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The 503nm pigment of Escherichia coli: properties and function Kamitakahara-Pearlstone, Joyce Reiko 1972

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THE  503nm PIGMENT OF ESCHERICHIA COLI: PROPERTIES AND FUNCTION  by  JOYCE REIKO KAMITAKAHARA-PEARLSTONE B.Sc,  Honours, U n i v e r s i t y  of T o r o n t o , 1967  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  the Department of BIOCHEMISTRY  We accept t h i s t h e s i s as conforming required  THE  t o the  standard  UNIVERSITY OF BRITISH COLUMBIA September, 1972.  In p r e s e n t i n g t h i s t h e s i s  in p a r t i a l  f u l f i l m e n t o f the r e q u i r e m e n t s  an advanced degree at the U n i v e r s i t y of B r i t i s h C o l u m b i a , I agree the L i b r a r y I further  s h a l l make i t  freely  available for  agree t h a t p e r m i s s i o n f o r e x t e n s i v e  r e f e r e n c e and copying of t h i s  of  this thesis for  written  ^  It  i s understood  thesis  „  ,  BIOCHEMISTRY  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8. Canada  Date  SEPT.  15.  1972  or  publication  f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my  permission.  Department or  that c o p y i n g or  that  study.  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department by h i s r e p r e s e n t a t i v e s .  for  i  ABSTRACT  The streptomycin (Sm)-dependent mutants of four Escherichia c o l i s t r a i n s (known to be catabolite-repression negative) were found to have a 25-35% lower aerobic e f f i c i e n c y ( y i e l d of protein from glucose) than the parent wild-type organisms. A non-dependent revertant derived from the Sm-dependent mutant of JE. c o l i B was also lower i n e f f i c i e n c y . In contrast, the anaerobic protein y i e l d s (although lower than that from aerobic growth) were i d e n t i c a l f o r both types of c e l l s . In both wild-type and mutant s t r a i n s the  glucose-reduced/peroxide-  oxidized difference spectra of whole c e l l s showed the same content of cytochromes and f l a v i n . However, Sm-dependent strains lacked the 503nm pigment which was present i n a l l wild-type s t r a i n s . These observations suggested that the 503nm pigment (P503) (of previously unknown function) might play a role i n energy metabolism. Addition of gluconate to a i r - o x i d i z e d c e l l s produced  the P503 peak  p r i o r to the appearance of cytochrome and f l a v i n absorption bands. Addit i o n of succinate, g l y c e r o l , lactate or acetate produced  cytochrome and  f l a v i n spectra but not P503. Addition of the amino acid L-methionine  to  a i r - o x i d i z e d c e l l s e l i c i t e d the P503 band rapidly but the other components of the difference spectrum did not appear u n t i l l a t e r . No other amino acid tested had t h i s s p e c i f i c e f f e c t on P503. When wild-type c e l l s were grown on l i m i t i n g glucose-salts medium containing 2,4-dinitrophenol (500 juM) , the y i e l d of c e l l protein decreased and formation of P503 was  was  i n h i b i t e d . Also, growth under these  conditions resulted i n derepressed levels of fumarase, aconitase and, unexpectedly, glucose 6-phosphate dehydrogenase.  ii  From these observations the general hypothesis was developed that P503 p a r t i c i p a t e s i n an oxidative energy-yielding pathway i n which the i n i t i a l substrate i s reduced nicotinamide adenine triphosphate (NADPH) , (the  f i r s t product of gluconate metabolism i n glucose-grown 12. c o l i ) . The synthesis of P503 was observed with glucose or gluconate as  carbon source. Less P503 was synthesized when succinate, g l y c e r o l or l a c t a t e was the carbon source, i n which cases generation of NADPH would be less e f f i c i e n t . When L-methionine was present i n medium containing glucose as carbon source, the synthesis of P503 was i n h i b i t e d . Other amino acids did not i n h i b i t synthesis of P503. The unique response of P503 to methionine suggests regulation of the pigment by this amino acid. In agreement with observations of other investigators, P503 was found to be transient and l a b i l e , and could not be detected i n c e l l s which had been frozen and thawed or i n c e l l extracts.  iii  TABLE OF CONTENTS Page ABSTRACT  i  TABLE OF CONTENTS  i i i  LIST OF TABLES  viii  LIST OF FIGURES  x  ABBREVIATIONS USED  xi  ACKNOWLEDGEMENTS  xiii  INTRODUCTION  1  METHODS AND MATERIALS  8  I.  8  Organisms i. ii. iii. iv.  II.  Wild-type strains  8  Glycogen-less mutant  8  Methionine-less mutant  8  Streptomycin mutants  8  a.  Dependent  8  b.  Non-dependent revertant  9  c.  Resistant ( i n d i f f e r e n t )  9  Growth of Cultures i. ii. iii.  9  Media  9  Measurement of c e l l growth  9  Growth and harvesting of c e l l s f o r difference spectra  10  a.  Aerobic growth with glucose  10  b.  Inhibitors  10  c.  Variation of carbon source  11  iv  Table of Contents  (continued) Page  d.  iv. III.  L-methionine  and i t s analogues as  growth supplement  12  e.  Other compounds as growth supplement. . .  13  f.  Anaerobic growth with glucose  13  Growth and harvesting  of c e l l s f o r enzyme assays.  Determination of E f f i c i e n c y of C e l l Growth i. ii.  Definition Amount of protein formed from glucose by c e l l s  14  Amount of protein formed from glucose by c e l l s growing anaerobically  iv.  14 14  growing a e r o b i c a l l y iii.  13  15  Amount of protein formed from glucose by c e l l s growing a e r o b i c a l l y and anaerobically on medium supplemented with 2,4-dinitrophenol  IV.  V.  Chemical Analyses  16  i.  Protein  16  ii.  Glucose  16  Determination of Difference Spectra i. ii.  VI.  16  C e l l s grown with l i m i t i n g carbon source  18  C e l l s grown with excess carbon source .  19  Enzyme Assays i. ii.  17  20  Fumarase Aconitase  20 '.  20  V  Table of Contents (continued) Page iii. iv. v. VII.  Glucose 6-phosphate dehydrogenase. . . .  21  Glucokinase  21  I s o c i t r i c dehydrogenase  21  Attempts to Obtain a Peak at 503nm i n Crude C e l l Extracts or Permeabilized  PART A:  Cells  Y i e l d of C e l l Protein from Glucose  RESULTS I.  23 23  Amount of Protein Formed from Glucose by C e l l s Growing Aerobically  II.  23  Amount of Protein Formed from Glucose by C e l l s  III.  Growing Anaerobically  23  The "Aerobic Increment" of Protein Y i e l d from Glucose .  28  DISCUSSION PART B;  21  28  Difference Spectra  31  RESULTS  31  DISCUSSION  36  PART C:  Characterization of P503  I.  II.  Attempts to Isolate P503  37  RESULTS  37  DISCUSSION  37  Production 1.  37  of the 503nm Peak  39  E f f e c t of sugars and acids on production of the 503nm peak  39  vi  Table of Contents  (continued)  Page  2.  RESULTS  39  DISCUSSION  43  E f f e c t of L-amino acids on production of the  3.  503nm peak  46  RESULTS  46  DISCUSSION  46  E f f e c t of L-methionine analogues on production i  4.  III.  of the 503nm peak  52  RESULTS  52  DISCUSSION  52  E f f e c t of other compounds on production of the 503nm peak  55  RESULTS  55  DISCUSSION  55  Formation 1.  2.  3.  (Synthesis) of P503  59  E f f e c t of 2,4-dinitrophenol on formation of P503. . .  59  RESULTS  59  DISCUSSION  70  E f f e c t of anaerobic growth on formation of P503 . . .  82  RESULTS  82  DISCUSSION  82  N u t r i t i o n a l e f f e c t s on formation of P503  87  RESULTS  87  DISCUSSION  '  90  vii  Table of Contents  (continued) Page  4.  E f f e c t of L-methionine  and i t s analogues as  growth supplement on formation of P503  96  RESULTS  96  DISCUSSION 5.  105  E f f e c t of other compounds as growth supplement on formation of P503  115  RESULTS  115  GENERAL DISCUSSION AND CONCLUSIONS  116  BIBLIOGRAPHY  126  viii  LIST OF TABLES Page Table  Table  Table  Table  Table  Table  Table Table Table Table  Table  I. Protein y i e l d s from glucose for wild-type and streptomycin-dependent (and non-dependent revertant) mutant of Escherichia c o l i s t r a i n s B, CRX, K12 and UL grown aerobically  24  I I . Protein y i e l d s from glucose for streptomycinr e s i s t a n t mutants (1 dihydrostreptomycin) of E_. c o l i strains B, CRX, K12 and UL grown aerobically  25  I I I . Protein y i e l d s from glucose f o r wild-type and streptomycin-dependent strains of E_. c o l i B and CRX during aerobic and anaerobic growth  26  IV. Calculation of "aerobic increment" of protein y i e l d f o r the wild-type and streptomycindependent strains of E_. c o l i B  29  V.  Height (or depth) of pigment peaks (or trough) obtained from the reduced/oxidized difference spectra of wild-type and streptomycin-dependent (or non-dependent revertant) strains of E_. c o l i B, CRX, and K12  VI. Height (or depth) of pigment peaks (or trough) obtained from the reduced/oxidized difference spectra of wild-type and streptomycin-resistant (_ dihydrostreptomycin) strains of E_. c o l i B, CRX and K12 VII. E f f e c t of various sugars and acids on production of the 503nm peak i n wild-type E_. c o l i B and B-SG1 . VIII.  E f f e c t of L-amino acids on production of the 503nm peak i n wild-type E_. c o l i B  IX. E f f e c t of L-methionine analogues on production of the 503nm peak i n wild-type E_. c o l i B X.  E f f e c t of various concentrations of supplement on the e f f i c i e n c y of growth for wild-type E. c o l i B  XI. E f f e c t on e f f i c i e n c y during aerobic and anaerobic growth on 2,4-dinitrophenolsupplemented medium f o r wild-type E_. c o l i B  33  35 42 47 53  62  63  ix  L i s t of Tables  (continued) Page  Table  Table  Table  Table  Table  Table  Table  X I I . E f f e c t of various concentrations of supplement on the height of P503 i n wild-type E_. c o l i B . . . XIII.  XIV.  XV.  XVI.  XVII.  XVIII.  68  S p e c i f i c a c t i v i t i e s of enzymes from wild-type, wild-type supplemented with 2,4-dinitrophenol, streptomycin-dependent, and non-dependent revertant s t r a i n s of E_. c o l i B  69  S p e c i f i c a c t i v i t i e s of enzymes from wild-type E_. c o l i B grown on glucose supplemented with 1,3,5-tribromophenol or chloramphenicol  71  E f f e c t of various carbon sources as growth supplement on P503 formation (synthesis) i n wild-type E_. c o l i B  88  E f f e c t of concentration of carbon source i n medium (glucose or gluconate) on P503 formation (synthesis) i n wild-type E_. c o l i B . . .  89  E f f e c t of L-methionine and i t s analogues as growth supplement on P503 formation (synthesis) i n wild-type E_. c o l i B  97  E f f e c t of L-methionine and i t s analogues as growth supplement on rate of growth (doubling time) of wild-type E. c o l i B  100  X  LIST OF FIGURES  Page Figure  Figure  Figure  Figure  Figure  Figure  Figure  Figure  Figure  1.  2.  3.  4.  5.  6.  7.  8.  9.  