a t o r y pathways i n the absence of a f u n c t i o n a l f o r m a t e - n i t r a t e r e d u c t a s e pathway. The presence of cytochrome b , , and the high l e v e l s of cytochrome o, suggested that 562 v the low-aeration oxidase pathway had become derepressed. This idea 116 TABLE 10 CYTOCHROME 0 CONTENT AND CYTOCHROME REDUCIBILITY BY ASCORBATE AND PMS FOR CHL MUTANTS1 S t r a i n Phenotype Cytochrome o Cytochrome o" Cytochrome b % of cytochrome b reduced by ascorbat and PMS HfrH LCB61 Wild-type 0.073 0.064 0.16 0.17 25 34 RK7-16 CGSC 4459 Class I II 0.250 0.161 0.31 0.21 22 22 LCB79 RK7-19 Class II 0.134 0.182 0.37 0.57 16 19 LCB68 CGSC 4444 Class I I I 0.024 0.035 0.07 0.08 61 58 LCB517 Fdh 0.073 0.16 55 NH20 NH40 NH50 Double Mutant 0.265 0.293 0.226 0.78 0.66 0.47 53 s40 40 1. C e l l s were grown anaerobically with n i t r a t e and spectra taken of membranes as described i n "Materials and Methods'. 2. nmol/mg protein 3. mol cytochrome o/mol cytochrome b 4. Determined as described i n 'Materials and Methods'. 117 suggests that the fdhA mutant should al s o have an increased l e v e l of cytochrome o, since i t too had more cytochrome b than the 562 wild-type. However, l i k e the wild-type i t had a Soret band peak which was much more pronounced than the trough (Figure 29c), and not the spectrum c h a r a c t e r i s t i c of c l a s s I mutants. Moreover i t s l e v e l of cytochrome o appeared to be the same as that found in the wild-type. (Table 10) Separation of the Cytochromes of c h i Mutants into High and Low  Potential Species Using Ascorbate and PMS The reduction of cytochrome by ascorbate and PMS i n a series of ch i mutants and the fdhA mutant i s shown i n F i g u r e 30 and the amount of cytochrome reduced i n these s t r a i n s i s summarised in Table 10. Because the t i t r a t i o n data had implied a simpler cytochrome content for these mutants i t was believed that t h i s approach should allow the c o r r e l a t i o n of the r e s u l t s of d i f f e r e n c e spectroscopy and redox t i t r a t i o n . Only approximately 20% of the cytochrome i n the class I st r a i n s , f o r instance CGSC 4459. was r e d u c i b l e by ascorbate and PMS. As discussed before, t h i s value r e f l e c t s only that cytochrome which i s readily reducible by ascorbate and PMS and i s not a l l of the cytochrome having a p o t e n t i a l higher than the imposed e q u i l i b r i u m potential of +10mV. The cytochrome which was reduced (Figure 30a) had an absorption maximum of 556nm with a h i n t of a shoulder on the high wavelength side. That cytochrome which was not reduced by ascorbate and PMS in t h i s mutant had a s l i g h t l y sharper &-peak centred at 555nm with a 118 Wavelength, nm 118a FIGURE 30. D i f f e r e n t i a l reduction of b-type cytochromes by ascorbate ( and PMS i n c h i and fdh mutants. C e l l s were grown a n a e r o b i c a l l y with n i t r a t e . Ascorbate- (2.5mM) and PMS ( 25/iM)-reduced minus ferricyanide-oxidised (ap-f) and dithionite-reduced minus ascorbate and PMS-reduced (d-ap) difference spectra were recorded at 77K. The strains used were (a), CGSC 4459 (AA=0.03 f o r ap-f and 0.1 for d-ap; protein, 3.4mg/ml); ( b ) , RK7-19 (AA = 0.01; p r o t e i n , 4.5mg/ml); (c) , LCB68 (AA=0.03; protein, 4.7mg/ml); (d), LCB517 (AA=0.03; protein, 3.1mg/ml). 119 detectable amount of cytochrome c. T h i s pattern of d i f f e r e n t i a l reduction was very r e m i n i s c e n t of the parent and supports the c o n t e n t i o n t h a t c l a s s I mutants have the same complement of cytochromes. Class II mutants such as L C B 7 9 a l s o showed the reduction of very l i t t l e of their cytochrome by ascorbate and PMS (Figure 30b). In th i s case the amount reduced agreed with that which would be predicted from the redox t i t r a t i o n . The cytochrome that was reduced had an absorption maximum at 558nm. The cytochrome which was not reduced had an absorption maximum at 556nm but w i t h a s i g n i f i c a n t content of cytochrome absorbing at 558nm to 562nm. In the case of class I I I mutants such as LCB68 approximately 60% of the t o t a l b-cytochrome was reduced by ascorbate and PMS (Table 10). This i s a r e f l e c t i o n of the high content of high-potential cytochrome revealed by t i t r a t i o n . There was only a s l i g h t difference between the spectrum of the cytochrome which was reduced and that which was not reduced (Figure 30c). The species absorbing at 558.5nm was s l i g h t l y more reduced by ascorbate and PMS than that absorbing at 555nm. There was also a clear pattern of d i f f e r e n t i a l reduction i n the fdhA mutant (Figure 30d). The cytochrome reduced by ascorbate and PMS absorbed with a sharp peak centred at 556nm while the material that was not reduced had a much broader a b s o r p t i o n peak centred at approximately 554nm and c l e a r l y had a s i g n i f i c a n t c o n tent of cytochromes b and c. 556 In a l l of these mutants, as i n the w i l d - t y p e , the l e v e l of cytochrome b • was greater i n the cytochromes reduced by ascorbate 562 and PMS than i n the cytochromes which were not reduced. By contrast, 120 cytochrome c, as revealed by the r e l a t i v e absorbance at 550nm, seemed more prominent i n that cytochrome which was not reduced. Electrophoretic Analysis of Mutants for the Presence of Nitrate  Reductase and Formate Dehydrogenase' Polypeptides SDS polyacrylamide gels of the membrane and soluble fractions of a s e r i e s of c h i mutants, the fdhA mutant and wild-type s t r a i n s are shown in Figure 31. I t was hoped that the presence of the ^ -subunits of formate dehydrogenase and n i t r a t e reductase would correlate with fdh nr the proposed presence or absence of cytochromes b and b respectively. The high molecular weight of the Ct-subunits of these two enzymes, 155,000 f o r n i t r a t e r e d u c t a s e and 110,000 for formate dehydrogenase, made their presence easy to detect in SDS-polyacrylamide gels of whole membranes. P a r t i a l l y p u r i f i e d n i t r a t e reductase and formate dehydrogenase preparations were run on the same gels to act as markers f o r these subunits. The s o l u b l e f r a c t i o n from a l l of the mutants studied was a l s o run on g e l s to detect the possible presence of formate dehydrogenase or n i t r a t e r e d u c t a s e s u b u n i t s i h the cytoplasm. The migration p o s i t i o n s of the n i t r a t e reductase and formate dehydrogenase at-subunits are indicated on the gels. The class I mutants had cytochromes of the properties expected of fdh nr cytochromes b and b .^ In a d d i t i o n they showed a prominent membrane-bound protein with a m o b i l i t y the same as that of the nitrate reductase c t - s u b u n i t . They d i d not appear to have the formate dehydrogenase pc-subunit ( F i g u r e . 31 )• The i n t e n s i t y of the band believed to be the formate dehydrogenase Cfc-subunit was rather 121 121a FIGURE 31« SDS-polyacrylamide gels of membrane and soluble fractions of w i l d - t y p e , c h i and fdh s t r a i n s of Ej_ c o l i . The s t r a i n s were grown anaerobically with n i t r a t e , d i s r u p t e d i n a French press and the soluble and membrane fractions separated by centrifugation at 178,000g for 2h. These fractions were run on 5% to 22.5% exponential acrylamide gradient g e l s i n the b u f f e r system of Laemmli ( 8 8 ) . The bands corresponding to the n i t r a t e reductase (nr ) C t-subunit are marked with open triangles whilst those corresponding to the formate dehydrogenase (fdh) #-subunit are marked by c l o s e d t r i a n g l e s . The migration position of an unidentified band found only i n LCB79 i s indicated with a c r o s s . Lanes a through u are membrane f r a c t i o n s w h i l s t lanes a' through u' are soluble f r a c t i o n s . The samples i n lanes a and a', b and o b 1, etc. are from the same s t r a i n : ( a ) , TS9A grown at 42 C; (b), TS9A grown at 29°C; ( c ) , NH30; ( d ) , RK5274; (e) , RK4353; ( f ) , NH20; (g), LCB517 grown at 42°C; (h) , LCB517 grown at 29°C; ( i ) , RK7-19; (j) , LCB61; ( k ) , CGSC 4459; ( 1 ) , RK7-16; (m), RK7 -36; ( n ) , 356-15; (o), 356-24; (p), LCB68; (q), LCB79; ( r ) , LCB61-22; ( s ) , NH40; ( t ) , CGSC 4444; (u), RK7-36. ( 122 variable between d i f f e r e n t batches of the same s t r a i n . Moreover i t s electrophoretic m o b i l i t y i n t h i s gel system barely distinguished i t from another more intense band. Therefore t h i s type of analysis was only of l i m i t e d v a l u e i n r e v e a l i n g the presence or absence of particular proteins. The s p e c t r a l a n a l y s i s showed c l a s s I I I mutants not to produce fdh nr cytochromes w i t h the p r o p e r t i e s of cytochromes b and b Therefore, they were predicted to lack both the formate dehydrogenase and n i t r a t e reductase od-subunits. Gel analysis of several such strains supported t h i s contention (Figure 31). The exception i s CGSC 4444 which showed a small amount of a band which could be the a£-subunit of ni t r a t e reductase. This would be c o n s i s t e n t with the low l e v e l of nitrat e reductase a c t i v i t y found in t h i s s t r a i n (Table 6). Class II mutants showed v a r i a b l e patterns of production of these polypeptides (Figure 31). The #-subunit of formate dehydrogenase was especially prominent i n many of these s t r a i n s , i n keeping with their high formate oxidase a c t i v i t y (Table 6 ) . The a n a l y s i s of TS9A after o o growth at 29 C or 42 C s u p p o r t e d t h e i d e n t i f i c a t i o n of the c^-subunit of n i t r a t e reductase. .• The high molecular weight band was o more prominent i n the membranes of c e l l s grown at 29 C than i t was o i n c e l l s grown at 42 C. This would be p r e d i c t e d from the higher nitrat e reductase s p e c i f i c a c t i v i t y i n the membranes of c e l l s grown at o 29 (Table 8 ) . I t has p r e v i o u s l y been shown t h a t c h l l mutants possess a normal n i t r a t e reductase a c t i v i t y with benzyl viologen and retain the ^ -subunit of n i t r a t e reductase (59.65). This was evident in the gel a n a l y s i s of s e v e r a l such mutants, for instance NH30. The chlC mutant RK7-19 by c o n t r a s t l a c k e d n i t r a t e reductase a c t i v i t y 123 with any e l e c t r o n donor and had no trace of the df-subunit. S t r a i n LCB79 was of p a r t i c u l a r i n t e r e s t . I t had both membrane bound and sol u b l e p o l y p e p t i d e s of apparent m o l e c u l a r weight approximatly 135,000. These bands were not seen i n the a n a l y s i s of any other s t r a i n s . These bands may be / S - g a l a c t o s i d a s e , a p a r t i a l n i t r a t e reductase protein or a fusion p r o t e i n . In view of the interest i n the mechanism of i n s e r t i o n ' of membrane p r o t e i n s t h i s might be a p a r t i c u l a r l y interesting mutant to study i n more d e t a i l . The single fdhA mutant that has been s t u d i e d , LCB517, was found to lack detectable formate dehydrogenase of-subunit whilst retaining the nitr a t e reductase c£-subunit (Figure 31). These data might be predicted from i t s lack of low-potential cytochromes which are l i k e l y to include cytochrome b Kinetics of Cytochrome Reduction i n chi Mutants The k i n e t i c s of cytochrome r e d u c t i o n i n the wild-type a f t e r anaerobic growth with n i t r a t e were d i f f i c u l t to analyse. I t was hoped that by performing s i m i l a r experiments on c h i mutants, some of which appear to have a smaller number of cytochromes, that k i n e t i c phases could be correlated with particular cytochromes. The results of some of these experiments are shown i n Figure 32. In a l l of the mutants NADH was used as reductant since many of .them have no formate oxidase a c t i v i t y . For none of them did the a d d i t i o n of nitrate have an effect d i f f e r e n t from the addition of oxygen. The apparent absorbance changes which occur on nitrate a d d i t i o n . r e f l e c t only the effect of the oxygen which i s introduced with the n i t r a t e . This would be expected from the 124a FIGURE 32. K i n e t i c s of cytochrome r e d u c t i o n i n c h i mutants. The redox state of the cytochromes was monitored at 428nm using 410nm as reference wavelength. Membranes were prepared from the s t r a i n s indicated after anaerobic growth with n i t r a t e . They were suspended in 50mM potassium phosphate b u f f e r , pH 7.0 i n a f i n a l volume of 3ml. At the times indicated 6/umol of NADH (N), a trac e of oxygen by s t i r r i n g the cuvette (0^) , or 25/imol of n i t r a t e (N0^) were added. LCB162 (AA=0.03; p r o t e i n , 7.1mg/ml); RK7-36 (AA=0.03; p r o t e i n , 2.0mg/ml); RK7-16 (AA=0.03; protein, 2.9mg/ml). 125 lack of n i t r a t e reductase a c t i v i t y in the mutants. In class I I I mutants such as RK7-36 the reduction k i n e t i c s were quite simple. Approximately 15% of the cytochrome was reduced i n the aerobic steady s t a t e and a l l of the remainder was reduced i n what appeared to be a single phase f o l l o w i n g oxygen depletion. This might have been predicted from the simple pattern of cytochrome production found by t i t r a t i o n . The s i n g l e phase i s repeated upon the addition of oxygen or n i t r a t e . The reduction k i n e t i c s observed f o r the c l a s s I strains such as RK7-16 were s i m i l a r to those found f o r the wild-type. However, the slow phase following oxygen d e p l e t i o n involved a larger proportion of the t o t a l cytochrome reduced by NADH. This was repeated upon the a d d i t i o n of oxygen or n i t r a t e . The c l a s s I I mutants, for example LCB162 also have k i n e t i c s resembling those of the wild-type. However in t h i s case the 'aerobic steady state was rather higher at 20% of the t o t a l cytochrome reduced , and a l l of the slow phase following oxygen depletion was not repeated upon the i n t r o d u c t i o n of more oxygen or n i t r a t e . " ' F r a c t i o n a t i o n of Anaerobic Cytochromes from c h i Mutants The fractionation procedure applied previously to the membranes of wild-type c e l l s grown a n a e r o b i c a l l y with n i t r a t e appeared to separate two types of cytochrome c o p u r i f y i n g with either formate dehydrogenase nr or n i t r a t e reductase which were presumed to be cytochromes b and fdh b ' . Therefore i t was hoped t h a t t h i s method would provide an an a l y t i c a l procedure to detect the presence of these two cytochromes 126 i n mutants. When applied to the cytochromes of several c h i mutants and the fdhA mutant the column p r o f i l e s shown i n Figure 33 were obtained. There was only one peak of cytochrome eluted from the column when the cytochromes from LCB79 were fr a c t i o n a t e d by the standard* procedure (Figure 3 3 a ) . This was peak A which corresponded e x a c t l y to the e l u t i o n p o s i t i o n of formate dehydrogenase. Peak B was completely nr absent. Since these mutants l a c k cytochrome b t h i s species i s probably the only cytochrome n o r m a l l y p r e s e n t i n peak B. Redox t i t r a t i o n of the membrane bound cytochrome of TS9A after growth at the non-permissive temperature (Figure 28) gave data similar to that nr found f o r LCB79. This suggested t h a t TS9A lacked cytochrome b This hypothesis i s confirmed by the data shown i n Figure 33b. When the o so l u b i l i s e d membranes of TS9A a f t e r growth at 42 C were fractionated on DEAE Bio-Gel A, peak B was very small i n r e l a t i o n to peak A. This nr r e f l e c t e d the low content of cytochrome b e x i s t i n g i n TS9A when grown at t h i s temperature. I t would be expected for a stra i n retaining a small residual n i t r a t e reductase a c t i v i t y to re t a i n a small amount nr of cytochrome b , and the small s i z e of peak B r e l a t i v e to peak A confirmed t h i s . The cytochrome e l u t i n g i n peak A for TS9A was subjected to a spectrophotometric redox t i t r a t i o n with the result shown i n Figure 34. A curve d e s c r i b i n g the b e h a v i o u r of two components f i t the e x p e r i m e n t a l data b e t t e r t h a n f o r one component. These two hypothetical cytochromes would have mid-point potentials of -2mV and -102mV with the low p o t e n t i a l s p e c i e s c o n t r i b u t i n g 60% of the absorbance d i f f e r e n c e . An even better f i t of the experimental data 127 Fraction Number 127a FIGURE 33. E l u t i o n p r o f i l e s from DEAE B i o - G e l A of cytochromes so l u b i l i s e d from r e s p i r a t o r y mutants. C e l l s were grown anaerobically with n i t r a t e and membranes were prepared, so l u b i l i s e d and fractionated as described in 'Materials and Methods'. A li n e a r gradient of 0 to 0.3M NaCl was applied between fractions 30 and 85. The elution of cytochrome was monitored by measuring the absorbance at 410nm of the fractions. The absorbance u n i t s for each p r o f i l e are a r b i t r a r y ; they have been p l o t t e d on the same axes to emphasize t h e i r s i m i l i a r i t y . The cytochromes were derived from ( a ) , LCB79; (b) , TS9A grown at 42°C; (c) , CGSC 4459; and (d), LCB517. 128 o ho CM o •o T— I < < I 1 a • O 128a FIGURE 34. T i t r a t i o n of cytochromes a s s o c i a t e d with a p a r t i a l l y p u r i f i e d p r e p a r a t i o n of formate dehydrogenase. Membranes were solu b i l i s e d and fractionated from TS9A grown anaerobically with nitr a t e o at 42 C (see Figure 33b). The major peak of cytochrome, eluting with formate dehydrogenase a c t i v i t y was pooled, concentrated and buffered as described i n 'Materials and Methods'. The f i n a l protein concentration was 4.3mg/ml. This m a t e r i a l was subjected to redox t i t r a t i o n . A A represents an absorbance d i f f e r e n c e of 0.01. (A), theoretical curve describing the redox behaviour of one cytochrome; (B) curve describing the redox behaviour of two cytochromes giving the best f i t of the data. 129 could be obtained i f t h r e e components were assumed. In the two component f i t shown there i s a c l e a r divergence of the data from the theoretical curve i n the high p o t e n t i a l region of the t i t r a t i o n . In spite of t h i s , the t i t r a t i o n q uite c l e a r l y shows that peak A contains more than one species of cytochrome. The c o - p u r i f i c a t i o n of formate dehydrogenase a c t i v i t y w i t h peak A suggests that these include fdh cytochrome b which would be the species of p o t e n t i a l about -100mV. The standard f r a c t i o n a t i o n procedure was also a p p l i e d to the class I mutant CGSC 4459. which maps to the chlE locus (Figure 3 3 ) . Like the wild-type; t h i s mutant shows two approximately equal peaks of elution of cytochrome. Although neither n i t r a t e reductase or formate dehydrogenase a c t i v i t y can be detected i n these peaks, the conductivity of the fractions in which they eluted suggested that they corresponded to peaks A and B from the wi l d - t y p e . This suggested that cytochromes fdh nr b and b were r e t a i n e d . The fdhA mutant, LCB517 has been analysed by the same procedure (Figure 3 3 d ) . For t h i s s t r a i n both peak A and B were detected but i n t h i s case peak A was rather smaller. Peak B was shown to correspond to the e l u t i o n p o s i t i o n of n i t r a t e reductase a c t i v i t y and the c o n d u c t i v i t y of the f r a c t i o n s of peak A suggested that i t corresponded to the expected e l u t i o n position of formate dehydrogenase. However LCB517 had been suggested to lac k fdh cytochrome b . This means t h a t peak A must c o n t a i n other cytochromes. The data for the t i t r a t i o n of the peak A cytochrome from TS9A discussed above supports t h i s i d e a . This als o means that the existence of peak A i n the column p r o f i l e for CGSC 4459 did not prove fdh the presence of cytochrome b i n c l a s s I s t r a i n s . Peaks A and B 130 were of equal i n t e n s i t y i n the c l a s s I mutant w h i l s t i n the fdhA mutant peak A was r e l a t i v e l y s m a l l e r . This may mean that the fdhA mutant lacked a cytochrome which CGSC 4459 possessed. Gels of f r a c t i o n s from the column separation of st r a i n s HfrH, CGSC 4459 and LCB517 are shown i n Figure 35. For a l l of these strains peak B c l e a r l y corresponded to the e l u t i o n p o s i t i o n of the nit r a t e reductase of-subunit, and i t i s evident that n i t r a t e reductase i s a major component of t h i s peak. However only i n the case of HfrH can peak A unequivocally be said to correspond to the elution position of the formate dehydrogenase tf-subunit. Neither CGSC 4459 nor LCB517 have bands of the expected m o b i l i t y e l u t i n g i n peak A. These stains were suggested above (Figure 31), and have been shown previously (71). to lack formate dehydrogenase subunits. nr The t i t r a t i o n data on TS9A showed that cytochrome b could be inserted into the membrane only i n the presence of a normal#-subunit. The g e l a n a l y s i s suggested t h a t CGSC 4459 l a c k e d the formate dehydrogenase ^-subunit. By analogy i t can be argued that t h i s s t r a i n fdh should lack cytochrome b . The column p r o f i l e s did not establish fdh < t h i s . To determine i f cytochrome b was present the cytochromes eluting i n peaks A and B for s t r a i n s HfrH, CGSG 4459 and LCB517 were analysed by difference spectroscopy with the result shown i n Figure 36. The cytochrome eluting i n peak B appeared to be the same for both mutants (CGSC 4459 and LCB517) and the wild-type s t r a i n (HfrH). It was approximately 30% reduced by a s c o r b a t e and PMS, and had an absorption maximum at 556nm, but appeared i n a d d i t i o n to contain a small amount of cytochrome absorbing at about 560nm. The cytochrome eluting i n peak A was quite d i f f e r e n t i n the fdhA mutant than i n the 131 b c d e f 9 h i J HfrH 94K 67K»M 43 K >l i 3 0 K » ~ 20K>~ 14K — nr — fdh —m -nr -fdh CGSC 4459 f g h i j I I mm -<94K «<67K H<43K — «30K | i <20K >•» o14K LCB517 20K • 14K b c d e f 9 h i J k 94K>— j j i 67K •<§ 4 3 K - | 30K m-II 1 i i • y 1 i - I «3> « 3 » «fc • -nr -fdh 131a FIGURE 35. SD S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of n i t r a t e reductase and formate dehydrogenase complexes fractionated from chi mutants. The strains i n d i c a t e d were grown anaerobically with nitrate and s o l u b i l i s e d membranes were f r a c t i o n a t e d on DEAE Bio-Gel A. The column fractions were run on 5% to 22.5% l i n e a r acrylamide gradient gels. The lanes a through k were loaded with samples from alternate column fractions from number 75 i n descending order (see Figure 33). The expected migration p o s i t i o n of the n i t r a t e reductase (nr) and formate dehydrogenase (fdh)a-subunits are indicated. The Ot-subunit of formate dehydrogenase i s i n d i c a t e d by a s o l i d t r i a n g l e . The molecular weights of the standard proteins are shown on the gels. 1 3 2 a FIGURE 36. Difference spectra of cytochromes of respiratory mutants fractionated on DEAE Bio-Gel^ A. The s t r a i n s i n d i c a t e d were grown a n a e r o b i c a l l y on n i t r a t e . Cytochromes were s o l u b i l i s e d from the membranes and fractionated on DEAE Bio-Gel A (see Figure's 16 and 33). The f i r s t (peak A) and second (peak B) peaks of cytochrome eluted from the column were pooled, concentrated and bu f f e r e d . The samples (in sucrose) were then s p l i t i n two and one h a l f oxidised by the addition of ferricyanide and was used as ref e r e n c e . The other half was f i r s t reduced by 2.5mM ascorbate with 25/JM PMS and l e f t 15min before freezing in l i q u i d nitrogen and scanning against the reference at 77K (ap-f). Subsequently, i t was thawed, f u l l y reduced by dithionite and rescanned against the reference sample (d-f) . For CGSC 4459,AA=0.1 at a protein concentration of 1.5mg/ml f o r peak A and A A = 0.1 at 0.8mg protein/ml for peak B. For HfrH, peak A had a p r o t e i n concentration of 1.1mg/ml and A A was 0.1, whilst peak B at a p r o t e i n concentration of 0.4mg/ml hadAA=0.03 for 'ap-f' and 0.1 for ' d - f . For LCB517, peak A had a protein concentration of 1. Omg/ml and A A was 0.03 whilst peak B had a 0.3mg protein/ml andAA was 0.03-133 chlE s t r a i n or the wi l d - t y p e . The peak A cytochrome of LCB517 had a broads-peak with absorption maxima at 557nm and 561nm. This material was completely reducible by ascorbate and PMS. However the cytochrome eluting i n peak A i n the wild-type was only 30% reducible by ascorbate and PMS. When p a r t i a l l y reduced i t had a broad #-peak with a d e f i n i t e b component, although not as broad as in^ the peak A cytochrome 562 seen for the fdhA mutant. Upon complete reduction a cytochrome b 556 component became more prominent and t h i s i s suggested to be fdh cytochrome b . In the case of CGSC 4459 the peak A cytochrome was 50% reduced by ascorbate and PMS and when f u l l y reduced had an #-peak centred at 556nm l i k e the wild type (Figure 36). This suggested that i t contained a low p o t e n t i a l cytochrome b r r , which was present i n the 556 wild-type but not the fdhA mutant. I t confirmed the p r e l i m i n a r y conclusion t h a t ,the c h l E mutant, and by i n f e r e n c e a l l c l a s s I fdh mutants, r e t a i n cytochrome