Structures of chemical or photoreduced porphyrins  6  Y i e l d of c e l l protein from glucose f o r wild-type E_. c o l i B and the streptomycindependent mutant  27  Reduced/oxidized difference spectra a f t e r addition of glucose to a i r - o x i d i z e d c e l l suspensions of E_. c o l i B  32  Reduced/oxidized difference spectra a f t e r addition of gluconate to a i r - o x i d i z e d suspensions of wild-type E_. c o l i B  40  Reduced/oxidized difference spectra a f t e r addition.of glucose or succinate to a i r oxidized suspensions of wild-type E_. c o l i B . . . .  42.  Reduced/oxidized difference spectra a f t e r addition of L-methionine to a i r - o x i d i z e d suspensions of wild-type E_. c o l i B  48  Growth of wild-type E_. c o l i B on minimal s a l t s - l i m i t i n g glucose medium supplemented with various concentrations of 2,4-dinitrophenol. .  60  Reduced/oxidized difference spectra a f t e r addition of glucose to a i r - o x i d i z e d suspensions of wild-type E_. c o l i B grown i n medium supplemented with 2,4-dinitrophenol  65  Reduced/oxidized difference spectra a f t e r addition of glucose to wild-type E_. c o l i B grown anaerobically under a nitrogen atmosphere . .  83 98  Figure  10.  Structural analogues of methionine  Figure  11.  Biphasic nature of growth, glucose consumption, and protein y i e l d from glucose for the met" mutant, E. c o l i K12W-6  102  Pathways of carbohydrate metabolism i n E_. c o l i . . .  120  Figure  12.  ABBREVIATIONS USED  P503  pigment absorbing maximally at 503nm  A420 (600) or absorbance^20  absorbance at 420nm (600nm)  &A  (i) difference i n absorbance between two suspensions or solutions, one oxidized and the other reduced or ( i i ) change i n absorbancy e f f i c i e n c y of growth (ug/ml increase  E  i n protein per ug/ml glucose consumed) Sm  streptomycin  DHSm  dihydrostreptomycin nicotinamide adenine dinucleotide  ITADP+ (NADPH)  phosphate  (reduced)  nicotinamide adenine dinucleotide NAD  +  (NADH)  (reduced) f l a v i n adenine dinucleotide  FAD'  f l a v i n mononucleotide  (reduced)  FMN (FMNH )  adenosine triphosphate  ATP  inorganic phosphate  Pi  pyrophosphate  PP  2,4-dinitrophenol  DNP  1,3 ,5-tribromophenol  TBP  chloramphenicol  CM  ethylenediamine t e t r a a c e t i c acid  EDTA  phosphoenolpyruvate  2  PEP  xii  Abbreviations Used (continued)  G6-P  glucose 6-phosphate  TCA  t r i c a r b o x y l i c acid cycle  HMP  hexosemonophosphate  E-D  Entner-Doudoroff  E-M  Embden-Meyerhof vitamin B^2  f-met  formyl methionine  acidmethionyl 5^ ^  tRNAS M met tRNAlr F  transfer ribonucleic  aa's  amino acids  Km  Michaelis Menton constant  Vmax  maximum v e l o c i t y  et  me  oni  e  r -ii • ..methionine transfer ribonucleic acid formyl  ACKNOWLEDGEMENTS  I would l i k e to express my sincere gratitude to Dr. W.J. Polglase for h i s excellent guidance and h e l p f u l c r i t i c i s m throughout the course of both the research and w r i t i n g of this thesis. His keen i n t e r e s t and enthusiasm w i l l long be remembered. To my colleague, Don J . Rainnie, I extend my warmest thanks f o r h i s encouragement and informative discussions during the past four years. To my parents, s i b l i i n g s , parents-in-law  and e s p e c i a l l y to my  husband Paul, whose patience and encouragement have been a great comf o r t , I am most appreciative. F i n a l l y I would l i k e to acknowledge the Medical Research Council of Canada f o r f i n a n c i a l  support.  1  INTRODUCTION  The occurrence of a pigment absorbing maximally around 503nm has been reported i n several organisms. Its presence i n baker's yeast (Saccharomyces cerevisiae) was noted by Lindenmayer  (1959), Lindenmayer  and Smith (1964) , Nosoh (1964) and Nosoh and Itoh (1965). Several s t r a i n s of b a c t e r i a , including B a c i l l u s megaterium  (Kepes, 1964), Azotobacter  chroococcum and A. agile (Goucher and Kocholaty, 1957), Escherichia c o l i (Kepes, 1964; Olden and Hempfling, 1970), Rhizobium japonicum (Appleby, 1969) , and f i n a l l y the photosynthetic bacterium Rhodopseudomonas sp. (Cooper, 1956) were also found to possess t h i s pigment. In a l l cases, the  presence of P503 was detected v i a spectrophotometry methods. In  view of the existence of these observations, i t seems rather odd that l i t t l e attention has been given to this pigment. Perhaps an explanation for t h i s paucity of information can be found when one studies the conditions under which P503 i s found. The pigment i s absent i n c e l l s grown on an enriched or complex medium; only when a r e s t r i c t i v e  environment (minimal s a l t s ) surrounds the organism  i s P503 formed. Thus Labbe, Volland and Chaix (1967) noted the lack of P503 when yeast c e l l s were grown on nutrient agar or peptone broth, and i t s appearance when a synthetic medium was used. S i m i l a r l y , Nosoh and Itoh (1965) reported that E_. c o l i and B a c i l l u s s u b t i l i s grown i n nutrient broth and excess glucose f a i l e d to exhibit a 503nm peak. Furthermore, the i n t e n s i t y of i t s absorption has been found to be dependent upon the physiol o g i c a l state of the culture. Lindenmayer  (1959) observed the peak to vary  greatly i n height with the age and treatment of the c e l l s . Whereas l o g a r i t h mically growing c e l l s showed a large P503 peak, stationary phase c e l l s had  2  a s u b s t a n t i a l l y decreased l e v e l of the pigment (Lindenmayer, 1959; Labbe et a l . , 1967; Nosoh, 1964). The l a t t e r author noted that addition of a reducing agent such as sodium d i t h i o n i t e to oxidized c e l l s resulted i n a 503nm band i n the absorption spectrum. However, with a more concentrated reducing agent, the 503nm band disappeared completely. I t i s not s u r p r i s i n g , therefore, that the pigment has received l i t t l e  attention  by investigators of microbial metabolism. In contrast to the cytochromes, the transient, unstable nature of P503 has discouraged attempted  i s o l a t i o n or s t r u c t u r a l determination.  Reduction of the pigment by glucose results i n i t s appearance along with the cytochromes and f l a v i n ; however, the P503 peak begins to decrease and eventually disappears i n 10-15  minutes while the spectrum of the  cytochrome components remains constant. Lindenmayer and Smith (1964) observed the disappearance of the pigment on rupture of the c e l l s ; t h i s r e s u l t discouraged more d i r e c t studies. The only successful attempt reported by Labbe et a l . (1967) who  was  claimed that P503 was present i n the  soluble f r a c t i o n i n yeast c e l l s homogenized under nitrogen. The structure of P503 has not yet been determined. The p o s s i b i l i t y of i t s being cytochrome-like or f l a v i n - l i k e has been ruled out due to several observations: (1) The disappearance of the P503 peak does not r e s u l t i n a concomitant  appearance (or increase) of any other absorption  band either i n the v i s i b l e (700-450nm) or Soret (~'425nm) region of the spectrum  (Lindenmayer, 1959); (2) The 503nm pigment does not have an  absorption spectrum of a hemoprotein; whereas the l a t t e r shows several bands (-^,j5, andY ) varying i n absorption i n t e n s i t y , P503 has a single peak (Lindenmayer and Smith, 1964); (3) The cytochrome system and f l a v o proteins react rapidly on exhaustion or addition of oxygen while changes  3  observed with P503 are much slower (Lindenmayer and Smith, 1964). These workers suggested that the absorbancy and r e a c t i v i t y of P503 was i n agreement with properties possessed by the semiquinone form of ubiquinone. Several findings are inconsistent with this view. Ubiquinones (or coenyme Q) are a class of homologues derived from  2,3-dimethoxy-5-methyl-benzo-  quinone; s u b s t i t u t i o n by varying lengths of side chain isoprenoid units at p o s i t i o n 6 of the quinone ring accounts f o r the d i f f e r e n t homologues formed. In microorganisms, the presence and amount of coenzyme Q has been correlated with respiratory rates. Thus the formation of coenzyme Q was found i n two f a c u l t a t i v e organisms, E_. c o l i and Saccharomyces  cerevisiae,  to be adaptive and induced only by growth under aerobic conditions (Lester and Crane, 1959; Sugimura and Rudney, 1960). The 503nm pigment, however, i s synthesized to the same extent under both aerobic and anaerobic atmospheres (Lindenmayer, 1959; Nosoh, 1964). A second point against the ident i t y of P503 as a semiquinone of ubiquinone i s that Labbe et a l .  (1967)  located the 503nm pigment i n the soluble f r a c t i o n of yeast c e l l s ; on the other hand, Cox et a l . (1970) indicated that ubisemiquinone exists i n the membrane f r a c t i o n i n E_. c o l i . Furthermore, the l a t t e r compound was stable, unlike P503. Several publications i n the l i t e r a t u r e have referred to the semiquinoid intermediate of f l a v i n possessing an absorption band around 490-510nm. Although a semiquinone of free FMN does absorb at 503nm i n a 1 M HC1 s o l u t i o n (pH 0), the form present under p h y s i o l o g i c a l conditions (neutral pH) absorbs at 565nm (Beinert, 1956a; 1956b). In 1957, Beinert extended h i s studies to include three enzyme-bound flavoproteins (a f a t t y acyl CoA dehydrogenase, L-amino acid oxidase of snake venom, and the "old yellow  4  enzyme of yeast"- acyl dehydrogenase,  to C-^) . Investigations of  difference spectra suggested to him that the s p e c t r a l charaderistics of free f l a v i n s during oxidation-reduction at neutral pH were almost i d e n t i c a l with those observed with fatty acyl CoA dehydrogenaseflavoprotein (the l a t t e r enzyme having been reduced by octanoyl CoA). Both spectra showed a broad absorption band between 500-650nm with a peak at 560-570nm. A noticeable difference was  that the band absorbing  maximally at 565nm had approximately 20 times the i n t e n s i t y with enzymebound FAD as with free FAD. The band having an absorption maximum at 565nm during reduction and re-oxidation of free f l a v i n s was ascribed to a semiquinone form of the f l a v i n (Beinert, 1956b). In addition, the intermediate formed upon reduction of the acyl dehydrogenase with d i t h i o n i t e as substrate was  concluded to be a semiquinone form of the enzyme-  bound f l a v i n (Beinert, 1957). The formation and disappearance of the l a t t e r intermediate was  found by Beinert to be inversely related to the  absorption c h a r a c t e r i s t i c s of the f l a v i n band at about 450nm. In the case of L-amino acid oxidase, the absorption band ranged from 520-650nm with a maximum value at about 545nm. Spectrophotometric analysis of the enzyme-substrate D-amino acid oxidase (Yagi and Ozawa, 1962; 1963)  complex of  indicated an absorption  spectrum possessing a c h a r a c t e r i s t i c shoulder at 490nm. In their work, benzoate was used as a substitute f o r the r e a l substrate since i t does not undergo dehydrogenation  to reduce FAD , thereby enabling them to +  study the complex of apo-enzyme + FAD + substrate-substitute. Since the shoulder at 490nm was observed only i n the enzyme-substrate  model, not  i n the