b i n s p i t e of t h e i r apparent lack of the other formate dehydrogenase subunits. Double c h i Mutants A l t e r n a t i v e ways of p r o v i n g t h e presence or absence "of fdh nr , - ' cytochromes b and b i n the v a r i o u s classes of mutants were sought. One promising approach was the c o n s t r u c t i o n of mutants co n t a i n i n g two c h i mutations which, on the basis of the r e s u l t s presented above, would be expected to lack p a r t i c u l a r cytochromes. fdh Since cytochrome b - appeared to be the major species i n c h l l mutants i t was believed that c h l l , c l a s s I double mutants should show the same phenotype as c h l l s i n g l e mutants provided that class I 134 mutants r e a l l y do r e t a i n cytochromes b and b The f i r s t i n v e s t i g a t i o n along these l i n e s Involved the chlE d e l e t i o n s t r a i n LCB61-357 and a ^ ( c h l l - l a c Z ) d e r i v a t i v e LCB79-357 (Table 1). The d i f f e r e n c e spectra of these s t r a i n s after anaerobic growth with n i t r a t e are shown i n Figure 37. The s i n g l e mutant had a cytochrome content t y p i c a l of c l a s s I mutants with a sharp4>peak f 0 r cytochrome b with a maximum at 556nm. However i t had a l e v e l of cytochrome b rather higher than the t y p i c a l class I mutanti which 558 might be expected from i t s higher cytochrome d l e v e l . T i t r a t i o n of the b-type cytochrome of t h i s s t r a i n gave a curve quite* similar to that found for any class I mutant with cytochrome t i t r a t i n g over the whole range of +200mV to -150mV (Figure 38). The double mutant had an even higher content of cytochrome d and a corresponding increase i n the > l e v e l of cytochrome b r e l a t i v e to b . I t s t o t a l cytochrome b 558 556 l e v e l was 0.28nmol/mg p r o t e i n , considerably lower than i t s parent which had a l e v e l of 1.00nmol/mg p r o t e i n . The level in LCB61-357 was higher than that found i n other c l a s s I mutants. Redox t i t r a t i o n of the double mutant showed a preponderance of low-potential cytochrome (Figure 38). In s p i t e of the poor 'data, which r e s u l t e d from the low sp e c i f i c content of cytochrome i n the membranes of these c e l l s , i t i s clear that i n the double mutant 60% of the cytochrome had a redox potential of l e s s than OmV while i n i t s chlE parent t h i s value was 39%. The paucity of cytochrome of p o t e n t i a l approximately +50mV i n a chlE , c h l l double mutant when compared to i t s parent suggested that the chlE parent s t r a i n possessed the, cytochrome s p e c i f i e d by nr the c h i I gene, t h a t i s cytochrome b . i n a d d i t i o n the large amount of cytochrome of p o t e n t i a l approximately -100mV i n the double 5 3 5 5 5 5 Wavelength, nm ,135a FIGURE 37. D i f f e r e n c e s p e c t r a o f membranes of a c h l E , c h i I double mutant and i t s i s o g e n i c c h l E p a r e n t . The two s t r a i n s indicated were grown a n a e r o b i c a l l y with n i t r a t e . Dithionite-reduced minus ferricyanide-oxidised d i f f e r e n c e spectra were measured at 77K. LCB79-357, AA=0.03 at a p r o t e i n concentration of 4.7mg/ml; LCB61-357. NAA=0.1 at 6.8mg protein/ml. Co ON 79-357 + 200 ~0 -200 E h ( m v ) 136a FIGURE 38. Redox t i t r a t i o n s of the b-type cytochromes of a chlE, c h l l double mutant and i t s i s o g e n i c chlE parent. T i t r a t i o n s were performed on membrane preparations of the s t r a i n s i n d i c a t e d after anaerobic growth with n i t r a t e . For LCB79-357,A A was 0.002 at a p r o t e i n c o n c e n t r a t i o n of U.9mg/ml. The s o l i d l i n e i s the three-component best f i t . For LCB61-357 . A A was 0.055 at 17.9mg protein/ml. The s o l i d l i n e i s the four component f i t (Table 9). 1 3 7 mutant suggested t h a t i t , and t h e r e f o r e i t s c h l E parent, both possessed cytochrome b*"^. Construction of c h l l " Double Mutants LCB61-357 has been called a class I mutant but i n one aspect, i t s high cytochrome d content, i t i s not r e a l l y t y p i c a l . Therefore, f u r t h e r c h i I double mutants were constructed from other c l a s s I mutants. The c h l E s t r a i n CGSC 4459' was t r a n s d u c e d to c h l l to give s t r a i n NH40 and the c h i A s t r a i n RK7-16 was transduced to c h l l to g i v e NH50. The p u r p o s e of t h e s e c o n s t r u c t i o n s , as described p r e v i o u s l y , was to confirm the presence of cytochromes fdh nr b and b i n the c l a s s I p a r e n t s . In a d d i t i o n , an fdhA, c h l l double mutant (NH20) was constructed from LCB517 i n order to determine what cytochromes are normally masked i n the wild-type by the presence of cytochromes b and b . F i n a l l y a Ach1E68. c h l l s t r a i n (NH10) was constructed from LCB68 to show that t h i s class I I I mutant nr was not producing cytochrome b . A s u i t a b l e donor for s t r a i n c o n s t r u c t i o n by P1 t r a n s d u c t i o n was RK5274 which contains a Tn10 insertion i n the c h l l gene. Therefore LCB68, RK7-16, CGSC 4459 and LCB517 were P1 transduced to t e t r a c y c l i n e resistance using RK5274 as donor. Since the c h l l mutation w i l l be c r y p t i c i n the most of the double mutants i t s presence was confirmed by marker rescue. In the case of the fdhA, c h l l double mutant (NH20) the c a p a c i t y to reduce n i t r a t e using reduced benzyl viologen but not ascorbate and PMS was demonstrated (65). 138 The Cytochrome Content of c h l l Double Mutants The chlE68, c h l l : :Tn10 mutant (NH10,) was shown by difference spectroscopy and redox t i t r a t i o n to produce cytochromes i d e n t i c a l to i t s p a r e n t . However the i n t r o d u c t i o n of the c h i I mutation i n t o the other t h r e e s t r a i n s produced a quite d i s t i n c t phenotypes. The d i f f e r e n c e spectra of the chlE, c h l l s t r a i n (NH40), the chlA, c h l l s t r a i n (NH50) and the fdhA, c h l l s t r a i n (NH20) are shown i n Figure 39. NH40 and NH50 had si m i l a r difference spectra with rather more cytochrome d than t h e i r c l a s s I parents (Figure 23). This has been mentioned p r e v i o u s l y as a property of c h l l mutants. NH20 had a cytochrome d l e v e l s i m i l a r to that of i t s parent. A l l three s t r a i n s had an d£-peak maximum at 555nm with a d i s t i n c t shoulder on the high wavelength s i d e . This was most evident i n the case of NH20 which had a very prominent shoulder at 562nm. NH40 and NH50 appeared to possess an appreciable content of cytochrome D^g2 but a l s o cytochrome b . As expected these two s t r a i n s had 5 5 8 cytochrome levels lower than t h e i r parents at 0.46nmol/mg protein. The l e v e l of cytochrome in NH20 was even lower at 0.35nmol/mg protein. Figure 40 compares the t i t r a t i o n data for these three strains with that of a t y p i c a l c l a s s I mutant CGSC 4459. Four component f i t s of these data are given in Table 9 (p109). In spite of the large apparent differences between the d i f f e r e n c e spectra of these strains and those of their parents, the d i f f e r e n c e s of the redox t i t r a t i o n s were much more s u b t l e . The double mutants NH40 and NH50 contained more cytochrome of high p o t e n t i a l (-> 150mV) than did the class I parent. They were also d e f i c i e n t i n cytochrome of low potential ( < -100mV). Wavelength, nm 139a FIGURE 39. Difference s p e c t r a of membranes of r e s p i r a t o r y chain double mutants. C e l l s were grown a n a e r o b i c a l l y w i t h n i t r a t e . Dithionite-reduced minus f e r r i c y a n i d e - o x i d i s e d difference spectra were measured at 77K. (A), NH40 (A.A = 0.1; p r o t e i n , 5.6mg/ml); (B), NH50 (AA=0.1; protein, 6.4mg/ml); (C), NH20 (AA=0.03; protein, 3.9mg/ml). \ 140 + 200 0 E h (mv) -200 140a FIGURE 40. Redox t i t r a t i o n s of the b-type cytochrome of double chi mutants. T i t r a t i o n s were performed on the membranes of c e l l s grown anaerobically with n i t r a t e . The s o l i d l i n e s are four-component best f i t s of the experimental data, except i n the case of NH20 where a three-component f i t was used (see Table 9).~ NH20 (AA=0.09; protein, 6.9mg/ml); NH40 (AA=0.016; p r o t e i n , 8.7mg/ml); NH50 (AA=0.19; protein, 8.9mg/ml); CGSC 4459 (AA=0.030; protein, 9.1mg/ml). 1 4 1 nr However i n the region of p o t e n t i a l where cytochrome b has been suggested to l i e (around +50mV) , the s i n g l e and double mutants appeared to t i t r a t e s i m i l i a r l y . The t i t r a t i o n of NH20 was rather unusual (Figure 40). I t contained l a r g e l y cytochrome of potential greater than OmV although t h e r e was one cytochrome of p o t e n t i a l approximately -50mV. There were at l e a s t two cytochromes of high potential but no cytochrome of potential -100mV. These double mutants have a l s o been i n v e s t i g a t e d by carbon monoxide difference spectroscopy (Figure 41). The difference spectrum found for NH50 was i d e n t i c a l to that shown for NH40. A l l three strains possessed very high levels of cytochrome o (Table 10, p116) - This correlates with t h e i r high l e v e l s of cytochrome b_. . Cytochrome o 562 was present i n such large amounts that i t obscured the carbon monoxide difference spctrum of cytochrome d which the difference spectrum showed to be present. Using published extinction coefficents i t was calculated that for a l l these s t r a i n s more than 50% of the OC-peak of tlie b-type cytochromes i s contributed by cytochrome o (Table 10). F i n a l l y the cytochromes of the double mutants have been i n v e s t i g a t e d by p a r t i a l r e d u c t i o n u s i n g a s c o r b a t e and PMS and spectroscopy at 77K. The spectra are shown i n Figure 42 and the amount of reduction obtained shown i n Table 10. The cytochromes which were reduced in NH40 and NH50 by ascorbate and PMS were primarily b and 562 b . Cytochrome b was reduced to a much l e s s e r e x t e n t . A 558 556 simil a r pattern of d i f f e r e n t i a l reduction of the cytochromes absorbing at a higher wavelength was seen for NH20 (Figure 42c). r 7 0 0 4 0 0 5 0 0 — i — 6 0 0 Wavelength, n m 142a FIGURE 41 • Carbon monoxide d i f f e r e n c e specrta of r e s p i r a t o r y chain double mutants. Carbon monoxide d i f f e r e n c e spectra were recorded for the membranes of c e l l s grown a n a e r o b i c a l l y with n i t r a t e . (A), NH40 (AA=0.03 i n dC-band region and 0.1 i n Soret band region; p r o t e i n , 7.2mg/ml); NH20 (AA = 0.03 i n <*-band r e g i o n and 0.1 i n Soret band region; protein, 6.8mg/ml). 143 AA Wavelength, nm 1 4 3 a FIGURE 42. Reduction of cytochrome by a s c o r b a t e and PMS i n the membranes of chi double mutants. C e l l s were grown anaerobically with n i t r a t e . Ascorbate (2.5mM) and PMS ( 50/JM)-reduced minus ferricyanide-o x i d i s e d d i f f e r e n c e s p e c t r a ( a p - f ) and d i t h i o n i t e - r e d u c e d minus ascorbate and PMS-reduced d i f f e r e n c e spectra (d-ap) were recorded at 77K. (a), NH50 (3.0mg p r o t e i n / m l ) ; (b) , NH40 (3.6mg protein/ml) (c), NH20 (3.4mg protein/ml).Ak represents an absorbance difference of 0.03. 