holoenzyme (apo-enzyme + FAD), they concluded that the band at 490nm was  c h a r a c t e r i s t i c f o r the a r t i f i c i a l complex composed of the  5  apo-enzyme, coenzyme and substrate-substitute. Labbe et a l . (1967) and Olden and Hempfling (1970) favoured a protoporphomethene  structure f o r P503 on the basis of the s t r i k i n g s i m i l a r i t y  i n the properties of both compounds. The photoreduction of porphyrins proceeds through two stages from porphyrin (absorption peaks at 400, 550, 590nm) to dihydroporphyrin (absorption peaks at 440, 735nm) to tetrahydroporphyrin or protoporphomethene (absorption peak at 500nm) (Figure 1). Certain reducing agents such as sodium d i t h i o n i t e can reduce the protoporphomethene beyond the second stage to the colourless hexahydroporphyrin or porphyrinogen (Mauzerall, 1962). This t h i r d stage of reduction accounted for the a b i l i t y of d i t h i o n i t e , s u l f i t e , potassium cyanide and cysteine to cause the disappearance of the 503nm peak (Labbe et a l . , 1967; Nosoh, 1964). The former group suggested that the mechanism involved the linkage of s u l f i t e and cyanide to the methene bridge to form the colourless structure. No evidence thus f a r has been established to refute this protoporphomethene i d e n t i t y of P503. A p o s i t i v e i d e n t i f i c a t i o n awaits the actual i s o l a t i o n and p u r i f i c a t i o n of the pigment. If the chemical nature of the 503nm pigment remains unknown, i t s function i n c e l l u l a r metabolism has been even more obscure. No functional hypotheses have been advanced p r i o r to the present i n v e s t i g a t i o n . Our i n t e r e s t i n P503 resulted from investigations on the decreased e f f i c i e n c y of growth i n Sm-dependent mutants compared to the wild-type parents observed only during aerobic growth. The growth of microorganisms i n r e l a t i o n to their energy supply (the amount of substrate u t i l i z e d ) under aerobic versus anaerobic conditions has been well  documented  (Bauchop and Elsden, 1960; Kormancikova, Kovac and Vidova, 1969; Coukell, 1969; Cox et at., 1970). A l i n e a r r e l a t i o n s h i p exists between the c e l l  V  H /  H  H  H  H  H  porphyrin  (absorption peaks at 400, 550, 590nm)  dihydroporphyrin or phlorin (absorption peaks at 440, 735nm)  V " 7  NH  NH  Figure 1.  / \  NH  HN H  tetrahydroporphyrin or porphomethene  hexahydroporphyrin or porphyrinogen  (absorption peak at 500nm)  (colourless)  Structures of chemical or photoreduced porphyrins. The structures were taken from Mauzerall  (1962). A l k y l substituents on the ring carbons and hydrogens on the methene bridges are omitted.  7  mass formed and amount of glucose consumed at low concentrations of substrate. This r a t i o (termed v a r i o u s l y : growth y i e l d constant, molar growth y i e l d , or simply y i e l d constant) i s substantially higher during aerobic growth than under anaerobic conditions i n wild-type E_. c o l i A preliminary survey of four E_. c o l i strains (B, K12, UL and  cells. CRX)  indicated that although the Sm-dependent mutants were just as e f f i c i e n t as t h e i r wild-type parents during anaerobic growth, t h e i r aerobic e f f i ciency was  as much as 25-35% less than the wild-type organisms. A d e f i -  ciency was  therefore implicated i n an aerobic route of energy metabolism.  The Sm-dependent mutants had previously been found to d i f f e r from wild-type c e l l s i n the following manner: (1) decreased aerobic e f f i c i e n c y (Coukell, 1969), (2) excretion of v a l i n e i n the medium when glucose  was  the substrate (Bragg and Polglase, 1962; Tirunarayanan, Vischer and Renner, 1962) , and (3) derepressed l e v e l s of the catabolite repressible enzymes c i t r a t e synthase, fumarase, and aconitase (Coukell, 1969). In view of the numerous aspects of deficiency exemplified by the Sm-dependent phenotype, i t was  of i n t e r e s t to do comparative  studies of  hydrogen metabolism i n the wild-type versus Sm-dependent mutant. An analysis of the reduced/oxidized difference spectra of wild-type, Sm-dependent, and non-dependent revertant (derived from the Sm-dependent mutant) indicated that the cytochromes a (600-650nm), cytochrome b (560nm), and f l a v i n trough (460nm) were present to the same extent i n a l l three cases. The only difference observed was at 503nm (P503) i n wild-type c e l l s was  that the large symmetrical peak absent or n e g l i g i b l e i n the  Sm-  dependent and revertant mutants. Since no role for this pigment, or i t s p a r t i c i p a t i o n i n any reaction was yet known, t h i s study was  directed to  i t s characterization i n an attempt to ascertain i t s function.  8  METHODS AND MATERIALS  I.  Organisms  i.  Wild-type s t r a i n s :  Most of the research was c a r r i e d out on E_. c o l i B (ATCC 11303) . For comparative purposes, E_. c o l i strains K12, UL, and CRX were also used. S t r a i n K12 was obtained from Dr. J . Stock, Department of Microbiology, U n i v e r s i t y of B r i t i s h Columbia, Vancouver; s t r a i n UL was i s o l a t e d at the  Department of Bacteriology, U n i v e r s i t y of Laval, Quebec C i t y ; s t r a i n  CRX was obtained from Laboratory of Hygiene, Ottawa, Ontario. Stock s t r a i n s were stored on minimal salts-agar slopes and subcultured every month.  ii.  Glycogen-less mutant:  E_. c o l i B-SG1, a mutant of E_. c o l i B lacking adenosine diphosphateglucose:o(-4-glucosyl transferase and thus incapable of synthesizing glycogen, was o r i g i n a l l y i s o l a t e d and characterized by Dr. J . P r e i s s , Department of Biochemistry, University of C a l i f o r n i a , Davis.  iii.  Methionine-less mutant:  E. c o l i K12-met(~) (ATCC 25019), a mutant of E. c o l i K12  requiring  L-methionine f o r growth, was used i n l a t e r experiments a f t e r the discovery that L-methionine affected the production and formation (synthesis) of P503 i n a unique manner.  iv.  Streptomycin mutants: a.  Spontaneous  Dependent:  Sm-dependent mutants of E_. c o l i s t r a i n s B, K12, CRX,  UL were i s o l a t e d from the wild-type organisms as described by Coukell  and  9  and Polglase (1965) . b.  Non-dependent  revertant:  E_. c o l i Br4 i s a Sm-sensitive revertant  obtained from Sm-dependent  E_. c o l i B by M.B. Coukell. c.  Resistant  (indifferent):  Sm-resistant mutants of E. c o l i B, K12, CRX, and UL were i s o l a t e d from the wild-type organisms as described by Coukell and Polglase (1965) .  II.  Growth of Cultures i.  Media:  A l l cultures were grown on a basal s a l t s medium, pH 7 . 0 , consisting of K HP0 2  4  (0.7%), KH P0 2  4  (0.3%),  (NH ) S0 4  2  4  (0.1%) and MgS0 .7H 0 (0.02%) 4  2  as described by Davis and M i n g i o l i (1950), but without c i t r a t e . The pH of the medium was adjusted to 7.5 with aqueous sodium hydroxide for growth of cultures overnight. succinate, and lactate)  The carbon sources (glucose, gluconate, and growth supplement (^-methionine),  glycerol, autoclaved  separately i n concentrated s o l u t i o n , were added to the medium to obtain the f i n a l concentration required. Sm-dependent strains were grown on medium supplemented with 1 mg of dihydrostreptomycin  (Merck, Sharp, and Dohme,  Montreal, Quebec) / m l .  ii.  Measurment of c e l l  growth:  A Beckman B spectrophotometer with l i g h t path of 1.0 cm was used to determine t u r b i d i t y of the c u l t u r e s . The change in absorbancy of the cultures was measured at 420nm (or at 600nm when c e l l s were grown on minimal medium supplemented with 2 , 4 - d i n i t r o p h e n o l ) . Throughout  D i s t i l l e d water was used as blank.  the growth p e r i o d , protein determination*:- were made (see  10  METHODS IV, i ) , and standard curves of plotted f o r the wild-type,  A420  versus protein (ug/ml) were  Sm-dependent, and Sm-resistant (jidihydrostrepto-  mycin) mutants of each s t r a i n . In addition, f o r wild-type E_. c o l i B, a graph r e l a t i n g A g  n n  to protein was prepared. In a l l cases, the absorbancy  was d i r e c t l y proportional to c e l l p r o t e i n .  iii.  Growth and harvesting a.  of c e l l s f o r difference spectra:  Aerobic growth with glucose:  Wild-type, non-dependent revertant, Sm-dependent, and Sm-resistant ( i n d i f f e r e n t ) c e l l s were grown under aerobic conditions as follows. A culture of c e l l s , grown a e r o b i c a l l y overnight  in a 37*  shaking water bath  on 0 . 2 % glucose-salts medium (plus supplementation where required i n part i c u l a r experiments) was inoculated into 500 ml of fresh s a l t s medium (plus supplementation when required) and grown with aeration on 0 , 2 % glucose from A ^ Q  0 . 1 to 1 . 0 . The suspension was then cooled on i c e ,  harvested by centrifugation  ( 1 2 , 0 0 0  g f o r 15 minutes) at 4 • , washed i n  0 . 0 1 M potassium phosphate buffer (pH 7 . 0 ) of fresh s a l t s medium to give  A ^ Q  and resuspended i n 2 l i t r e s  0 . 1 0 - 0 . 2 0 .  The c e l l s were grown on  l i m i t i n g glucose ( 0 . 0 4 % , w/v), harvested j u s t at completion of growth, washed once i n 2 0 0 ml of phosphate b u f f e r , and resuspended i n buffer at a protein concentration  of 6.25 mg/ml. To e f f e c t the depletion of substrate  as w e l l as to oxidize the electron transport system, the suspension of c e l l s was aerated  f o r 1 - 1 1 / 2 hr at 3 7 "  p r i o r to analysis of the difference  spectra. b.  Inhibitors:  Wild-type E. c o l i B was grown as before on l i m i t i n g glucose supplemented with i n h i b i t o r s as follows. Stock solutions of 1 0 " ^ M 2 , 4 - d i n i t r o -  11  phenol  (The B r i t i s h  Drug Houses L t d . , P o o l e , E n g l a n d ) , 1 . 3 x l 0 ~  1,3,5-tribromophenol  ( g i f t of D.J. R a i n n i e ) , 10~^ M  chloramphenicol  (Parke, Davis & Co., D e t r o i t , M i c h i g a n ) , and 10~1 M hydrochloride  M  z  hydroxylamine-  (Matheson Coleman & B e l l , Norwood, Ohio) were p r e p a r e d  and the a p p r o p r i a t e volume (ml) added to the medium c o n c e n t r a t i o n d e s i r e d i n each c. To determine  t o g i v e the f i n a l  case.  V a r i a t i o n o f carbon s o u r c e  :  the e f f e c t o f v a r i o u s carbon s o u r c e s as growth s u p p l e -  ment on P503 f o r m a t i o n (see T a b l e XV and T a b l e X V I ) , the c e l l s were first  adapted  (sodium  salt;  Chemical  f o r growth on the a p p r o p r i a t e compound. Thus 0.2% Eastman O r g a n i c Chemicals)  Co.) was s u b s t i t u t e d f o r g l u c o s e and t h e c e l l s  METHODS I I , i i i , In  the case of s u c c i n a t e , 10 ml o f glucose-grown  Fisher S c i e n t i f i c  water b a t h .  c e l l s were i n o c u l a t e d  (pH 6.0) c o n t a i n i n g 0.8% s u c c i n a t e (disodium  Co.) and grown o v e r n i g h t at 37' i n a s h a k i n g  were n e c e s s a r y ) . 10 ml o f t h i s o v e r n i g h t c u l t u r e were  t o r e - i n o c u l a t e 100 ml of f r e s h 0.8% s u c c i n a t e - s a l t s medium, and  the c e l l s of  t r e a t e d as i n  (The pH a f t e r o v e r n i g h t growth was 7.0 and t h e r e f o r e no  re-adjustments used  (The N i c h o l s  a.  i n t o 100 ml minimal medium salt;  o r 0.2% g l y c e r o l  gluconate  grown o v e r n i g h t a second  the second  2 litres  time. Without  o v e r n i g h t c u l t u r e was d i l u t e d  o f f r e s h medium  c o n t a i n i n g 0.067%  harvesting, a portion  to A ^ Q o f 0.10-0.20 i n ( o r 0.53%) s u c c i n a t e . The  pH d u r i n g t h e f i n a l growth (monitored a t 30 minute i n t e r v a l s )  remained  c o n s t a n t at 7.0. F o r experiments grown on 0.5% l a c t a t e  i n v o l v i n g l a c t a t e as carbon s o u r c e , 5 ml of c e l l s (sodium  l a t e d i n t o 100 ml o f minimal  salt;  Fisher S c i e n t i f i c  salts-lactate  Co.) were i n o c u -  (1.0%) medium  f o r overnight  12  growth. Without harvesting, a portion of the overnight culture was d i l u t e d to A^Q of 0 . 1 0 i n 2 l i t r e s of fresh medium containing 0.2% l a c t a t e . In a l l cases, the c e l l s were harvested as described i n METHODS I I , i i i ,  a, at the end of growth (when carbon source was  l i m i t i n g ) or during log phase (when excess carbon source was used). d.  L-methionine and i t s analogues as growth supplement :  Two  per cent stock solutions of L-methionine (Calbiochem, Los  Angeles), casein hydrolysate (vitamin and s a l t free; N u t r i t i o n a l Biochemicals Corp., Cleveland, Ohio), L-ethionine (Sigma Chemical Co., St. Louis, M i s s o u r i ) , DL-ethionine  (Sigma Chemical Co.), DL-  norleucine ( N u t r i t i o n a l Biochemicals Corp.), and DL-selenomethionine (Sigma Chemical Co.) were prepared, and the appropriate volume added to the f i n a l growth medium to give the desired concentration. The 1 8 L-amino acids (+ methionine) were weighed out i n the proportions given f o r casein hydrolysate ( i . e . , the 2 1 amino acids minus tryptophan, glutamine, and asparagine)  (West and Todd, 1 9 5 5 )  and dissolved i n 5 0 ml of heated medium. This amino acid mixture was subsequently made up to a f i n a l volume which resulted i n a t o t a l concentration of  0.10%.  Alanine, glutamic a c i d , v a l i n e , phenylalanine,  serine, aspartic acid, arginine, h i s t i d i n e , threonine, and isoleucine were purchased  from Calbiochem, Los Angeles. Glycine, tyrosine, and  l y s i n e monohydrochloride were supplied by N u t r i t i o n a l  Biochemicals  Corp., Cleveland, Ohio. Leucine, cystine, and hydroxyproline were obtained from Mann Research Laboratories Inc., New York, N.Y. The experimental conditions described i n METHODS I I , i i i ,  a were followed  13  for growth of cultures. e.  Other compounds as growth supplement •*  Wild-type E_. c o l i B was grown as before on l i m i t i n g glucose,(0.04%)s a l t s medium supplemented  with one of the following compounds: f o l i c  acid (0.01% and 0.10%) (Sigma Chemical Co.); ascorbic acid (0.01% and 0.05%) (Calbiochem, Los Angeles); glycine (0.01% and 0.10%) ( N u t r i t i o n a l Biochemicals Corp.); L-cysteine (10~3 M) (Mann Research Laboratories Inc., New York); 2-deoxy-D-glucose (0.04%) (20% aqueous solution; Sigma Chemical Co.) . f.  Anaerobic growth with glucose '  The c e l l s were prepared as described i n METHODS I I I , i i i . At the end of anaerobic growth, the 37" water bath was replaced by an i c e - s a l t bath and nitrogen gas flow continued f o r 1 hr. The c e l l s were then harvested, washed i n 0.01 M potassium phosphate b u f f e r , pH 7.0, and the difference spectra studied immediately. iv.  Growth and harvesting of c e l l s f o r enzyme assays :  Overnight cultures were centrifuged, washed, and resuspended i n fresh salts-glucose (0.2%) medium (supplemented with 250 uM 2,4-dinitrophenol i n p a r t i c u l a r experiments; see Table IX). Suspensions were grown from A420 of 0.10 to 0.80 (or A g  n n  of 0.06 to 0.50 f o r DNP-containing  media). The c e l l s were harvested, washed i n 0.01 M potassium  phosphate  b u f f e r , pH 7.0, then resuspended i n 500 ml of 0.2% glucose-salts medium i n separate flasks supplemented  with various concentrations of 2,4-  dinitrophenol, 1,3,5-tribromophenol, or chloramphenicol as indicated in Table XIII and Table XIV. The c e l l s were then grown with aeration at  14  37' u n t i l they reached A  of about 0.80 (or g A  n n  of 0.47) at which  point they were c h i l l e d i n i c e , harvested by centrifugation, washed i n 0.01 M potassium phosphate buffer (pH 8.0), and stored at 0' as packed  cells.  To obtain sonic extracts, the c e l l s were resuspended  to a f i n a l  concentration of 1 g wet weight of c e l l s / 15 ml of 0.10 M potassium phosphate buffer (pH 8.0) and the suspension kept i n i c e . Three ml of this c e l l suspension were pipetted into a small p l a s t i c tube. With the p l a s t i c tube held i n an ice-water bath, the c e l l s were disrupted by a 30 second treatment i n a Branson model W 1350 S o n i f i e r operated at 100 watts. III.  Determination of E f f i c i e n c y of C e l l Growth i.  Definition:  The e f f i c i e n c y of growth (E) on minimal s a l t s medium was defined as the ug/ml increase i n protein per ug/ml glucose consumed. ii.  Amount of protein formed from glucose by c e l l s  growing  aerobically: C e l l s were grown aerobically on 0.2% glucose-minimal s a l t s medium from  A420 °f 0.10 to 0.80, harvested during log phase, washed i n 0.01 M  potassium phosphate buffer, pH 7.0, then resuspended i n fresh medium (minus glucose) to give A ^ n °f 0.10 and incubated i n a 37 * shaking water bath f o r 30 minutes. After the absorbancy was re-measured, 400 ug/ml of glucose were added and a sample (5 ml) removed immediately f o r i n i t i a l glucose and protein determination. Thereafter, portions were removed at 15 minute i n t e r v a l s throughout the growth period, cooled immediately i n  15  an i c e - s a l t bath, and the c e l l s c e n t r i f u g e d down at 1 2 , 0 0 0 g f o r 2 0 minutes. The p e l l e t was resuspended i n d i s t i l l e d water f o r p r o t e i n determination w h i l e the supernatant was analyzed f o r glucose content (see METHODS IV, i and i i ) . To ensure complete exhaustion of glucose, sampling was continued f o r 1 h r a f t e r the absorbancy had become constant. iii.  Amount of p r o t e i n formed from glucose by c e l l s growing anaerobically:  Glucose-starved c e l l s were prepared as described i n METHODS I I I , i i . A f t e r the 3 0 minute i n c u b a t i o n p e r i o d , the c e l l s were f i r s t grown a e r o b i c a l l y from A ^ Q of 0.10 to 0.40 i n minimal s a l t s medium c o n t a i n i n g 650 ug glucose / ml, then t r a n s f e r r e d i n t o a 2 l i t r e Erlenmeyer  flask  f i t t e d w i t h a two-hole stopper. L-grade (99.99% pure) n i t r o g e n gas (Canadian L i q u i d A i r L t d . , Vancouver, B r i t i s h Columbia) was bubbled through an i n l e t tube v i g o r o u s l y f o r 1 0 minutes, then at a slower r a t e . A g l a s s e x i t tube, placed 1.3 cm above the c u l t u r e , allowed gases to escape. With anaerobic conditions being maintained, the c e l l s were grown at 3 7 * u n t i l the glucose was exhausted (A420 ~ 1 . 0 ) . Portions of 5 ml were removed at 1 5 minute i n t e r v a l s , i n i t i a l l y , and t h e r e a f t e r at 7 1/2 minute i n t e r v a l s (when growth was more rapid) throughout anaerobic growth. In the sampling procedure, a 1 0 ml Hamilton syringe w i t h p l a s t i c tubing attached to i t s needle was i n s e r t e d through the gas-exit tube a f t e r the trapped a i r had been e x p e l l e d from the s y r i n g e . This procedure was followed u n t i l the end of growth. A l l samples f o r p r o t e i n and glucose determinations were treated as previously described.  16  iv.  Amount of protein formed from glucose by c e l l s growing aerobically and anaerobically on medium supplemented with 2,4-dinitrophenol :  The experimental d e t a i l s were as described i n METHODS I I I , i i and iii,  the medium being supplemented with 250 pmoles 2,4-dinitrophenol.  The absorbancy was  followed at 600nm. The c e l l s were transferred to  anaerobic conditions at A^QQ of 0.20 absorbance of 0.40 IV.  (equivalent to c e l l s having an  at 420nm).  Chemical Analyses i.  Protein :  Protein was  determined by the method of Lowry, Rosebrough, F a r r ,  and Randall (1951). A standard protein curve r e l a t i n g the absorbancy at 500nm to concentration of bovine Y - g l o b u l i n from 0-400 ug/ml ( C a l i f o r n i a Corp. for Biochemical Research, Los Angeles, was  California)  prepared, and a l l samples (in duplicate) were d i l u t e d with  water so that the protein concentration was ii.  Glucose  Glucose was  distilled  i n the range of 20 to 300 ug/ml.  :  determined by using Glucostat, a commercial preparation  purchased from Worthington Biochemical Corp., Freehold, New  Jersey. The  l y o p h i l i z e d enzyme preparation and chromogen, contained i n separate v i a l s , were dissolved together i n 50 ml of d i s t i l l e d water (Glucostat reagent). A standard curve was  prepared of A^QQ versus glucose concen-  t r a t i o n , from 0 to 300 ug/ml. A l l samples ( i n duplicate) were d i l u t e d with d i s t i l l e d water so that the glucose concentration was  i n the range  17  of  0-300 ug/ml. A 0.5 ml volume o f each sample was p i p e t t e d i n t o a  t e s t - t u b e . P o t a s s i u m phosphate b u f f e r , 2.5 ml o f 0.10 M (pH 7.0), f o l l o w e d by 2.0 ml o f the G l u c o s t a t and t h e c o n t e n t s mixed of  reagent were added  t o t h e sample  t h o r o u g h l y on a V o r t e x m i x e r . A f t e r  incubation  t h i s m i x t u r e f o r 30 minutes i n a 37" water b a t h , 2 drops o f 6 N  HC1 were added  t o s t a b i l i z e the s o l u t i o n a t room temperature, f o l l o w e d  by 5.0 ml o f d i s t i l l e d water. Absorbancy  r e a d i n g s were taken a t 400nm  and t h e g l u c o s e c o n c e n t r a t i o n o b t a i n e d from the s t a n d a r d c u r v e ,  V.  Determination of D i f f e r e n c e  Spectra  A Cary 15 s p e c t r o p h o t o m e t e r w i t h an absorbance s c a l e o f 0.1 is,  showing  (that  a maximum absorbance o f 0.1) was used. I n most c a s e s , the  s p e c t r a were scanned from 700nm t o 430nm. I n the F i g u r e s o f d i f f e r e n c e s p e c t r a t h e curves were c o r r e c t e d f o r s l i g h t  deviation, of the b a s e l i n e .  