144 DISCUSSION The r e s u l t s presented i n t h i s t h e s i s i n conjunction with much previously published data, have allowed a model for the arrangement of the electron transport chains of E s c h e r i c h i a c o l i to be proposed. The data i n part 1 of the 'Results' s e c t i o n emphasized the complexity of the cytochromes present in c e l l membranes although the role of some cytochromes i n TMAO r e d u c t i o n c o u l d be e l u c i d a t e d . However the analysis of the cytochromes of c h i mutants gave an in s i g h t into the make-up of both the form a t e - n i t r a t e r e s p i r a t o r y pathway and the two aerobic r e s p i r a t o r y pathways. The scheme proposed r e s t s upon the results of spectrophotometric redox t i t r a t i o n s and so t h i s method w i l l be discussed in some d e t a i l f i r s t . In addition, the data obtained from the c h i mutants w i l l be d i s c u s s e d w i t h r e s p e c t to the membrane assembly and regulation of respiratory enzymes. The cytochrome content of chi and fdh mutants i s summarised i n Table 11. Spectrophotometric Redox T i t r a t i o n It was necessary to go to some lengths to ensure the v a l i d i t y of the redox t i t r a t i o n experiments. The data presented s a t i s f i e d the conditions set for r e l i a b i l i t y i n potentiometric t i t r a t i o n s (25). That i s : (a), the data were independent of the concentrations of mediators used over a wide range; (b) , there was e f f e c t i v e redox buffering over the whole range of po t e n t i a l studied and (c) , there was no hysteresis between the o x i d a t i v e ^ and r e d u c t i v e t i t r a t i o n s . The use of ferricyanide (100) and pyocyanin (27) has been avoided. At the end of a l l t i t r a t i o n s the redox p o t e n t i a l was reduced to below -300mV to TABLE 11 SUMMARY OF RESPIRATORY CHAIN CONTENT OF CHL AND FDH MUTANTS Phenotype Defect In: L o c i Cytochromes nr fdh N i t r a t e Formate Reductase Dehydrogenase d o A c t i v i t y -subunit A c t i v i t y -subunit Wild-type None + C l a s s I Mo-cofactor formation chlA  chlB  chlE  chlG narK Class I I N i t r a t e Reductase chlC c h l l C l a s s I I I Regulation chlB  chlC  chlE n i r R Fdh Formate Dehydrogenase fdhA + ensure that no cytochrome of very low p o t e n t i a l had been overlooked and that no cytochrome had become denatured during the lengthy t i t r a t i o n procedure. The v a l i d i t y of the procedure i s further supported by the good reproducibility of the f i t s derived from the t i t r a t i o n data (Table 4). The results of these experiments re q u i r e careful comparison with those reported elsewhere (26,27,28). Such a comparison i s given i n Table 3« Because of the l i m i t s of r e s o l u t i o n of t h i s procedure the values quoted for the r e l a t i v e amounts and mid-point p o t e n t i a l s of cytochromes are presented merely to f a c i l i t a t e a comparison with other work. It i s not suggested that they represent the properties of the cytochromes present. As mentioned p r e v i o u s l y , the r e s u l t s found in t h i s . s t u d y agree with those of Hendler and Shrager (27) but are dif f e r e n t from those reported by Pudek and Bragg (26) and Reid and Ingledew (28) . There are some problems associated with the chemical t i t r a t i o n technique employed i n these studies (100). These involve the d i f f i c u l t y of o b t a i n i n g redox e q u i l i b r a t i o n between the cytochromes and the electrode over the whole range of p o t e n t i a l being used and i n the c o l l e c t i o n and a n a l y s i s of d a t a . Hendler and Shrager (27) have developed an e l e c t r o d i c potentiometry system and c o l l e c t e d data on l i n e . This allowed a s o p h i s t i c a t e d mathematical analysis of the data to be made. That t h e i r r e s u l t s agree with those obtained here by a much more simple method supports the v a l i d i t y of both approaches. In a d d i t i o n the e l e c t r o d i c t i t r a t i o n described i n the present study (Figure 11) agreed with the r e s u l t of a chemical t i t r a t i o n of the same preparation. 146 The reason for the discrepancy between the r e s u l t s presented i n t h i s thesis and those of Reid and Ingledew (28) i s not known. I t i s considered unlikely that the d i f f e r e n c e s can be accounted for by the different strains or growth co n d i t i o n s being used. Certainly there are differences between the cytochrome content of some strains (Figures 8 and 9) but these are not great enough to account for the differences between these two sets of data. Cytochromes of. the potential +250mV observed by Reid and Ingledew were not found after exactly reproducing thei r growth conditions. On the basis of> their difference spectrum the aerobic c e l l s of exponential phase used i n t h i s study appear similar to the aerobic c e l l s they used. Two f a c t o r s may c o n t r i b u t e to the discrepancy i n the redox t i t r a t i o n r e s u l t s . The f i r s t i s the procedure by which they standardised t h e i r electrode which they do not describe. Since the platinum electrode used i n the present study was standardised by three independent procedures which agreed very closely i t i s believed to be c o r r e c t . Reid and Ingledew describe cytochromes of consistently higher p o t e n t i a l than those found here although there i s no systematic d i f f e r e n c e . Secondly, they do not describe how they derived the mid-point p o t e n t i a l of t h e i r cytochromes from their data. I t was found to be i m p o s s i b l e to do t h i s w i t h o u t r e s o r t to a computer, and i f they did not use one then t h i s would be a source of considerable error. The f i t t i n g procedure performed on the t i t r a t i o n data suggested the existence of at l e a s t four cytochromes i n the membranes of E. c o l i . Due to the error i n c o l l e c t i n g data the use of a f i v e -component f i t was not considered j u s t i f i a b l e . At t h i s l e v e l the values of the parameters being optimised by the f i t t i n g procedure used become 147 extremely s e n s i t i v e to noise and a s i n g l e wayward point had a large effect on the f i t obtained. I t i s p o s s i b l e to resolve more than four components i f a t h i r d v a r i a b l e , that of wavelength, i s also used. In f a c t by computational a n a l y s i s of s p e c t r a of Rhizobium t r i f o l i i taken at a s e r i e s of redox p o t e n t i a l s up to 12 components have been resolved (115). In an attempt to obtain a preparation with a more simple pattern of cytochrome p r o d u c t i o n a number of growth c o n d i t i o n s were investigated. None of these lead to the production of a small number of cytochromes. In a l l cases s t u d i e d the t i t r a t i o n data showed the presence of at l e a s t four' cytochromes. This value i s the minimum number of cytochromes. Other evidence^ points to the existence of even more. The C o n s t i t u t i v e Production of A l l R e s p i r a t o r y Pathways Much of the evidence presented i n Part I of t h i s thesis points to the fact that c o l i produces a l l of i t s r e s p i r a t o r y pathways at a discernable l e v e l regardless of the growth conditions. ^ v. Some evidence for t h i s can be d i s c e r n e d from the d i f f e r e n c e spectra (Figures 4 and 5) . Regardless of the growth conditions there i s always a detectable l e v e l of cytochrome d. This i s associated with cytochrome b although i n many d i f f e r e n c e spectra this cytochrome 558 i s masked by the higher l e v e l s of other b-type cytochromes. The c e l l s w i t h the s m a l l e s t c o n t e n t o f c y t o c h r o m e d are those grown anaerobically with n i t r a t e but even these have a l i t t l e . The l e v e l of cytochrome d under these c o n d i t i o n s i s markedly strain-dependent and 148 varies by a factor of f i v e . HfrH has an exceptionally low l e v e l . Only i n the case of c e l l s grown anaerobically, with TMAO can the f u l l d i v e r s i t y of cytochrome c o n t e n t be a p p r e c i a t e d from the difference spectrum. The b- and c-type cytochrome 06-peak i s very broad in the spectrum of membranes of these c e l l s and i t cl e a r l y shows the presence of s u b s t a n t i a l amounts of s e v e r a l cytochromes absorbing in the wavelength range 548nm to 562nm. The carbon monoxide difference spectra (Figure 12) further support the idea that at least a small amount of a l l species of cytochrome i s produced c o n s t i t u t i v e l y . R e g a r d l e s s of the c u l t u r e c o n d i t i o n s cytochrome o i s d e t e c t e d i n the. membranes by t h i s method. This r e f l e c t s the production of the 'high a e r a t i o n ' oxidase pathway which also includes cytochrome b . Therefore t h i s * cytochrome i s expected 562 in a l l of the difference spectra although again i t may be masked. The reduced minus oxidised difference spectrum and carbon monoxide difference spectrum show that c e l l s grown anaerobically with nitra t e possess the cytochrome d and cytochrome o oxidase pathways. This i s reflected i n t h e i r oxidase a c t i v i t i e s which are quite s i g n i f i c a n t , about one f i f t h of that of c e l l s grown a e r o b i c a l l y ; The scheme of Downie and Cox (30, Figure 2b), suggests that these two pathways would contain four cytochromes absorbing i n theOi-peak region. The membranes ^ , , fdh nr must also contain cytochromes b and b . Therefore a minimum estimate of the N number of b-type cytochromes present i n the membranes of c e l l s grown anaerobically with nitra t e i s s i x . By the same c r i t e r i a c e l l s grown a e r o b i c a l l y have both oxidase pathways regardless of the growth c o n d i t i o n s . In addition the c e l l s grown i n th i s study possess a formate oxidase a c t i v i t y , which implies 149 fdh that they possess cytochrome b . The exponential phase c e l l s grown on a semi-defined medium have a low but detectable nitrate reductase a c t i v i t y but t h i s i s much higher i n s t a t i o n a r y phase c e l l s grown on a nr minimal medium. This i m p l i e s the presence of cytochrome b Therefore i t can be proposed that c e l l s grown a e r o b i c a l l y have the same cytochromes as those grown a n a e r o b i c a l l y with nitrate a l b e i t at very different r e l a t i v e amounts. These results may seem p a r t i c u l a r to the growth conditions being used. For instance, i t i s not common to use c e l l s of stationary phase for physiological studies as they tend to become oxygen limited and t h i s results i n s t r i k i n g metabolic changes. Amongst these i s c l e a r l y the induction of n i t r a t e reductase although i t i s also detected i n lower l e v e l s i n c e l l s of e x p o n e n t i a l phase. I t i s probably not observed in some studies (74) due to the absence of molybdate from the growth medium. S i m i l a r l y the production of a formate dehydrogenase fdh a c t i v i t y , and so presumably cytochrome b i s dependent on the presence of s e l e n i t e and m o lybdate i n the medium ( 8 7 ) . The simultaneous production of the both oxidase pathways i s a problem that Kita and Anraku (20) have addressed but even the growth conditions developed by them to minimize the cytochrome d content do not lead to the exclusive production of the cytochrome o pathway. The c e l l s grown anaerobically in t h i s study were grown i n standing . culture, and not under s t r i c t l y anaerobic conditions. Neither were they harvested a n a e r o b i c a l l y . These f a c t o r s c o u l d have lead to the induction of some aerobic r e s p i r a t o r y pathways. However published data on c e l l s grown s t r i c t l y a n a e r o b i c a l l y (15,28) s t i l l show the presence of cytochrome d and oxidase a c t i v i t i e s . I t would seem then 150 that large changes can be e f f e c t e d i n the r e l a t i v e amounts of the various ' respiratory pathways but that they are a l l to some degree produced c o n s t i t u t i v e l y . The proposed synthesis of a number of species of cytochrome under a l l growth conditions agrees with the r e s u l t s of fourth-order f i n i t e d i f f e r e n c e a n a l y s i s of s p e c t r a . Shipp (23) reported f i n d i n g cytochromes of the same absorption maxima regardless of the growth conditions, although Scott and Poole (24) have f a i l e d to reproduce t h i s f i n d i n g . The value of taking higher d e r i v a t i v e s of spectra i s that i t can detect components present i n r e l a t i v e l y small amounts, which may be masked i n an ordinary difference spectrum (21). Therefore the presence of cytochromes b and b can be shown d i r e c t l y 55 8 562 rather than t h e i r presence be i n g i n f e r r e d from the presence of cytochromes d and o r e s p e c t i v e l y . However f o u r t h - o r d e r f i n i t e d i f f e r e n c e a n a l y s i s cannot d i s t i n g u i s h two cytochromes with very s i m i l a r absorption maxima such as the aerobic cytochrome b,_ ,. 