The nomenclature a s s i g n e d t o b a c t e r i a l r e s p i r a t o r y pigments has been reviewed e x t e n s i v e l y i n the l i t e r a t u r e  (Smith, 1961; B a r t s c h , 1968;  Kamen and H o r i o , 1970). The cytochrome pigments  (hemoproteins) have  been grouped i n t o t h e c l a s s e s a, b and c depending upon t h e type o f i r o n - p o r p h y r i n p r o s t h e t i c group and p r o t e i n as w e l l as upon the n a t u r e of  the bond formed between the two p o r t i o n s o f the m o l e c u l e . F u r t h e r  breakdown o f the nomenclature t o a-^, a2 and so on was n e c e s s i t a t e d i n the  b a c t e r i a l system t o d i f f e r e n t i a t e among t h e numerous forms found  i n various microorganisms. G e n e r a l l y , t h e a b s o r p t i o n maxima o f t h e cytochromes i n t h e l i t e r a t u r e have been determined from the reduced spectrum s i n c e sharp bands appear i n the v i s i b l e  r e g i o n , 500-650nm. I n c o n t r a s t , t h e o x i d i z e d  18  spectrum shows b r o a d , d i f f u s e bands (Smith, 1961). E_. c o l i  i s known to p o s s e s s the  f o l l o w i n g cytochromes which absorb  i n the v i s i b l e spectrum: (1) cytochrome a^, the a b s o r p t i o n maximum of i t s <k band o c c u r r i n g at 590nm; (2) cytochrome a^ maximum at 630nm; (3) cytochrome b^ showing and  530nm r e s p e c t i v e l y . In l o g - p h a s e c e l l s ,  E_. c o l i i s cytochrome o. The component i s s t i l l chromes a2 a  l  +  and  a  2 ^-H W  and ^  e  600-650nm) and  absolute  absorption  terminal oxidases.  ^  an  absorption  peaks at 560nm  the t e r m i n a l oxidr.se of  unknown. S t a t i o n a r y - p h a s e  o as the  having  spectrum of  this  c e l l s possess both  cyto-  In t h i s t h e s i s , cytochromes  r e f e r r e d to as cytochromes a ( a b s o r p t i o n peaks between cytochrome b^ as cytochrome b  In m i t o c h o n d r i a  ( a b s o r p t i o n peak a t 560nm).  of e u k a r y o t e s , s p e c t r o s c o p i c s t u d i e s o f the  from 430-500nm have i n d i c a t e d o v e r l a p p i n g p r o t e i n s and non-haem i r o n p r o t e i n s  absorption  (Ragan and  bands due  Garland,  1971).  region  to f l a v o The  s i m i l a r i t y of t h e i r s p e c t r a l c h a r a c t e r i s t i c s has made i t e x t r e m e l y difficult alone.  to determine the c o n t r i b u t i o n by  I f the s i t u a t i o n i n m i t o c h o n d r i a  e i t h e r group of  i s p o o r l y u n d e r s t o o d , even l e s s  i s known about the non-haem i r o n p r o t e i n s of p r o k a r y o t i c A l t h o u g h the t r o u g h a t 460nm, t h e n , may other  than f l a v i n ,  i t w i l l be  compounds  be  due  organisms.  i n p a r t t o compounds  r e f e r r e d to i n t h i s t h e s i s as  "flavin  trough". The two  reduced/oxidized  d i f f e r e n c e s p e c t r a were o b t a i n e d  i n one  of  the  f o l l o w i n g ways depending upon the growth c o n d i t i o n s o f the c u l t u r e s .  i.  C e l l s grown w i t h  l i m i t i n g carbon  source:  In most experiments c e l l s were grown on a l i m i t i n g  carbon s o u r c e  19  and harvested at the end of the log phase. The f i n a l suspensions were a i r - o x i d i z e d f o r 1-1 1/2 hr p r i o r to analysis of the difference spectra. C e l l suspension, 2.3 ml, was placed i n cuvettes i n both the upper and lower compartments of the spectrophotometer.  A baseline was obtained by  scanning the spectrum from 700 to 430nm. To establish.that c e l l s i n the upper cuvette were oxidized, 0.1 ml of a 0.3% solution of hydrogen peroxide was added to the lower cuvette, while 0.1 ml of d i s t i l l e d water was added to the upper cuvette (the l a t t e r addition being made to maint a i n an equal concentration of c e l l s i n both the upper and lower cuvettes). When the spectrum was again scanned from 700 to 430nm and the r e s u l t i n g baseline was i d e n t i c a l to that obtained p r i o r to addition of the hydrogen peroxide, i t was assumed that the upper cuvette did indeed contain oxidized c e l l s . An amount of 0.1 ml of a 10% solution of the substrate (sugar or acid) or 0.1 ml of a 0.2% solution when the substrate was an amino acid, was then added to the upper cuvette to produce the reduced/oxidized difference spectra, the concentration of the c e l l suspension i n the bottom cuvette having been adjusted appropriately by the addition of 0.1 ml of d i s t i l l e d water. Repeated, consecutive scanning (7 to 15 times) of the difference spectrum was carried out from 700 to 430nm f o r approximately 30 minutes.  ii.  C e l l s grown with excess carbon source:  In certain cases when c e l l s were grown on an excess of carbon  source  and harvested during log phase (see Table XVI), the reduced/oxidized difference spectra were obtained by addition of 0.1 ml of a 0.3% solution of hydrogen peroxide to the bottom cuvette. The concentration of the c e l l suspension i n the upper cuvette was adjusted by addition of 0.1 ml of  20  distilled  VI.  water. The spectrum was scanned repeatedly as i n METHODS V, i .  Enzyme Assays Enzyme a c t i v i t i e s were determined on freshly prepared crude sonic  extracts (see METHODS I I , iv) using a Cary 15 spectrophotometer  (direct  s c a l e ) . The reaction mixtures were kept i n a 25* water bath. The c e l l extracts were placed i n i c e . The s p e c i f i c a c t i v i t i e s , designated as units of enzyme / mg of protein, were obtained from several enzyme a.ssays and the average value taken.  i.  Fumarase:  Fumarase (EC 4.2.1.2) a c t i v i t y was determined using the method of Hanson and Cox (1967). C e l l extract, 0.025 ml, (diluted when necessary to contain 50-250 pg of protein) was added to 0.975 ml of reaction mixture consisting of 50 umoles potassium phosphate buffer (pH 7.2) and 15 umoles L-malic acid. The rate of formation of fumarate from L-malate was  followed at a wavelength of 240nm using a blank of 0.975 ml d i s t i l l e d  water mixed with 0.025 ml c e l l extract. A unit of a c t i v i t y i s defined as & A „ of 0.001 / min. 240 0 /  ii.  Aconitase:  Aconitase (EC 4.2.1.3) a c t i v i t y was determined as outlined by Hanson and Cox (1967). C e l l extract, 0.025 ml, (50-250 pg protein) was added to 0.975 ml of reaction mixture containing 50 umoles potassium phosphate b u f f e r (pH 7.2) and 15 umoles sodium D L - i s o c i t r a t e . The rate of formation of c i s - a c o n i t i c acid from i s o c i t r a t e was followed at a wavelength of 240nm against a b u f f e r - c e l l extract blank, A unit of a c t i v i t y i s defined as AA..  n  of 0.001 / min.  21  iii.  G l u c o s e 6-phosphate dehydrogenase:  Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) was measuring the r a t e o f r e d u c t i o n  o f NADP  +  determined by  by g l u c o s e 6-phosphate at 340nm.  The r e a c t i o n m i x t u r e c o n t a i n e d the f o l l o w i n g i n 1.0 ml: g l u c o s e  6-phos-  phate, 5 umoles; M g C ^ j 10 umoles; NADP , 0.41 umoles; g l y c y l g l y c i n e +  buffer  (pH 7.5), 50 umoles; crude c e l l e x t r a c t  A unit of a c t i v i t y  i s defined  (100-500 ug p r o t e i n ) .  as t h a t amount of enzyme e f f e c t i n g the  f o r m a t i o n o f 1 nmole o f NADPH / min.  iv.  Glucokinase:  Glucokinase reduction  (EC 2.7.1.2) was d e t e r m i n e d by measuring the r a t e of  o f NADP  +  i n a g l u c o s e 6-phosphate dehydrogenase-coupled  r e a c t i o n system at 340nm. The reacv.ion m i x t u r e c o n t a i n e d the f o l l o w i n g i n 1.0 m l : t r i s - H C l b u f f e r  (pH 7.0), 100 umoles; g l u c o s e , 4 umoles;  ATP, 2 umoles; MgSO^, 4.5 umoles; NADP , 420 nmoles; g l u c o s e 6-phosphate +  dehydrogenase, 1 u n i t ; crude c e l l e x t r a c t of a c t i v i t y  i s equivalent  v. Isocitric the  (100-500 ug p r o t e i n ) . A u n i t  to the f o r m a t i o n of 1 nmole o f NADPH / min.  I s o c i t r i c dehydrogenase: dehydrogenase (EC 1.1.1.42) was  r a t e of r e d u c t i o n  o f NADP  +  m i x t u r e c o n t a i n e d the f o l l o w i n g  determined by measuring  by i s o c i t r a t e a t 340nm. The i n 1.0 ml: t r i s - H C l b u f f e r  reaction (pH 7.5), 50  umoles; M g C ^ ^ ^ O , 10 umoles; sodium D L - i s o c i t r a t e , 10 umoles; NADP , +  0.41 pmoles; crude c e l l e x t r a c t i s defined  VII.  (100-500 ug p r o t e i n ) . A u n i t of a c t i v i t y  as t h a t amount of enzyme forming 1 nmole NADPH / min.  Attempts to O b t a i n a Peak at 503nm i n Crude C e l l E x t r a c t s Permeabilized C e l l s  or  22  The  following methods were used i n attempts to prepare c e l l extracts  or to increase c e l l permeability so that P503 might be studied d i r e c t l y : (i) u l t r a s o n i c a t i o n ; ( i i ) French press; ( i i i ) EDTA-tris- HCl;  (iv) toluene;  (v) lysozyme; (vi) warming^ ( v i i ) heat denaturation of proteins. Since a 503nm peak could not be detected following any one of these  treatments,  no d e t a i l s of methods w i l l be given here. The previously l i s t e d techniques  proved to be too harsh f o r the  s u r v i v a l of the 503nm peak; therefore, the l a b i l i t y of P503 was tested by subjecting the c e l l s to freezing and thawing as follows: 5 ml portions of the f i n a l a i r - o x i d i z e d c e l l suspensions were pipetted i n t o 3 separate test-tubes. The samples were quick-frozen i n dry ice-ethanol and subsequently quick-thawed i n a 37" water bath. The 3 test-tubes were thus treated 1, 3 and 5 times consecutively, followed immediately by a s p e c t r a l analysis. Although the c e l l s remained oxidized even a f t e r 5 consecutive exposures to freezing and thawing, reduction by glucose e l i c i t e d the control heights of only the cytochrome b and f l a v i n bands. Since the 503nm peak could not be detected a f t e r t h i s extremely mild treatment, further e f f o r t s to obtain this peak i n permeabilized c e l l s or i n crude c e l l extracts by an alternate treatment of whole c e l l s were abandoned. See PART C: I, DISCUSSION.  23  PART A:  Y i e l d of C e l l Protein from Glucose  RESULTS I. Amount of Protein Formed from Glucose by Cells Growing Aerobically When grown on minimal s a l t s and l i m i t e d glucose under aerobic conditions, the streptomycin (Sm)-dependent mutant of E_. c o l i B was found to produce 35% less c e l l weight than the wild-type organism (Coukell and Polglase, 1969). A proportional difference was observed when the e f f i c i e n c i e s (ug/ml increase i n c e l l u l a r protein per ug/ml glucose consumed) were determined f o r the Sm-dependent and wild-type cultures of E. c o l i strains B, CRX, K12 and UL (Table I ) . The per cent decrease i n e f f i c i e n c y ranged from 24.3 to 37.6. A non-dependent revertant (SBr4), derived from the Sm-dependent mutant of E_. c o l i B, showed a 42.7% decrease, s l i g h t l y less e f f i c i e n t than i t s parent. The corresponding Sm-resistant (indifferent) mutants of these strains grown on minimal s a l t s - l i m i t i n g glucose medium  dihydrostrepto-  mycin) did not show a consistent pattern of e f f i c i e n c y with respect to the wild-type organism (Table I I ) , but rather a different one f o r each s t r a i n . In addition, the presence or absence of dihydrostreptomycin did not a f f e c t a l l strains i n the same manner. II.  Amount of Protein Formed from Glucose by Cells Growing Anaerobically When the wild-type and Sm-dependent mutant of E_. c o l i strains B and  CRX were grown on minimal s a l t s - l i m i t e d glucose medium under an anaerobic nitrogen atmosphere, both strains of organisms were found to have the same decreased protein y i e l d r e l a t i v e to aerobic growth of approximately 0.10  (Table I I I ) . Figure 2 further emphasizes the e f f e c t of changing  Table I.  Protein yields from glucose for wild-type and streptomycin (Sm)-dependent (and non-dependent  revertant) mutant of Escherichia c o l i strains B, CRX, K12 and UL grown aerobically  E*  St rain  wild-type  Sm-dependent  Decrease i n E (%)**  non-dependent revertant  Sm-dependent  +  non-dependent revertant  B  0.314  0.196  0.180  37.6  42.7  CRX  0.292  0.200  -  31.5  -  K12  0.289  0.210  -  27.3  -  UL  0.300  0.227  24.3  _  *E i s the e f f i c i e n c y of growth, defined as the ug/ml increase i n protein per ug/ml glucose consumed. Values of E are the averages of several experiments. ft ft Per cent decrease i n E was calculated using the wild-type values as reference i n each case, "^Non-dependent revertant (SBr4) i s a sensitive s t r a i n derived from Sm-dependent E. c o l i B.  Table I I .  Protein yields from glucose for Sm-resistant (indifferent) mutants  Ct dihydrostreptomycin, DHSm) of E_. c o l i strains B, CRX, K12 and UL grown aerobica  E  Strain  Sm-resistant (-DHSm)  Decrease i n E (%)  Sm-resistant (+DHSm)  Sm-resistant (-DHSm)  Sm-resistant (+DHSm)  B  0.216  0.183  31.2  41.8  CRX  0.205  0.230  29.8  21.2  K12  0.255  0.248  11.8  14.2  UL  0.260  0.225  13.3  25.0  E i s the e f f i c i e n c y of growth as defined i n Table I . **Per cent decrease i n E was calculated using wild-type values of Table I as reference i n each case.  26  Table I I I .  Protein y i e l d s from glucose f o r wild-type and Sm-dependent  strains of E. c o l i B and CRX during aerobic and anaerobic growth  E*  Strain B:  CRX:  Aerobic  Anaerobic  wild-type  0.314 ;  0.111  Sm-dependent  0.196  0.100  wild-type  0.292  0.114  Sm-dependent  0.200  0.110  *E i s the e f f i c i e n c y of growth as defined i n Table I. Values are the averages of several experiments.  Log-phase c e l l s were harvested, washed, and oxidized i n a i r (see METHODS). The c e l l s were resuspended  i n minimal salts-glucose (0.065%)  medium and grown a e r o b i c a l l y . When the A420 reached 0.40, the culture was transferred to anaerobic conditions as described under METHODS. Samples were removed throughout both the aerobic and anaerobic growth periods f o r glucose and protein determinations.  27  150  0  100  200  300  400  500  600  700  decrease in glucose (pg/ml)  Figure 2. Y i e l d of c e l l protein from glucose f o r wild-type E_. c o l i B (» • ) and the streptomycin-dependent mutant (o The i n i t i a l concentration of glucose was 650 ug/ml. After a preliminary period of aerobic growth, the atmosphere was changed (at the arrows) to N gas. Samples of culture were removed at i n t e r v a l s throughout the aerobic and anaerobic stages f o r protein and glucose determinations. Experimental d e t a i l s are given i n the METHODS. 2  a).  28  E_. c o l i B from aerobic to anaerobic growth conditions. The  slopes  obtained a e r o b i c a l l y of the wild-type and Sm-dependent mutant are divergent while the slopes obtained anaerobically are p a r a l l e l . Ill.  The "Aerobic Increment" of Protein Y i e l d from Glucose Since the difference i n e f f i c i e n c y of growth between the  dependent and wild-type organisms was  Sm-  observed only under aerobic  conditions, the "aerobic increments" were compared i n the two  cases  (Table IV) . The aerobic protein y i e l d for Sm-dependent E_. c o l i B exceeded the anaerobic y i e l d by an increment of 0.096. For wild-type c e l l s , the aerobic increment was  0.203, approximately  twice the value  of the Sm-dependent mutant.  DISCUSSION A l l four Sm-dependent E_. c o l i strains tested resulted i n a s i m i l a r pattern of 25-35% decreased  aerobic growth e f f i c i e n c y compared to t h e i r  corresponding wild-type parent. Under anaerobic conditions, however, both c e l l types produced i d e n t i c a l protein y i e l d s . The  metabolic  deficiency of the Sm-dependent mutants therefore implicated an aerobic energy-yielding process. In view of these findings, i t was  of interest  to c a l c u l a t e the difference i n the "aerobic" portion of growth for the two types of organisms. From Table IV, i t can be seen that the r a t i o of the aerobic increments of wild-type to Sm-dependent E_. c o l i B i s  2.11,  i n d i c a t i n g that the former possesses the a b i l i t y of producing double the aerobic energy of the l a t t e r . Several Sm-dependent strains had previously been characterized i n  Table IV. Calculation of "aerobic increment" of protein y i e l d f o r the wild-type and Sm-dependent strains of E_. c o l i B  ^ig/ml protein increase / ug/ml glucose consumed  Strain  Aerobic  Anaerobic  Increment  wild-type  0.314  0.111  0.203  Sm-dependent  0.196  0.100  0.096  wild-type Ratio of "aerobic increments"  _  2.11  Sm-dependent  The aerobic increment  i s defined as the d i f f e r e n c e i n protein  y i e l d obtained from c e l l s grown a e r o b i c a l l y versus anaerobically on glucose.  30  t h i s laboratory and were found to have the following properties i n common: (1) an impairment i n their aerobic energy metabolism (Coukell, 1969) ; (2) excretion of valine into the medium when grown on glucose (Bragg and Polglase, 1962; Tirunarayanan, Vischer and Renner, 1962); (3) de-repression of the catabolite repressible enzymes, c i t r a t e synthase, fumarase and aconitase (Coukell, 1969). In order to understand  the reason f o r t h i s difference i n energy  y i e l d , i t was necessary to choose which d i r e c t i o n further studies would follow- substrate l e v e l energy-yielding reactions, or hydrogen metabolism. For the following reasons, hydrogen metabolism seemed more appropriate: (1) Since the anaerobic energy y i e l d s on l i m i t i n g glucose were the same for the wild-type and Sm-dependent organisms, i t was unlikely that the impairment was at the substrate l e v e l ; (2) The c e l l s were grown not on an excess, but rather a l i m i t i n g amount of glucose, so that intermediate metabolites would not be expected to accumulate to the extent that would be expected when an excess of substrate i s present; (3) Sm-dependent c e l l s grown aerobically on l i m i t i n g succinate as carbon source also resulted in a decreased y i e l d of c e l l s when compared to the wild-type. Further investigations were therefore focussed on the metabolism of hydrogen.  31  PART B:  Difference Spectra  RESULTS The mechanism underlying the production of a peak at 503nm may involve reduction of the pigment by a substrate or another  process  (such as ligand formation between some compound and P503). However, since the c e l l suspensions were a i r - o x i d i z e d i n i t i a l l y and the difference spectra obtained by addition of a substrate to the top cuvette (while the bottom reference cuvette contained c e l l s oxidized by hydrogen peroxide), the appearance of the 503nm peak was i n t e r preted as being the r e s u l t of reduction of the pigment (P503) by substrate. When the reduced/oxidized difference spectra of the wild-type, Sm-dependent, and non-dependent revertant of E_. c o l l B were studied (Figure 3), the usual complement of cytochromes was found to be presentthe cytochromes a (600-650nm), cytochrome b (560nm) and the f l a v i n }  trough (460nm). (An explanation f o r the nomenclature of the pigments i s given i n METHODS V). But i n addition, the wild-type s t r a i n had a very large, symmetrical peak at 503nm which was missing or n e g l i g i b l e i n both the dependent and revertant mutants. S i m i l a r l y the wild-type strains of E_. c o l i CRX and K12 possessed  the 503nm pigment, while the  corresponding Sm-dependent mutants showed only a trace. Measurements  of the peaks (or trough) are tabulated i n Table V.  For purposes of comparison, the cytochromes b of a l l strains were normalized to that of wild-type E_. c o l i B (x 1000) . By c a l c u l a t i n g the r a t i o s of heights of peaks (or trough) i n the wild-type to the Smdependent mutant, i t i s seen that most of the values do not deviate  32  + 0-05  -O05-  400  450  500  550  600  wavelength (nm)  650  700  Figure 3. Reduced/oxidized difference spectra a f t e r addition of glucose to a i r - o x i d i z e d c e l l suspensions of E_. c o l i B. C e l l s were grown on minimal s a l t s medium and prepared as described under METHODS.  , wild-type;  dependent revertant  (SBr4)  , Sm-dependent;  non-  Table V.  Height (or depth) of pigment peaks (or trough) obtained from the reduced/oxidized difference  spectra of wild-type (S) and Sm-dependent (D) (or non-dependent revertant, SBr4) strains of E_. c o l i B, CRX, and K12  B  CRX  K12  Strain  SB  DB (b)  SBr4 (c)  a/b  SCRX (d)  DCRX (e)  ratio d/e  SK12 (f)  DK12 (g)  ~W~  13.4  15.4  0.74  0.64  9.3  10.4  0.90  12.1  11.7  1.03  cytochrome b (560nm)  29.2* 29.2  29.2  1.00  1.00  29.2  29.2  1.00  29.2  29.2  1.00  P503 (503nm)  55.8  1.2  11.40  46.60  26.4  1.0  26.40  15.6  3.6  4.33  -36.0**30.2 -25.8  1.19  1.44  -51.8  -51.5  1.01  -21.0  -29.6  0.71  (aY  a cytochromes (600-650nm)  f l a v i n trough (about 460nm)  9.9  4.9  ratio a/c  ratio  ft The value for cytochrome b i n wild-type E_. c o l i B was used as reference to standardize the cytochrome b of a l l the other s t r a i n s . **A minus sign (-) indicates a trough i n the reduced/oxidized difference spectrum (see Figure 3). u>  34  from 1.00  to the same extent as that found i n the case of the 503nm  pigment. The height of the P503 peak i n wild-type E_. c o l i B exceeds that found i n the Sm-dependent organism by a factor of 11.40, and that i n the non-dependent revertant by 46.60. The r a t i o of P503 peak heights i n wild-type versus Sm-dependent E_. c o l i varied with the s t r a i n . The s i t u a t i o n i s not as clear-cut for the Sm-resistant  (indifferent)  organisms of the same E_. c o l i strains (Table VI) . No consistent pattern emerges f o r the presence or absence of a 503nm peak, either with or without dihydrostreptomycin. The P503 peak, when present, was more persistent (less transient) than i n the wild-type s t r a i n s . A comparison between Tables II and VI indicates a c o r r e l a t i o n , nevertheless, of decreased e f f i c i e n c y accompanied by a substantial decrease i n height of the 503nm pigment. It should be mentioned at t h i s point that the 503nm pigment i s transient i n the wild-type organism, and i t s decrease and eventual disappearance can be followed with time. Under the same conditions, the cytochromes and f l a v i n trough remain unchanged i n the reduced/oxidized difference spectra. Addition of more glucose a f t e r the disappearance of the 503nm band does not cause the subsequent re-appearance  of this  peak. The reversible nature of the reduction-oxidation of P503 was shown by a i r - o x i d i z i n g the c e l l s a f t e r glucose reduction had  produced  the difference spectra. Reduced P503 underwent re-oxidation during aeration again of the c e l l s , and could be reduced a second time by addition of glucose.  