55o fdh nr b and b . Therefore the f a i l u r e of Shipp to see the complete repression of any one cytochrome may be spurious. Scheme for the Arrangement of Cytochromes i n the Membranes of  E. c o l i The spectroscopic a n a l y s i s of f r a c t i o n a t e d cytochromes and the membranes of r e s p i r a t o r y mutants f a c i l i t a t e d the proposal of the scheme given i n Figure 43 f o r the arrangement of cytochromes in the respiratory chains and t h e i r p o s s i b l e i n t e r a c t i o n . No data have been collected on the involvement of quinones in these pathways and so they "200 -r 0 + 200 Potential (mV) 1 5 1 a FIGURE 43« Proposed arrangement of cytochromes i n the aerobic and anaerobic r e s p i r a t o r y chains of E^ c o l i . The cytochromes i d e n t i f i e d in t h i s study are located at p o s i t i o n s corresponding to their redox potenti a l . The'probable major routes of electron transfer are shown by arrows. Question marks i n d i c a t e an u n c e r t a i n t y about the redox potential of the components following them. V 152 are not included i n these schemes. On the basis of the r e s u l t s of others (9.30), i t might be expected for quinones to be involved at several positions. Cytochromes of the TMAO Reductase Pathway Dual-wavelength spectroscopy i n conjunction with trapping of ki n e t i c intermediates has proven a us e f u l approach i n the analysis of the cytochromes of c e l l s grown ana e r o b i c a l l y with TMAO. The membranes of these c e l l s had a very broad <£-peak absorption band and t i t r a t e d over an exceptionally wide range of p o t e n t i a l (Figures 5 and 7). This suggested that a l a r g e number of cytochromes were present. The presence of s u b s t a n t i a l amounts of both of the aerobic respiratory pathways was shown by carbon monoxide differ e n c e spectroscopy (Figure 12). The c-type cytochromes evident i n the d i f f e r e n c e spectrum are suggested to be the very low p o t e n t i a l species evident i n t i t r a t i o n . They were not reduced by ascorbate and PMS and were only p a r t i a l l y reduced by NADH and formate ( F i g u r e s 13 and 22). I f the TMA/TMAO couple r e a l l y does have the high redox p o t e n t i a l , as proposed e a r l i e r i n t h i s thesis, then i t should p r e f e r e n t i a l l y oxidise high potential cytochrome. Since the a d d i t i o n of TMAO to a reduced membrane preparation r a p i d l y r e o x i d i s e d cytochromes c . and c , these 548 552 species must be in r a p i d e q u i l i b r i u m with the TMAO reductase and the cytochrome b s p e c i e s t h a t i s a l s o r a p i d l y r e o x i d i s e d . The 55 6 membranes of these c e l l s had s u b s t a n t i a l NADH and formate oxidase s p e c i f i c a c t i v i t i e s . Neither of these compounds was a good electron donor for TMAO reduction. The NADH-dependent TMAO reductase a c t i v i t y 153 of membrane preparations was l e s s than 5nmol/min/mg protein. Neither did NADH or formate achieve a s u b s t a n t i a l reduction of cytochrome c (Figure 22). Therefore i t i s proposed that a respiratory pathway from an unidentified low p o t e n t i a l e l e c t r o n donor passes electrons through cytochromes c , c and b to TMAO reductase. The e l e c t r o n 548 552 556 donor has not been i d e n t i f i e d but i t i s not NADPH or any amino acid. This anaerobic r e s p i r a t o r y pathway i s proposed to be quite separate from the formate and NADH oxidase pathways. However electrons can be transferred slowly from the NADH and formate dehydrogenases into the TMAO reductase pathway. This accounts f o r the low NADH-TMAO reductase a c t i v i t y observed. Alt e r n a t i v e l y the involvement of a soluble electron c a r r i e r p a s s i n g e l e c t r o n s between the dehydrogenases and TMAO reductase complex could be proposed. The existence of th i s might be demonstrated by ki n e t i c measurements and trapping experiments on whole c e l l s rather than membranes. One problem encountered i n the dual-wavelength studies i s the indication that TMAO ox i d i s e d a s i m i l a r amount of cytochrome after reduction by e i t h e r formate or ascorbate and PMS (Figure 21). These data may be a consequence of the wavelengths chosen. It i s clear that ascorbate and PMS do not reduce the c-type cytochromes, which are the major species r e o x i d i s e d upon TMAO a d d i t i o n when formate i s the reductant. At 555nm, the measuring wavelength employed, the reduction of both b- and c-type cytochromes w i l l be measured. The apparently equal amount of cytochrome r e o x i d i s e d by TMAO a f t e r reduction by either formate or ascorbate and PMS i n fact represents the reoxidation of two quite d i f f e r e n t species. 154 Cytochromes of the Formate-Nitrate Reductase Respiratory Pathway The key to r e s o l v i n g the cytochromes of the for m a t e - n i t r a t e reductase pathway was the use of c h l l ( n a r l ) mutants. These strains nr have been proposed elsewhere (59.65) to lack cytochrome b on the basis of t h e i r e l e c t r o n donor s p e c i f i c i t y f o r n i t r a t e r e d u c t i o n . However no detailed analysis of t h e i r cytochromes has been published. Subsequently several chlC (narG) and a narH mutant were found to produce the same set of cytochromes. Mutants of" t h i s phenotype were named c l a s s I I mutants. This p h e n o t y p i c c l a s s included TS9A, a ' t s c h l C s t r a i n i n w h i c h t h e ' b e n z y l v i o l o g e n l i n k e d n i t r a t e reductase a c t i v i t y i s unusually thermolabile. This stra i n had provided the f i r s t unambiguous proof t h a t c h l C i s the s t r u c t u r a l gene for nitrate reductase (64). In the difference spectrum of c l a s s I I mutants the b-cytochrome #-peak was asymmetrical w i t h a maximum at 555nm and a d e f i n i t e shoulder at 558nm (Figure 2 3 c ) . This i s quite d i s t i n c t from the wild-type i n which i t i s symmetrical and centred at 555.5nm (Figure 5). The amount of cytochrome b i n the c l a s s I I mutants i s also lower than in the wild-type (Table 6). These changes are consistent with the loss nr of the major b-type cytochrome. This must be cytochrome b , which i s known to absorb maximally at about 556nm (15,53). The mutants also show a higher le v e l of cytochrome .d than i s customarily found in the wild-type. As i s usual t h i s cytochrome i s accompanied by an increased l e v e l of cytochrome b which c o n t r i b u t e s to the asymmetry of the 558 #-peak. Titrations of the class I I mutant, L C B 7 9 , showed the presence of 155 large amounts of low p o t e n t i a l cytochrome (Figure 26). In both the three- and four-component f i t s performed on the t i t r a t i o n data of LCB79 a cytochrome of p o t e n t i a l -105mV c o n s t i t u t i n g 59% of the t o t a l b-type cytochrome was i d e n t i f i e d . This i s interpreted to mean that the observed cytochrome i s a r e a l s p e c i e s . The strains of this class have a high formate oxidase, a c t i v i t y (Table 6) and a prominent formate dehydrogenase cX-subunit when analysed on gels (Figure 31). These data suggest that the major l o w - p o t e n t i a l cytochrome evident i n t h e i r membranes was cytochrome b . This conclusion was supported by the studies of cytochrome reduction using ascorbate and PMS (Figure 30). The cytochrome not reduced, which must be primarily of low pot e n t i a l , had an absorption maximum at 556nm i n -agreement with that expected for fdh cytochrome b (15,53). I The mutants of class I I are^also extremely deficient i h cytochrome of potential more than OmV. The t i t r a t i o n data i s consistent with the n r proposal that they lack cytochrome b . T i t r a t i o n of the cytochrome associated with a p a r t i a l l y p u r i f i e d n i t r a t e reductase preparation suggested that i t was composed of two species contributing equally to the absorption at 556nm having p o t e n t i a l s of +20mV and +120mV (Figure 17). This i s precisely the cytochrome which class II mutants appear to be m i s s i n g . A pro o f of t h i s i d e n t i t y came from ion-exchange chromatography of the extracted cytochromes from the wild-type and class I I mutants. The s o l u b i l i s e d cytochrome from the wild-type eluted from a DEAE BijD-Gel A column as two peaks.> The f i r s t , peak A, was associated with formate dehydrogenase a c t i v i t y and the second, peak B, with n i t r a t e reductase a c t i v i t y . Peak B, which contained two cytochromes as described above, was completely absent from the class I I 156 mutants (Figure, 33). Therefore both of the cytochromes associated with nitra t e reductase are absent from the membranes of the mutant. This could mean that one gene s p e c i f i e s two bio c h e m i c a l l y d i s t i n c t cytochromes, as has been shown i n yeast m i t o c h o n d r i a (114). Alternatively, i t may mean that the two cytochromes are genetically d i s t i n c t but one cannot insert i n t o the membrane i n the absence of the other. The analysis of c l a s s I I mutants then, suggested that there are three b-type cytochromes i n the r e s p i r a t o r y chain between formate and n i t r a t e . A low p o t e n t i a l species (E = -100mV), cytochrome b f d h i s m proposed to pass electrons to two high p o t e n t i a l species (E = +20mV m and +120mV) which are associated with n i t r a t e reductase. A l l of these cytochromes appear to absorb at 556nm. This arrangement of the chain seems be t t e r than that proposed by Reid and Ingledew (28). They nr suggested that cytochrome b has a p o t e n t i a l of +10mV since they saw a cytochrome of t h i s p o t e n t i a l only i n c e l l s induced for nitrate r eduction. This cytochrome has the lowest p o t e n t i a l of the three reported by them. I t seems pr e f e r a b l e however to have cytochromes of higher potential associated with the terminal reductase as proposed here. The components of the proposed pathway between formate and nit r a t e have been confirmed using an fdhA mutant. When grown anaerobically on n i t r a t e t h i s s t r a i n produced cytochromes which were i n many ways indistinguishable from those of the wild-type (Figure 22). However, redox t i t r a t i o n showed the mutant to have very l i t t l e cytochrome of p o t e n t i a l l e s s than -100mV w h i l s t the wild-type had a s u b s t a n t i a l amount (Figure 27). The absence of cytochrome of t h i s potential in an 157 J fdhA mutant confirmed the idea that cytochrome b i s a component of potential approximately -100mV. The conclusion that the fdhA mutant lacks cytochrome b^^ i s at variance with the i n t e r p r e t a t i o n of r e s u l t s from Salmonella typhimurium by Barret and Riggs (72). In an fdhA mutant they found 43% of the b-type cytochrome to be r e d u c i b l e by ascorbate. This implied that there was a s u b s t a n t i a l c o n tent of low p o t e n t i a l cytochrome i n these mutants which, on the basis of the r e s u l t s of fdh Ruiz-Herrera and DeMoss (53) they suggested to be cytochrome b They believed that t h e i r fdn mutants, i n which a l l of the cytochrome fdh was reducible by ascorbate, lacked- cytochrome b . The problem with the interpretation of Barret and Riggs (72) i s that other cytochromes fdh as w e l l as cytochrome b are not reduced by ascorbate. Their method of analysis i s i n s u f f i c i e n t to detect the absence of one of a number of low po t e n t i a l cytochromes, s p e c i f i c a l l y cytochrome b^* 1 i n the fdhA mutant. Their fdn mutants have v a r i a b l e cytochrome levels (72) and are suggested to be mutants of the phenotypic c l a s s I I I i d e n t i f i e d i n t h i s study. It, i s noted that they map close to chlB which i s included i n the set of l o c i g i v i n g the c l a s s I I I phenotype (Table 7). The apparent s i m i l a r i t y of the cytochromes of the fdhA mutant nr with those of the w i l d - t y p e suggested t h a t cytochrome b was present. Nitrate reductase a c t i v i t y measured with ascorbate and PMS as e l e c t r o n donors was detected. This a c t i v i t y i s absent i n s t r a i n s n r l a c k i n g cytochrome b ( 6 5 ) . A fdhA, c h l l double mutant (NH20) was constructed to t e s t t h i s hypothesis. This mutant was expected to have extremely low cytochrome l e v e l s since the major cytochrome i n 1 5 8 c h l l s t r a i n s i s cytochrome b . A l s o the double mutant should show the presence or absence of t h i s species i n the fdhA parent. In f a c t , NH20 had quite a high l e v e l of cytochrome, about half that found in the w i l d - t y p e . The shape of the d i f f e r e n c e spectrum was rather unexpected being very reminiscent of c e l l s grown a e r o b i c a l l y to the exponential phase but with an e x c e p t i o n a l l y prominent shoulder at 562nm (Figure 39) . T i t r a t i o n showed NH20 to lack a l l cytochrome of potential -100mV (Figure 40) . This i s taken as further evidence that fdh nr the fdhA mutant la c k s cytochrome b ( w h i l s t r e t a i n i n g b ) and ^ fdh that cytochrome b i s the major species i n c h l l mutants. The Cytochrome o Pathway The major cytochromes of NH20 when grown a n a e r o b i c a l l y with n i t r a t e were those of the 'high a e r a t i o n ' oxidase pathway. The cytochrome o and b content of the membranes of these c e l l s was 562 e x c e p t i o n a l l y high, higher even than that of aerobic c e l l s of the exponential phase. Res'ults obtained on t h i s s t r a i n were consistent with the scheme proposed by K i t a and Anraku (20, Figure 2a) who suggested that three cytochromes are involved in this pathway. However the data to be discussed suggest that a d i f f e r e n t order of these same cytochromes i n the pathway might be preferable (Figure 43). The cytochrome o content of many c h i mutants was found to be higher than in the wild-type grown a n a e r o b i c a l l y with nitrate (Table 6). However the exact shape of the carbon monoxide difference spectrum seemed to be v a r i a b l e between s t r a i n s (Figure 29) . The depth of the trough at 432nm r e l a t i v e to the height of the peak at 417nm was not 159 constant. The existence of an a l t e r n a t i v e cytochrome o, or at least a change i n the p r o p e r t i e s of one species has been shown to occur in response to oxygen l i m i t a t i o n (105). The membranes of the c l a s s I mutants and the double mutants, NH20 and NH40, have the conventional cytochrome o which i s found i n aerobic c e l l s of the exponential phase with an equally intense peak and trough i n the Soret region. However the membranes of the w i l d - t y p e , the fdhA mutant and the c l a s s I I mutants have a variant i n which the peak i s much more prominent than \he trough. The cytochrome o content of the double c h i mutants, NH20, NH40 and NH50 i s exceptionally high (Table 6). Since their t o t a l cytochrome l e v e l , especially in the case of NH20, i s quite low, cytochrome b_-_ 5o2 i s especially prominent. This species always appears to be associated with the presence of cytochrome o. For NH20 the apparent cytochrome o content i s 0.26nmol/mg p r o t e i n w h i l s t the cytochrome b le v e l i s only 0.34nmol/mg protein. This im p l i e s that cytochrome o should contribute 78% of ,the C£-peak of cytochrome b i n these c e l l s . The t i t r a t i o n data does not show any s i n g l e component i n t h i s amount. Rather i t can be resolved into three components of p o t e n t i a l +194mV, +80mV and -79mV c o n t r i b u t i n g 48%, 33% and 19% of the t o t a l cytochrome r e s p e c t i v l y (Table 9 ) . T h i s suggests t h a t the e x t i n c t i o n c o e f f i c e n t s for cytochrome o or cytochrome b are i n e r r o r . Since those cytochrome b species which have been p u r i f i e d have e x t i n c t i o n coefficients between -1 - 1 - 1 10 mM cm and 20 mM cm i t i s more l i k e l y t h a t the extinction c o e f f i c i e n t for cytochrome o may be wrong. Alternatively, cytochrome o may have an e x c e p t i o n a l l y low ot -peak absorption r e l a t i v e to i t s Soret band a b s o r p t i o n . These two p o s s i b i l i t i e s cannot be 160 distinguished from the present data. Regardless of the possible e r r o r y i n extinction c o e f f i c i e n t s i t i s clear that cytochrome o i s a major species in the membrane of NH20. The dif f e r e n c e spectrum als o shows a large amount of cytochrome b . 562 Cytochrome d i s only present at a low l e v e l suggesting that cytochrome b i s only a minor component . The t i t r a t i o n data shows that at 558 least three cytochromes are present. By d i f f e r e n t i a l reduction using ascorbate and PMS the low p o t e n t i a l component (E = -69mV) was shown m to be b . This i s presumably the same as the cytochrome b , of 555 556 potential -45mV reported by K i t a e_t a l . (36) from aerobic c e l l s of exponential phase. By the same procedure these authors report finding a complex of two cytochromes absorbing at 555nm and 562nm which binds carbon monoxide (20) . This probably corresponds to the cytochrome of NH20 which i s reduced by ascorbate and PMS and contains at least a b and a b . One of these must be cytochrome o. 555 562 To determine which, i t i s of value to compare the t i t r a t i o n and \ d i f f e r e n c e spectrum of NH20 a f t e r anaerobic growth with n i t r a t e (Figures 39 and 40) with those of MR43L grown a e r o b i c a l l y on a semi-defined medium to the exponential phase (Figures 4 and 7). NH20 has much more cytochrome b r,„ and more cytochrome of p o t e n t i a l 5 62 +200mV. In a d d i t i o n i t has a higher content of cytochrome o. This leads to the tentative suggestion that cytochrome o has a potential of +200mV and an absorption maximum at 562nm. Since cytochrome o i s a terminal oxidase i t i s reasonable f o r i t to be the species i n the membrane with highest p o t e n t i a l . This c r i t e r i o n i s not s a t i s f i e d by the suggestion of Reid and Ingledew (28) that cytochrome o has a potential of +80mV and an absorption maximum of 556nm. There i s no 161 concensus on the properties of cytochrome o although i t i s known that the #~band absorption minimum i n a carbon monoxide difference spectrum i s 557nm (19). This of course does not give the absorption maximum of the reduced form of cytochrome o. The complex p r o p e r t i e s of the extensively studied cytochrome o of V i t r e o s c i l l a (116,117). which has two redox centres suggest that i t may be unwise to t r y and attribute a single absorption peak and redox p o t e n t i a l to that of c o l i . The Cytochrome d Pathway The other aerobic respiratory pathway i s that ending in cytochrome d. I t s components have become apparent from a n a l y s i s of the cytochromes of the mutants i n class I I I . The most useful has been LCB68 whose cytochromes c o n s i s t almost e n t i r e l y of j.ust two species of potential +220mV and +110mV (Figures 23 and 26). It i s proposed that the high p o t e n t i a l species i s cytochrome b . This i s because the 55o ' d i f f e r e n c e spectrum shows i t to be the major species and the most prominent species in the t i t r a t i o n i s of^high p o t e n t i a l . In addition there i s a small degree of p r e f e r e n t i a l reduction of cytochrome b 558 over b by ascorbate and PMS s u g g e s t i n g that the former has a 556 higher p o t e n t i a l . Cytochrome b ' presumably passes electrons to the 558 oxidase cytochrome d which has an even higher potential (26,28, Figure 11). The possible r o l e of cytochrome a^ i n t h i s pathway i s not known but i t i s noted to be derepressed with cytochrome d i n the class I I I mutants. Aerobically grown c e l l s of s t a t i o n a r y phase were fractionated by the procedure of K i t a et; a l . (36) and found to contain substantial 162 amounts of cytochromes b r and b of h i g h p o t e n t i a l which 556 558 c o - p u r i f i e d with cytochrome d ( F i g u r e s 14 and 15). These are the cytochromes overproduced by c l a s s I I I s t r a i n s . In a d d i t i o n the cytochromes f r a c t i o n a t e d i n c l u d e d a cytochrome of p o t e n t i a l OmV absorbing at 557nm. Th i s was thought to be e q u i v a l e n t to the cytochrome b of K i t a et a l . which from t h e i r data appears to 556 be involved i n the cytochrome o pathway. I t s presence at high levels i n stationary phase c e l l s which have a low content of cytochrome o suggests that i t i s also involved i n the cytochrome d pathway. The way in which the cytochrome o and cytochrome d pathways may i n t e r a c t through t h i s cytochrome i s shown i n Figure 43. The t i t r a t i o n data suggested that LCB68 contained p r i n c i p a l l y two species of cytochrome. These would have the absorption maxima of 559nm and 556 nm found f o r t h i s s t r a i n , the peak at 559nm being the most prominent. The membranes of c e l l s grown a e r o b i c a l l y to s t a t i o n a r y phase, which a l s o have high l e v e l s of cytochrome d have a d i s t i n c t difference spectrum with two l e s s c l e a r l y d i s t i n g u i s h e d absorption maxima at 556nm and 558nm. The d i f f e r e n c e i n absorption maxima between these two preparations may r e f l e c t the presence i n the aerobically grown c e l l s of another cytochrome absorbing at around 557nm, probably the cytochrome b - of K i t a et a l . ( 3 6 ) . The redox t i t r a t i o n 556 — ; shows t h i s species to be present i n only minor amounts i n LCB68. However i f i t i s a mandatory e l e c t r o n c a r r i e r of the cytochrome d pathway then i t must be present at some l e v e l in the mutant since the membranes of these^ c e l l s have a s u b s t a n t i a l NADH oxidase a c t i v i t y . It i s c l e a r that the low p o t e n t i a l cytochrome b__. i s under separate 556 control from the cytochrome d pathway which i s found at a high level • 163 in LCB68. This f i n d i n g i s c o n s i s t e n t with i t s r o l e i n both oxidase pathways. Kinetic Demonstration of the I n t e r a c t i o n of the Cytochromes of  Different Respiratory Chains In the case of the membranes of the c e l l s grown anaerobically with n i t r a t e there was much k i n e t i c evidence for the existence of multiple cytochrome species. The k i n e t i c s of cytochrome reduction by formate i n the wfld-type were extremely complex and could not be f u l l y explained. It was only a f t e r a k i n e t i c a n a l y s i s of mutants that some i n s i g h t i n t o the phases of r e d u c t i o n was gained. The a n a l y s i s of similar data by Sanchez-Crispen ot_ a l . (8) i s questioned since the scheme they propose overlooks some of the b-type cytochromes known to be involved i n electron transport to oxygen. For example their scheme excludes cytochrome o, which has been shown i n t h i s work to be present i n the membrane. In a d d i t i o n some of the detailed findings of Sanchez-Crispen e_t a l . have not been reproduced i n the present study. The extent of cytochrome reduction by ascorbate and PMS which they report i s greater than that found here, as i s the extent of reoxidation of cytochrome upon n i t r a t e a d d i t i o n . Sanchez-Crispen et  a l . i n c l u d e azide i n the a n a e r o b i c growth medium to induce high levels of n i t r a t e reductase i n t h e i r c e l l s . This has been found to produce an unusually high l e v e l ' of cytochrome. Thus, i t i s possible that they may be studying a d i f f e r e n t group of cytochromes to those in the present experiments. The increased n i t r a t e reductase a c t i v i t y would explain the large degree of cytochrome reoxidation that they see 164 upon n i t r a t e a d d i t i o n s i n c e i t has been shown e a r l i e r that' the reduction l e v e l obtained represents a true steady-state. In agreement with Sanche z-Cr i spen et a h , some d i f f e r e n t i a l reduction of cytochrome by a s c o r b a t e and PMS was found i n these studies. The cytochrome which was reduced had an absorption maximum at 556nm while that not reduced had an absorption maximum at 555nm. This probably r e f l e c t s the separation of high and low, potential cytochrome although, as discussed p r e v i o u s l y , not a l l high potential cytochrome need be reduced by ascorbate and PMS. No such d i f f e r e n t i a l reduction of cytochrome absorbing at a d i s t i n c t wavelength could be found by trapping intermediates of the o x i d a t i o n and reduction c y c l e . This suggests that the phases seen represent pseudo-steady states i n which a mixture of cytochrome species are reduced to varying extents.. The a n a l y s i s of mutants was more i n s t r u c t i v e . Compare f o r instance, the reduction k i n e t i c s found for the wild-type (Figure 18) and RK7-16 (Figure 32). In the mutant the slow phase following oxygen depletion accounts for much more of the t o t a l cytochrome than i n the wild-type. This' phase i s repeated when traces of oxygen are introduced suggesting i t represents the reduction of cytochrome i n one of the oxidase pathways. The mutant and the wild-type have the same l e v e l of cytochrome d. Therefore t h i s phase i s suggested to represent the reduction of the cytochromes of the cytochrome o pathway. Certainly the carbon monoxide difference spectrum predicts this phase should be larger i n the mutant. This argument can also be applied to the class II mutants. The reduction k i n e t i c s f o r the c l a s s I I I mutant upon oxygen depletion were apparently monophasic. This supported the results of 165 t i t r a t i o n studies which suggested that these mutants produced only two major cytochromes. The f a s t phase i m m e d i a t e l y f o l l o w i n g oxygen depletion i s suggested to represent the reduction of the cytochromes of the cytochrome d pathway. This idea concurs with the interpretation of S a n c h e z - C r i s p e n e_t a l . . However the w i l d - t y p e and c l a s s I mutant, RK7-16, have very l i t t l e cytochrome d but r e t a i n t h i s fast phase. Therefore the phase has o t h e r components and these are nr suggested to be cytochromes b F i n a l l y , the aerobic steady s t a t e i s suggested to represent the p r e f e r e n t i a l reduction of cytochrome b . This^ i s because i t i s highest i n the c l a s s I I mutants which have most of t h i s cytochrome. However i t must also contain other species since c l