Table VI.  Height (or depth) of pigment peaks (or trough) obtained from the reduced/oxidized  difference  spectra of wild-type (S) and Sm-resistant (R) (+DHSm) strains of E_. c o l i B, CRX, and K12 (The same format i s used as i n Table V)  B  CRX  K12  Strain SB  RB (-DHSm)  RB (+DHSm)  SCRX  RCRX (-DHSm)  RCRX (+DHSm)  SK12  RK12 (-DHSm)  RK12 (+DHSm)  a cytochromes (600-650nm)  9.9  10.3  11.8  9.3  8.7  13.2  12.1  4.7  5.2  cytochrome b (560nm)  29.2  29.2  29.2  29.2  29.2  29.2  29.2  29.2  29.2  P503 (503nm)  55.8  11.3  15.8  26.4  0.0  6.1  15.6  8.5  9.2  -36.0  -39.2  -37.4  -51.8  -51.2  -21.0  f l a v i n trough (about 460nm)  -48.0  -56.4  -54.3  36  DISCUSSION  In a l l comparisons between the wild-type and Sm-dependent s t r a i n s , only the P503 band was  found to d i f f e r s i g n i f i c a n t l y while the cytochrome  and f l a v i n bands remained f a i r l y constant. These results suggested that the lack of P503 (or i t s decrease) and impaired energy metabolism observed i n Sm-dependent mutants of E_. c o l i  (as well as i n the non-dependent  revertant) were related phenomena. The 503nm pigment could not have been a precursor of another component i n the reduced/oxidized difference spectra since i t s disappearance with time d i d not affect the size of other peaks (or trough). In f a c t , re-runs of the spectra resulted i n p e r f e c t l y superimposable  peaks with  the exception of the 503nm band. The property of reversible reductionoxidation shown to be inherent i n P503 would imply i t s p a r t i c i p a t i o n i n an electron transport system. Wild-type E. c o l i B was used i n subsequent detailed studies of P503 since appropriate conditions had already been worked out f o r the consistent appearance of a large and symmetrical peak at 503nm.  37  PART C: I.  Characterization of P503  Attempts to Isolate P503  RESULTS The 503nm peak was  not detectable i n c e l l s which had been treated  as stated i n METHODS VII ( i . e . , disrupted, permeabilized  or frozen-  thawed c e l l s ) . DISCUSSION The evidence thus f a r has only i n d i r e c t l y implicated a r e l a t i o n ship between P503 and energy metabolism. It would have been advantageous at t h i s point to study the c h a r a c t e r i s t i c s of P503 d i r e c t l y i n crude c e l l extracts or i n permeabilized otherwise do not penetrate  c e l l s . Several compounds which  the E_. c o l i c e l l membrane could then be  added to v e r i f y or disprove our theory. In addition, i f P503 proved to be stable upon c e l l disruption, one might succeed i n i t s i s o l a t i o n , p u r i f i c a t i o n , and s t r u c t u r a l characterization. In 1964,  Lindenmayer and Smith t r i e d disrupting yeast c e l l s but  found the 503nm pigment to be very l a b i l e . S i m i l a r l y , our endeavours proved to be f u t i l e . The reason f o r the disappearance of the 503nm band i s not known. It may  be that the cofactor(s) necessary for i t s  s t a b i l i t y is(are) d i l u t e d out upon c e l l permeabilization or Neu,  rupture.  Ashman and Price (1967) found that EDTA treatment released large  molecular weight substances such as nucleotides into the medium. Another p o s s i b i l i t y may  be that disruption of the c e l l s changes the stable  conformation of the pigment. Whether the structure of the pigment i s  38  destroyed, or changed to a non-absorbing fora i s also unknown. I f the structure of P503 were s i m i l a r to protoporphomethene  (Labbe, Volland  and Chaix, 1967), then i t s absorption and fluorescence i n v i s i b l e  light  could depend on the resonating character of the conjugated double bonds, as i s the case f o r porphyrins (Mauzerall, 1962). When the double bonds of the methylene bridges are reduced, the porhyrins are converted to colourless porphyrinogens. In view of the extreme l a b i l i t y of P503, our emphasis was s h i f t e d to a more productive approach, and further investigations involved the characterization of P503 i n whole c e l l s .  39  II.  Production  1.  Effect  of the 503nm Peak  of sugars and  a c i d s on p r o d u c t i o n  o f the 503nm peak:  RESULTS In w i l d - t y p e  15. c o l i B c e l l s  c o n t a i n i n g P503, v a r i o u s sugars  a c i d s were t e s t e d f o r t h e i r a b i l i t y shows the s e q u e n t i a l e f f e c t m a t e l y 2 minutes to scan scanning  to produce the 503nm peak. F i g u r e  of g l u c o n a t e  addition. It requires  the spectrum from 700-430nm. Repeated  of the d i f f e r e n c e spectrum showed i n i t i a l l y  at 503nm. S u b s e q u e n t l y , f l a v i n steady-state  and  f o l l o w e d by  the e n t i r e spectrum on  detected  Other compounds as r e d u c t a n t s E_. c o l i B and ( P r e i s s and  B-SG1. S t r a i n B-SG1  Greenberg, 1965;  added would be m e t a b o l i z e d l a t e r use. reflect  a f f e c t e d by  glucose  run.  glycogen  and none would be s t o r e d as g l y c o g e n B-SG1  route a f t e r  for  might  back-reaction  o c c u r r e d . However, s i n c e b o t h s t r a i n s were s i m i -  were d i r e c t l y m e t a b o l i z e d .  G l y c e r o l and  the  Harvey, 1970), so t h a t a l l s u b s t r a t e s  the s u b s t r a t e s , one  peak. With g l u c o n a t e  after  i s incapable of synthesizing  d i f f e r e n c e , then, between E_. c o l i B and  s y n t h e s i s ) has  substrate  are shown i n T a b l e V I I f o r w i l d - t y p e  the u t i l i z a t i o n of another m e t a b o l i c  (glycogen larly  Any  the f i r s t  their  cytochrome b  appearance o f the o t h e r pigments. In c o n t r a s t , r e d u c t i o n by (as c o n t r o l ) e l i c i t e d  consecutive  o n l y a l a r g e peak  l e v e l s . F i g u r e 5 shows the e f f e c t of s u c c i n a t e as  reduced immediately, o n l y a t r a c e of P503 was  4  approxi-  cytochrome b were reduced to  under the same c o n d i t i o n s . Whereas the f l a v i n was  and  Reduction  as r e d u c t a n t ,  a c e t a t e d i d not  elicit  can  assume t h a t the added compounds  by g l u c o s e  produced a h i g h 503nm  the peak of P503 was  slightly  a P503 band. With s u c c i n a t e  smaller. and  40  Figure 4.  Reduced/oxidized difference  spectra after  addition  of gluconate to air-oxidized suspension of wild-type E. c o l i B: *  a f t e r 2 minutes;  , after 5.2  minutes.  41  + 0-05-  -0-05400  450  Figure 5.  500  550  wavelength (nm)  650  600  Reduced/oxidized difference spectra a f t e r  addition of glucose (  ) or succinate (  air-oxidized suspensions of wild-type E. c o l i B.  ) to  700  42  Table VII.  E f f e c t of various sugars and acids on production of the  503nm peak i n wild-type E. c o l i B and B-SG1  Strain  B-SG1  Sugar or acid  Height of 503nm peak (units)  Order of appearance of 503nm peak with respect to cytochrome peaks  gluconate  43.8  first  glucose  55.8  simultaneous  glycerol  0.0  succinate  4.3  lactate  2.6  acetate  0.0  A AA  AA  AA  after simultaneous A  gluconate  35.9  first  glucose  56.6  simultaneous  .  succinate  , A  3.4 A  lactate  2.9  acetate  0.0"  after simultaneous A  "In each case, the height of cytochrome b was normalized to the control value (29.2 units) obtained v i a reduction by glucose (see Table V). AA  The 503nm peak was small or absent a f t e r 10-15 minutes. Subsequent reduction by glucose resulted i n a large peak at 503nm. A- The 503nm peak was absent although the cytochromes were present. C e l l s containing P503 were grown on minimal salts-glucose (0.04%) medium, harvested, washed once i n phosphate buffer, and a i r - o x i d i z e d for 1 hr p r i o r to analysis of the difference spectrum. Various sugars and acids were tested as indicated f o r t h e i r a b i l i t y to reduce the 503nm pigment.  43  l a c t a t e reduction of the c e l l s , P503 was hardly detectable i n both s t r a i n s . After allowing 10-15 minutes to elapse following the addition of g l y c e r o l , succinate, l a c t a t e , or acetate, glucose was added. A large peak at 503nm appeared r a p i d l y , i n d i c a t i n g that the c e l l s possessed to  indeed  P503, but that the substrates added i n i t i a l l y were unable  cause the immediate appearance of i t s spectrum. DISCUSSION There appears to be a high l e v e l of s p e c i f i c i t y involved i n  reduction to produce a peak at 503nm. If the absorbance peaks or trough obtained 5.2 minutes a f t e r the addition of gluconate are considered to represent steady-state reduction l e v e l s of 100%, then gluconate  as reductant  (involving NADP as the f i r s t electron +  acceptor) caused the rapid appearance of 76% of the 503nm peak within the f i r s t 2 minutes; i n contrast, only 19% of the cytochrome b peak and 14% of the f l a v i n trough were observed. The s i t u a t i o n with succinate as reductant  (involving f l a v i n as the f i r s t electron  acceptor) showed d i f f e r e n t absorption c h a r a c t e r i s t i c s . Whereas the cytochrome b and f l a v i n bands appeared immediately, the 503nm band, observed only a f t e r several scans through the spectrum, was small compared to the height e l i c i t e d by glucose  (control). These results  suggest that the oxidation-reduction p o t e n t i a l of P503 i s lower (more negative)  than that of cytochrome b or f l a v i n , and that the 503nm  pigment accepts electrons at the l e v e l of pyridine nucleotide (NADPH). T h i r t y per cent of the glucose and gluconate, taken up by phosphorylation v i a the phosphoenolpyruvate phosphotransferase  system  (Simoni, Levinthal, Kundig, Kundig and Roseman, 1967; Fraenkel, 1968;  44  Roseman, 1969), follow the hexosemonophosphate shunt (Model and Rittenberg, 1967; Wang et a l . , 1958). In this pathway, glucose would give a maximum y i e l d of 2 molecules of NADPH per molecule of glucose, while gluconate would y i e l d only 1 molecule of NADPH per molecule gluconate. The steady-state l e v e l of NADPH might be lower and less rapidly attained with gluconate so that the extent of reduction to form the 503nm peak would be correspondingly l e s s . Succinate has been shown to be transported i n E_. c o l i by a highly s p e c i f i c inducible system (Kay and Kornberg, 1971). Since there was no accumulation of the dicarboxylic acid concomitant with i t s uptake from the medium, these workers concluded that the energy required f o r t h i s translocation was supplied by i t s rapid removal, through oxidative metabolism, subsequent to i t s entry into the c e l l s . I t s immediate  oxi-  dation to fumarate i s linked to a f l a v i n coenzyme. As discussed l a t e r (when the n u t r i t i o n a l aspects concerning P503 formation are considered), g l y c e r o l enters the metabolic scheme at the l e v e l of dihydroxyacetone phosphate; being uncharged, i t was  thought  i n i t i a l l y to freely penetrate b a c t e r i a l c e l l s (Packer and Perry, 1961; Koch, Hayashi and L i n , 1964) before being rapidly metabolized. More recent investigations (Sanno, Wilson and L i n , 1968; Berman and L i n , 19 71) have established that entry of g l y c e r o l into E_. c o l i i s not by simple d i f f u s i o n but rather by f a c i l i t a t e d d i f f u s i o n whereby a s p e c i f i c membrane c a r r i e r (permease) catalyzes the equilibrium of i n t r a c e l l u l a r and extrac e l l u l a r substrate concentrations without energy coupling. Lactate i s converted to pyruvate v i a the NAD -dependent l a c t i c dehydrogenase +  (Kline  and Mahler, 1965) and further to phosphoenolpyruvate v i a phosphoenolpyruvate synthase (Kornberg and Smith, 1967; Cooper and Kornberg, 1967).  