a s s I I I mutants which lack cytochrome b have some reduction of cytochrome at t h i s phase. As was found for the w i l d type, these kin e t i c phases cannot be assumed to represent the reduction of d i s c r e t e species but rather the p r e f e r e n t i a l reduction of some . These could have been f u r t h e r investigated by trapping techniques and more k i n e t i c studies on the unusual cytochromes of NH40 and NH20. The Retention of an Inactive Formate-Nitrate Reductase Pathway i n  Many c h i Mutants A large number of s t r a i n s c o l l e c t i v e l y known as class I mutants had cytochromes which were i n many ways indistinguishable from those of the wild-type. Their reduced minus o x i d i s e d difference spectra, t i t r a t i o n data and pattern of cytochrome reduction by ascorbate and PMS were a l l s i m i l a r to those of the w i l d - t y p e . The mutants of t h i s 166 class mapped to several l o c i , i n p a r t i c u l a r those known to be involved i n production and i n s e r t i o n of the molybdenum cofactor (54,57,59),. Therefore most c l a s s I mutants p l e i o t r o p i c a l l y lack both n i t r a t e reductase and formate dehydrogenase. There were s l i g h t differences between the cytochromes of the class I mutants and those of the w i l d - t y p e . The #-peak of b-cytochrome in these mutants i s broader than i n the wild-type due to the increased l e v e l of cytochromes c - and b (Figure 24). This i s r e f l e c t e d ' 552 562 1 i n the t i t r a t i o n curve (Figure 26) by the higher le v e l of cytochrome of potential less than -150mV which was suggested above to be cytochrome c . The c l a s s I mutants a l s o have a h i g h e r t o t a l l e v e l of 552 cytochrome than does the w i l d - t y p e . The carbon monoxide difference spectrum (Figure 29) shows t h e i r cytochrome o to be much larger in amount and d i f f e r e n t ' i n type from that of the wild-type. An understanding of the meaning of these observations came from the study of a narK mutant, RK5271 . This s t r a i n was selected on the basis of i t s derepression f o r TMAO r e d u c t i o n i n the presence of nitr a t e (59). Although t h i s i s u s u a l l y a property of nitrate reductase deficient mutants, RK5271 produces an active formate-nitrate reductase pathway. The cytochromes of t h i s mutant were found to be id e n t i c a l i n a l l respects to those of the c l a s s I mutants. Therefore i t appeared that'the c l a s s I mutants, although not a c t i v e i n the reduction of nitrate by formate, produce the cytochromes of t h i s pathway i n the normal way. In a d d i t i o n they would be derepressed for the TMAO reductase pathway. T h i s i s r e f l e c t e d i n t h e i r high content of cytochrome c . which has been shown above to be c h a r a c t e r i s t i c of 552 c e l l s grown with TMAO. They also appear to be derepressed for the high 167 aeration oxidase pathway which includes cytochromes o and b_, • 5o2 Several t e s t s of t h i s h y p o t h e s i s have been done. The f i r s t involved the running of SDS-polyacrylamide gels of the membranes of class I mutants and l o o k i n g f o r the presence of the &-subunits of formate dehydrogenase and n i t r a t e reductase (Figure 3D. On the basis of data on class II mutants to be discussed l a t e r i t was argued that fdh nr cytochromes b and b would o n l y i n s e r t i n t o the membrane i n the presence of their respective o j-subunits. Electrophoretic analysis of a l l c l a s s I mutants c l e a r l y showed the presence of the n i t r a t e reductase #-subunit. However they a l s o appeared to lack the formate dehydrogenase Of-subunit. The method employed i s admittedly not a s e n s i t i v e t e s t . However more thorough searches using antibodies against formate dehydrogenase have been published (71.118). In such a study formate dehydrogenase subunits were not detected i n the class I mutants CGSC 4459 and RK7-16 (71). but small amounts were found i n strains 356-15 and 356-24 (118). In the present study a better idea of the presence or absence of s u b u n i t s might have been obtained by removing e x t r i n s i c membrane p r o t e i n s by s u i t a b l e washes or by purifying the inner membrane p r i o r to e l e c t r o p h o r e s i s . The limited conclusion of these experiments was that there i s some evidence for n r the presence of cytochrome b but none f o r the presence of fdh cytochrome b i n the membranes of class I mutants. The f r a c t i o n a t i o n of s o l u b i l i s e d cytochromes on DEAE Bio-Gel A discussed previously was applied to the c l a s s I mutant CGSC 4459. Two peaks were eluted from the column corresponding to the peaks A and B found i n the wild-type (Figure 33)• This was a c l e a r proof of the nr presence in t h i s mutant of cytochrome b since peak B i s absent i n 168 s t r a i n s l a c k i n g t h i s cytochrome . However i t did not prove the -' fdh fdh presence of cytochrome b s i n c e cytochrome b had not been shown-to be the only species of cytochrome i n peak A. In an attempt to demonstrate t h i s f a c t the cytochromes of the fdhA mutant were fractionated by the same procedure. In t h i s case there were once again two peaks of cytochrome e l u t e d from the column, the second corresponding to the e l u t i o n p o s i t i o n of n i t r a t e reductase (Figure 33). Peak A was, s t i l l present ( a l b e i t at a iower l e v e l than i n the wild-type) although the spectroscopic evidence,had shown t h i s strain to lack cytochrome b . This was taken as evidence that peak A contained more cytochromes than j u s t b f d h . The t i t r a t i o n of peak A cytochrome from TS9A (Figure 34) , which showed the presence of more than one cytochrome, confirmed t h i s . Therefore i t had not been proven fdh that class I mutants r e t a i n cytochrome b The presence of s e v e r a l cytochromes i n peak A was confirmed by i taking spectra of the cytochromes el u t e d from the column for the wild-type (HfrH), c l a s s I mutant (CGSC 4459) and the fdhA (LCB517) s t r a i n s ( F i g u r e 3 6 ) . Peak A from the fdhA mutant c o n t a i n e d p r i n c i p a l l y high p o t e n t i a l cytochrome species b and b . The 557 562 same peak from the wild-type contained i n a d d i t i o n a low potential fdh cytochrome b which must be cytochrome b . CGSC 4459 also 5 56 -contained t h i s low p o t e n t i a l cytochrome b , 1 ° peak A a n d i s 556 fdh therefore proposed to have retained cytochrome b nr Another test of the presence of cytochromes b and especially x. .fdh cytochrome b i n the membranes of c l a s s I mutants was c l e a r l y ^ fdh called f o r . I t has been mentioned before that cytochrome b i s most prominent' i n c l a s s I I s t r a i n s , f o r instance c h l l mutants. 169 I f the cytochromes of c l a s s I mutants r e a l l y were l i k e those of the parent, which a l l of the spectroscopic data suggested, then a class I, c h l l double mutant should be s i m i l a r to a c h l l s t r a i n . Therefore the cytochrome content of LCB61-357 ( c h l E ) and LCB79-357 (chlE, ^ ( c h l l - l a c Z ) ) was compared. The double mutant was found to have more low-potential cytochrome than did the parent (Figure 38), r e f l e c t i n g i t s deficiency of cytochrome of p o t e n t i a l greater than OmV. The double mutant i n fact resembled other c h l l mutants with a major cytochrome of p o t e n t i a l around -100mV absorbing at 555nm. This i s cytochrome fdh b . Therefore the double mutant and presumably i t s chlE parent fdh nr possess cytochrome b . Cytochrome b must also be present i n the single mutant for the i n t r o d u c t i o n of the c h l l marker to affect the phenotype. Attempts to perform a s i m i l a r a n a l y s i s on two other c l a s s I mutants gave d i f f e r e n t d a t a . A c h i A 1 6 , chll::Tn10 mutant (NH50) and a chlE5, chll::Tn10 mutant (NH40) gave very s i m i l a r phenotypes of cytochrome production. The l e v e l of the 'high a e r a t i o n ' oxidase pathway was e v i d e n t l y h i g h s i n c e the carbon monoxide d i f f e r e n c e spectrum ( F i g u r e 41) i n d i c a t e d the presence of h i g h l e v e l s of cytochrome o, and cytochrome b,__ was very prominent amongst those 562 cytochromes reduced by ascorbate and PMS (Figure 42). T i t r a t i o n of NH40 and NH50 showed the double mutants to have'less low potential ( < OmV) cytochrome than t h e i r parents (Figure 40) . This i s not the expected pattern with c h l l mutants. NH40 and NH50 do however have a cytochrome fdh of p o t e n t i a l -100mV which could be cytochrome b . T i t r a t i o n of aerobic c e l l s of exponential phase does not reveal a cytochrome of thi s p o t e n t i a l , and therefore i t i s not considered to be part of the 170 cytochrome o pathway which i s prominent i n these mutants. So NH40 and fdh NH50 are suggested to r e t a i n cytochrome b and so by implication do thei r class I parents CGSC 4459 and RK7-16. nr These two double mutants must lack cytochrome b since they are c h l l mutants. Unlike t y p i c a l c h l l mutants they do have s i g n i f i c a n t amounts of cytochrome t i t r a t i n g i n the range of ^ potential +200mV to nr OmV. Since these cannot be cytochrome b they, are suggested to be the cytochromes of the cytochrome o pathway. Judging from thei r level of cytochrome d, which i s higher than i n t h e i r parents, NH40 and NH50 must also contain cytochrome b _ _ . This too i s of high p o t e n t i a l 558 nr and i t s increased l e v e l w i l l mask the absence of cytochrome b i n the t i t r a t i o n . The d i f f e r e n c e between the two c h l E , c h l l s t r a i n s , NH50 and LCB79-357 cannot be explained. I t i s suggested to a r i s e from the d i f f e r e n c e between the two c h l E parent s t r a i n s i n v o l v e d . The difference spectrum of LCB61-357 was found to be different from that of CGSC 4459. It had a higher concentration of cytochromes b and d. 55o This i s unlikely to represent a d i f f e r e n c e between the parent strains of the two chlE mutants. The parent of LCB61-357 i s the same strain (MC4100) as RK4353 which i s t h e ' p a r e n t of other t y p i c a l c l a s s I mutants. Therefore AchlEI6 mutants are suggested to be atypical class .1 mutants. The re a d i l y t e s t a b l e a l t e r n a t i v e i s that the ^ ( c h l l - l a c Z ) marker i s d i f f e r e n t from chll::Tn10, but there i s no evidence for t h i s . 171 The Membrane Assembly of Respiratory Complexes The data on c h i mutants has some i n t e r e s t i n g i n f o r m a t i o n bearing on the mechanism of i n s e r t i o n of formate dehydrogenase and n i t r a t e reductase i n t o the membrane . The s i t u a t i o n appears to be rather different for these two enzymes. The f i r s t mutants of t h i s study found to have the c l a s s I I phenotype were LCB160 ( c h l l : : Mu) and LCB162 ( c h l C : : M u ) . The nr absence of cytochrome b from the membrane of the chlC mutant was interpreted as a polar e f f e c t of the Mu i n s e r t i o n into the chlC gene on c h i I gene expression. This had p r e v i o u s l y been shown by other means (66). The subsequent a n a l y s i s of TS9A showed that even i n the absence of a polar e f f e c t chiC mutants do not possess cytochrome nr b i n t h e i r membrane ( F i g u r e 28). Because i t s benzyl viologen l i n k e d n i t r a t e reductase a c t i v i t y i s t h e r m o l a b i l e , TS9A must be a missense mutant i n the n i t r a t e reductase #-subunit. After growth at the n o n - p e r m i s s i v e temperature t h i s mutant was found to lack . . nr cytochrome b in the membrane although i t does have t h i s cytochrome after" growth at the permissive temperature. Since there cannot be a polar effect i n t h i s case i t i s proposed that n i t r a t e reductase can insert into the membrane only as a complex. This must include both the nr Ui-subunit and the cytochrome b . This has previously been suggested on the basis that mutants unable to produce cytochromes accumulate the (£-subunit i n the cytoplasm (119). Mutants of c l a s s I lac k n i t r a t e nr r e d u c t a s e a c t i v i t y but p o s s e s s b o t h cytochrome b and the t^-subunit. This means that the mutational absence of the Mo-cofactor does not cause a s u f f i c i e n t s t r u c t u r a l abnormality for the insertion 172 of the complex to be prevented. Rapid p r o t e o l y t i c cleavage of the uninserted subunits would also account for the absence immunologically detectable fragments i n any chlC mutants although t h i s observation alone may r e f l e c t a bias towards the s e l e c t i o n of e a r l y nonsense mutants (71). This mechanism of i n s e r t i o n of the n i t r a t e reductase complex i s not applicable to the formate dehydrogenase complex. The spectroscopic fdh evidence for the presence of cytochrome b i n the membranes of the class I mutants i s quite good. However there i s equally good evidence that they lack the #-subunit of formate dehydrogenase, both from the e l e c t r o p h o r e t i c a n a l y s i s p r e s e n t e d here and by an immunological fdh nr a n a l y s i s (71). Therefore cytochrome b , u n l i k e cytochrome b , can insert into the membrane i n the absence of the other subunits of the appropriate enzyme. This i s only a problem i f i t i s assumed that formate dehydrogenase i s a complex l i k e n i t r a t e reductase which can only i n s e r t i n t o the membrane when complete. Although several explanations for the observed data are possible, one plausible idea i s that formate dehydrogenase associates with another respiratory complex in the membrane. This constitutes peak A when the solub i l i s e d membranes of anaerobic c e l l s are separated on DEAE Bio-Gel A. The cytochrome can associate' with t h i s i n the absence of the r e s t of the dehydrogenase, but-the dehydrogenase can a s s o c i a t e o n l y when s t r u c t u r a l l y and f u n c t i o n a l l y normal and only then with the cytochrome. However the data i n t h i s t h e s i s provide l i t t l e information to support t h i s or indeed any other hypothesis of t h i s type. . 173 The Regulation of Cytochrome Production i n Class I I I Mutants Whilst the members of the phenotypic classes I and I I belong to recognisable genetic groups the same cannot be said for the members of c l a s s I I I . The l o c i represented i n t h i s c l a s s are chlB, chlC and chlE and there are mutants of other phenotypes at a l l of these l o c i (Table 7). An important observation i n i n t e r p r e t i n g the phenotype of these mutants was that the same pattern of cytochrome production was seen for LCB61-22, a nirR mutant. This- s t r a i n has a mutation causing the p l e i o t r o p i c i n a b i l i t y to e x p r e s s many anaerobic r e s p i r a t o r y systems (81). I t i s believed to be equi v a l e n t , to the fnr and nirA l o c i described elsewhere (76,80) and i s thought to specify a positive regulator required for the expression of the genes for a l l anaerobic respiratory systems. The other c l a s s I I I mutants were tested for their formate hydrogenlyase and t h e i r n i t r i t e reductase a c t i v i t i e s . A l l of them lacked both a c t i v i t i e s (Table 6) and are suggested to also be pleiotropic regulatory mutants. Some of the data presented on these s t r a i n s i s at variance with that reported by other workers. RK7-36 has been reported elsewhere (63, 71) to have the subunits of n i t r a t e reductase and retain the capacity to reduce n i t r i t e . The present i s o l a t e was tested i n an i_n v i t r o complementation system (60) and was found to complement RK7-16 and 356-15. Therefore i t retains at l e a s t some of the characteristics of a chlB mutant. I t i s proposed t h a t the present i s o l a t e i s a chlB, fnr double mutant. A tendency* f o r c h i mutants to mutate a second" time i n the fnr gene has been noted previously (59)-The phenotype of CGSC 4444 i s not t y p i c a l of any other chlC 174 mutant. I t r e t a i n s some n i t r a t e reductase a c t i v i t y but has a low formate dehydrogenase a c t i v i t y . A mutant of genotype narL also maps close to chlC and has a low n i t r a t e reductase a c t i v i t y (59). CGSC 4444 may be of t h i s genotype a l t h o u g h i t could e q u a l l y w e l l be a chlC, f n r double mutant. I t i s most u n l i k e l y that LCB68 i s a double mutant, since the marker responsible for the phenotype has been transduced into several strains without any change of phenotype (68). The properties of the AchlE68 a l l e l e have been s t u d i e d i n a <j){ c h l l - l a c Z ) s t r a i n (68). The /3-galactosidase of the parent i s under the control of nitrat e and oxygen. In the double m u t a n t ^ - g a l a c t o s i d a s e i s s t i l l repressed by oxygen but i s not repressed i n the absence of n i t r a t e when grown anaerobically. S i m i l a r l y i n t h i s study the le v e l of cytochrome d was unaffected by the presence of nitrate i n the anaerobic growth medium. The e f f e c t of t h e Achl_E68 a l l e l e i n the <j){ c h i I - l a c Z ) background suggested that LCB68 should produce nitrate reductase under anaerobic conditions regardless of the presence or absence of n i t r a t e . In f a c t , i t produces no detectable #-subunit or n i t r a t e reductase a c t i v i t y (Table 6, F i g u r e 31) . I t does however produce a high potential cytochrome with an absorption maximum at 556nm (Figures 23 and 25). The c o n s t r u c t i o n of a AchlE68, chll::Tn10 s t r a i n proved nr that t h i s was not cytochrome b since the double mutant produced the same cytochromes as LCB68. Thus the mutation has a d i f f e r e n t effect on the fusion than on the normal n i t r a t e reductase gene and i t i s tempting to speculate that there i s a degree of autoregulation of the nitrate reductase gene. This p o s s i b i l i t y has been proposed on the basis of genetic studies i n A s p e r g i l l u s nidulans (77). 175 There i s c e r t a i n l y a good deal of evidence in the results of th i s study for a complex r e g u l a t o r y system over cytochrome production. It i s clear that i n the wild-type both n i t r a t e and n i t r i t e repress the synthesis of the cytochrome d pathway by a mechanism independent of oxygen. The mutants of c l a s s I a l s o e f f e c t t h i s repression of the cytochrome d pathway by n i t r a t e but are d e r e p r e s s e d for other pathways. For instance, they have a higher cytochrome o le v e l than the wild-type. Class I mutants are marked by the presence of an inactive n i t r a t e reductase complex. Mutants of c l a s s I I lack t h i s complex, although they may possess i n d i v i d u a l s u b u n i t s . Like the c l a s s I mutants, these mutants are derepressed for cytochrome o but they are incapable of e f f e c t i n g the repression of cytochrome d by n i t r a t e . i Moreover they produce l e s s cytochrome d than the c l a s s I I I mutants which form abnormally high l e v e l s of the cytochrome d pathway and apparently very l i t t l e of any other r e s p i r a t o r y pathway. Some class I I I mutants are known to be p l e i o t r o p i c a l l y r e p r e s s e d f o r the expression of many anaerobic r e s p i r a t o r y pathways. This suggests that the system which a c t i v a t e s the expression of these i n the wild-type als o represses the cytochrome d pathway. The complex re g u l a t o r y mechanism d i c t a t i n g the expression of the various pathways would be quite amenable to study by the modern t e c h n i q u e s of b a c t e r i a l genetics. This would be a promising l i n e of research now that the components of the pathways are becoming better understood. 176 BIBLIOGRAPHY 1. Garland, P.B. (1977) PP 1-22 In Haddock, B.A. and Hamilton, W.A. (eds.), ' M i c r o b i a l E n e r g e t i c s ' , S o c i e t y for General Microbiology Symposium 27, Cambridge University Press. 2. Wikstrom, M. and Krab,K. (1980) Curr. Topics. Bioenerg. 20:51-101 . 3. Haddock, B.A. and Jones, C.W. (1977) Bact e r i o l . Rev. 4J_: 4 7 - 9 9 . 4. Bragg, P.D. 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The following subroutine was compiled into FORTRAN and renamed F4. It was used i n conjunction with the U.B.C. Computing Centre l i b r a r y program BMDP:3R to perform four component f i t s of t i t r a t i o n data. Similar programs were written for one, two or three components and used as desired. SUBROUTINE P3R FUN(F,DF,P,X,KASE,NVAR,NPAR,IPASS,XLOSS,IDEP) IMPLICIT REAL*8 (A-H.O-Z) C DOUBLE PRECISION DIMENSION DF(NPAR),P(NPAR),X(NVAR) T1=DEXP((X(T)-P(2))/25.6) T2=DEXP((X('1)-P(4))/25.6) T3=DEXP((X(1)-P(6))/25.6) T4=DEXP((X(1)-P(8))/25.6) C VARIABLES DEFINED TO EXPIDITE CALCULATION F=P(1)/(1+T1)+P(3)/(1+T2)+P(5)/(1+T3)+P(7)/(1+T4) C THE FUNCTION DF(1)=1/(1+T1) DF(2)=(P(1)*T1)/(25.6*((1+T1)**2)) DF(3)=1/(1+T2) DF(4)=(P(3)*T2)/(25.6«((1+T2)**2)) DF(5)=1/(1+T3) DF(6) = (P(5)*T3)/(25.6*«1+T3)**2)) DF(7)=1/(1+T4) DF(8)=(P(7)*T4)/(25.6*((1+T4)**2)) C DERIVATIVES W.R.T. EACH PARAMETER RETURN END i 184 APPENDIX I I . A Control Program (RUNPRO) f o r Curve F i t t i n g Using  BMDP:3R with the Michigan Terminal System i n Batch Mode. The following program was run i n batch mode to perform f i t s on t i t r a t i o n data. I t suppl i e s a l l the information required by the non-linear least squares program, P:3R (Reference 90). Data are read from a f i l e DATA between s p e c i f i e d l i n e numbers and f i t t e d to a function i n the subroutine F4 (see Appendix I ) . The output i s temporarily stored i n a f i l e OUTPUT from which i t i s p r i n t e d on a l o c a l terminal under control of the program ADDL:COPYPR. $SIGN0N PDBA $PASSWORD $EMPTY OUTPUT $RUN F4+PI3R 8=DATA(45,72) 6=0UTPUT /PROB TITLE IS '82-05-18'. /INP VAR=2. FORM='F4.0,F6;0)'. CASE=28. UNIT=8. /VAR NAM=MV,ABS. MAX=150,400. MIN=-600,0. /REGR DEPEND=ABS. PARAM=8. ITER=12. /PARAM INIT=20,0,10,-100,30,-200,20,-300. /END /FINISH $RUN ADDL:COPYPR SCARDS=OUTPUT $SIGN0FF v 185 APPENDIX I I I . Sample Run of The Curve P l o t t i n g Program FAKDAT  to Plot the Result of a Redox T i t r a t i o n Experiment. FAKDAT i s an i n t e r a c t i v e program w r i t t e n i n BASIC which i s designed to a s s i s t i n the a n a l y s i s of data. I t was w r i t t e n by R.A. Scott (California I n s t i t u t e of Technology, 1978) and was modified by Lome Reid of the Biochemistry Department at U.B.C. for use on a Data General Nova 2/10. The program was used to p l o t t i t r a t i o n data on a Hewlett-Packard model 7225 d i g i t a l p l o t t e r . Since t h i s i s a mul t i -purpose program not a l l of i t s features are relevant to the present a p p l i c a t i o n and these l i n e s a r e marked To f a c i l i t a t e understanding of the use of the program comments have been inserted into the conversation and these l i n e s are marked '!'. The parts supplied by the user are underlined. ENTER THE NUMBER OF POINTS DESIRED: 200 X(I) = X2*I (1=1,2 200) ENTER X2 (TIME INTERVAL): 1 # UNCERTAINTIES IN THE DATA WILL BE GIVEN BY: # ERROR = +/- W2*(YMAX-YMIN) .# ENTER W2: 0 # IF SIGMAS ARE TO BE OUTPUT, TYPE 1; OTHERWISE TYPE 0: 0 SELECT FUNCTION: # 1 - N-TH ORDER POLYNOMIAL # 2 - EXPONENTIAL (FIRST ORDER) DECAY # 3 - SECOND ORDER DECAY # 4 - SUM OF EXPONENTIALS 5 - EXP EQN (REDOX TITRATION) ENTER NUMBER (1-5): 5 INPUT P(1) r- P ( 8 ) : 19.7 ? 2A2 ? 23-5 ? _TM ? 34. 1 ? -7 ? 2 2^7 ? -HO These are the parameters returned by BMDP:3R i n units of percentage of t o t a l cytochrome and corrected redox po t e n t i a l . INPUT X START & X FINISH: -200 ? 250 The range of corrected p o t e n t i a l over which the function w i l l be calculated. NUMBER OF DATA POINTS: 28 186 X AXIS CORRECTING FACTOR: J_95 The p o t e n t i a l of the measuring electrode r e l a t i v e to the standard hydrogen electrode, which w i l l be added to the values of potential given as experimental points. Y AXIS SCALING FACTOR: 77.1 Absorbance peak height f o r a f u l l y reduced sample i n arbitrary units, by which the absorbance of the experimental points w i l l be divided. X(1) , Y(1) : +25 ? 0.7 X(2) , Y ( 2 ) : -3 ? 2.5 etc. • X(28) , Y ( 2 8 ) : -450 ? 76.9 The data i n u n i t s of uncorrected redox potential and arbitrary absorbance units. TO CHECK AND/OR EDIT DATA, TYPE 1; OTHERWISE TYPE 0: 1 INPUT DATA STEP VARIABLE (ALL POINTS = 1): 50 01 -200 99-78 50 -87-5 93-56 100 25 57-12 150 137-5 - 16.99 200 250 1.47 These are amongst the points to be plotted as the t i t r a t i o n curve which are reviewed at t h i s stage to ensure no mistakes have been made. TO SEE PLOT TYPE ( 1 ) ; OTHERWISE TYPE (0): 1 CHOOSE EITHER X-Y (1) OR DIGITAL PLOTTING (0): 0 X RANGE: -200 ? 250 Y RANGE : 0 ? 1 10. These variables dictate the portion of the results to be plotted, in th i s case, the whole range of potential and absorbance difference. X AXIS LABEL: POTENTIAL Y AXIS LABEL: ABSORBANCE Y AXIS SCALING,FACTOR: 260 PLOT TITLE: TITRATION DEFAULT CONDITIONS OK? YES(0) OR NO (1): 1 HP PLOTTER COORDINATES LX.LY.UX.UY: 2000 ? 1000 ? 8500 ? 7050 This step sets the size of the graph plotted. DATA POINT CHARACTER TYPE C-1 , S-2, T-3, D-4: 2 NUMBER OF DIVISIONS ON X AXIS & Y AXIS: 3 ? 4 INPUT DATA STEP VARIABLE; ALL POINTS = 1 : 1 SOLID LINE (0) OR USER SYMBOLS (1): 0 PAPER IN & READY TO GO? TYPE ANY KEY WHEN READY: P 

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