45  The  former group found pyruvate  transport to be controlled by a gene  (or genes) i n E_. c o l i , but noted that regulation of uptake for pyruvate was  not the same as for lactate. The entrance of lactate into c e l l s i s  most l i k e l y unhindered to acetate. The  by the membrane. The c e l l s are f r e e l y permeable  compound as sole carbon source results i n the induc-  t i o n of the glyoxylate bypass for i t s d i s s i m i l a t i o n (Kornberg,  1966).  However, t h i s anaplerotic pathway i s repressed during growth on glucose (Holms and Bennett, 1971), so that subsequent oxidation of acetate occurs by operation of the TCA  cycle. This was  the experimental  condition used  i n our experiments. The three compounds- g l y c e r o l , l a c t a t e , and acetate, entering the metabolic path at a lower l e v e l and presumably not generating high concentrations of NADPH during their breakdown or further metabolism would not be expected results.  to produce a large peak at 503nm as shown by the  46  2.  E f f e c t of L-amino acids on production  of the 503nm peak  RESULTS The s p e c i f i c i t y involved i n the appearance of the 503nm peak caused by sugars and acids prompted an i n v e s t i g a t i o n of the e f f e c t of L-amino acids as reductants of a systematic  under s i m i l a r conditions. The r e s u l t s  analysis are shown i n Table VIII. The L-amino acids  l i s t e d from alanine to valine (added i n s o l u t i o n to the c e l l s ) gave the absorption spectrum of P503, the cytochromes and f l a v i n simultaneously a f t e r an i n i t i a l lag period of 10-14 minutes. In each case, the 503nm peak was small compared to that of cytochrome b (29.2 u n i t s ) . Lysine produced no 503nm peak; a f t e r a 10 minute l a g , the f l a v i n and cytochromes were reduced. H i s t i d i n e produced no spectrum at a l l , even a f t e r 20 minutes. Addition of L-methionine (Figure 6) or casein hydrolysate  (which contains  the 21 amino acids excluding  tryptophan,  glutamine and asparagine) resulted i n the reduction of P503 within 0.9 minutes; the f l a v i n and cytochromes were reduced a f t e r 10 minutes. Of the L-amino acids tested, only methionine and casein  hydrolysate  were able to cause immediate appearance of the peak at 503nm. The other amino acids had no s i g n i f i c a n t e f f e c t on this pigment.  DISCUSSION Wild-type E_. c o l i i s capable of synthesizing a l l the necessary amino acids when grown on a minimal salts-glucose medium, and i s therefore independent of an exogenous supply of amino acids. However, when an amino acid i s added to the growth medium, the organism u t i l i z e s the appropriate transport system(s) f o r i t s uptake. Piperno and Oxender (1968) reported four d i s t i n c t transport systems to be operative i n E. c o l i K12 f o r  47  Table VIII. E f f e c t of L-amino acids on production of the 503nra peak in wild-type E_. c o l i B ^The format i s used as i n Table VII) Amino acid  Height of 503nm peak (units)  alanine  10.8  arginine  12.0  aspartic acid  9.0  glutamic acid  14.4  glycine  16.2  leucine  10.0  phenylalanine  9.5  proline  8.4  serine  7.3  tyrosine  7.8  valine  9.9  lysine  0.0  histidine  -  Order of appearance of 503nm peak with respect to cytochrome peaks  simultaneous  A* - (no spectrum)  methionine  32.9  first  casein hydrolysate  29.5  first  *See Table VII. Methionine and casein hydrolysate gave the absorption peak of P503 within 0.9 minutes, followed by the cytochromes after a 10 minute l a g . The L-amino acids from alanine to v a l i n e gave the absorption peaks of both P503 and the cytochromes simultaneously after a 10-14 minute l a g . Lysine gave no absorption peak at 503nm; the cytochromes were reduced a f t e r 10 minutes. H i s t i d i n e produced no spectrum at a l l .  48  + 0 0 5 -  0 c  D _Q  O  c/>  _Q D  - 0-05-  400  Figure 6. addition  450  500  wavelength  Reduced/oxidized difference  550  (nm)  600  spectra after  of L-methionine to air-oxidized suspensions  of wild-type E_. c o l i B: * * * *, after 1 minute; after 6 minutes;  , after 10 minutes.  ,  49  neutral amino acid uptake, with very l i t t l e overlap between them. Each system transported  the following s p e c i f i c groups of amino acids:  ( i ) leucine, isoleucine and v a l i n e ; ( i i ) alanine, glycine and serine; ( i i i ) phenylalanine,  tyrosine and tryptophan; (iv) methionine. The  only neutral amino acid found to have a s p e c i f i c transport system was L-methionine. The addition of L-amino acids to whole c e l l s of wild-type E_. c o l i B resulted i n a lag varying from 10-14  minutes p r i o r to the appearance  of the difference spectra. This delay might r e f l e c t the induction of the appropriate  transport system and/or accumulation of the amino  acid into pools p r i o r to i t s metabolism. Ames (1964) reported  the  existence of s p e c i f i c transport systems i n Salmonella typhimurium f o r h i s t i d i n e and some of the aromatic amino acids, i n addition to a less s p e c i f i c system which could transport a l l the aromatic amino acids. The eventual reduction of the cytochromes (except i n the case of h i s t i dine) indicated, then, that the c e l l s were permeable to a l l the L-amino acids added, and that the l a t t e r were metabolized a f t e r t h e i r uptake into the c e l l s . One might assume that h i s t i d i n e was  accumulated but  not metabolized, so that no reduction of the cytochromes In general, the uptake of C^4-  aT11  -j  no  acids was  occurred.  found by Piperno and  Oxender (1968) to be very rapid. Most of the radioactive l a b e l taken up within 30 seconds, and steady-state  was  levels were reached i n  1-2 minutes. The amino acids, L-leucine and glycine, were transported f o u r - f o l d f a s t e r than L-alanine and L-methionine. After 2 minutes, however, 98% of the radioactive L-isomers of leucine, i s o l e u c i n e , phenylalanine  and v a l i n e were s t i l l i n t a c t inside the c e l l s ; i n contrast,  only 50% of the r a d i o a c t i v i t y was  recovered  i n glycine, L-alanine  and  50  L-methionine. These workers concluded from the results that the former group of amino acids were not metabolized rapidly, while the l a t t e r group of amino acids underwent extensive metabolism. Since the time f o r transporting L-methionine into E. c o l i c e l l s i s longer than for other amino acids, the r e l a t i v e l y minute quantity that enters the c e l l s within the f i r s t minute must be a f f e c t i n g the P503 system either p r i o r to i t s metabolism or at an early stage i n i t s metabolism. Thus f a r , glucose, gluconate, and L-methionine have been found to cause the appearance of a 503nm band quickly. While glucose and gluconate also caused reduction of the cytochrome system f a i r l y quickly, L-methionine behaved d i f f e r e n t l y i n that a long lag persisted p r i o r to the appearance of the cytochromes. A p l a u s i b l e explanation can be found when one studies the metabolic fate of the three compounds. L-methionine i s converted into S-adenosylmethionine at the expense of ATP. As stated e a r l i e r , glucose and gluconate are phosphorylated p r i o r to t h e i r uptake by b a c t e r i a l c e l l s . The transport mechanism, well documented by several workers (Simoni, L e v i n t h a l , Kundig, Kundig and Roseman, 1967; Fraenkel, 1968; Roseman, 1969), has been shown to proceed v i a the phosphoenolpyruvate (PEP) phosphotransferase system. In both cases, PEP i s produced at the expense of ATP (Roseman, 1969). In addition, the metabolism of these two sugars involves the early production of hydrogen atoms, r e s u l t i n g i n the reduction of the cytochromes. This i s not the case f o r L-methionine; hydrogen atoms are not produced immediately, and consequently cytochromes are not reduced u n t i l much l a t e r . As speculated l a t e r , i t i s conceivable that NADPH i s oxidized v i a P503 to generate ATP. Then the addition of methionine would lower the  51  ATP  concentration  and s t i m u l a t e as a compensatory r e s p o n s e , t h e f o r m a t i o n  o f ATP v i a P503, thus g e n e r a t i n g c o n d i t i o n s used f o r p r e p a r i n g  t h e spectrum o f t h i s pigment. Under the  c e l l s , i . e . , growth on l i m i t i n g  glucose,  h a r v e s t i n g , and a e r a t i o n i n b u f f e r f o r 1 h r at 37*, NADPH was found to be p r e s e n t  a t a c o n c e n t r a t i o n i n excess o f 0.20 umoles o f NADPH p e r  gram o f c e l l p r o t e i n ( P o l g l a s e , 1972).  52  3.  E f f e c t o f L-methionine analogues on p r o d u c t i o n of the 503nm peak  RESULTS In view o f the s i n g u l a r e f f e c t of L-methionine on e l i c i t i n g a peak at 503nm, s e v e r a l o f i t s analogues  ( F i g u r e 10) were used to t e s t  t i o n a l groups and s t e r e o s p e c i f i c i t y o f a c t i o n g-methionine behaved s i m i l a r l y  (Table I X ) , The  isomeric  to L - l y s i n e , c a u s i n g r e d u c t i o n o f the  f l a v i n and cytochromes b u t not o f P503 a f t e r 12 minutes. The L - e t h i o n i n e , D L - n o r l e u c i n e , DL-methionine and DL-selenomethionine had  the f u n c -  analogues,  s u l f o n e , L-methionine s u l p h o x i d e  the same e f f e c t as the m a j o r i t y o f the o t h e r  amino a c i d s ; o n l y a f t e r a l a g o f 10 minutes  d i d the e n t i r e  difference  s p e c t r a appear s i m u l t a n e o u s l y w i t h a 503nm band. N - a c e t y l L-methionine produced no spectrum a t a l l .  A d d i t i o n o f L - e t h i o n i n e d i d not produce  peak a t 503nm immediately. A d d i t i o n of L - e t h i o n i n e f o l l o w e d by gave the same r e s u l t P503 was  as L - m e t h i o n i n e a l o n e . The e f f e c t  thus shown t o be v e r y s p e c i f i c . No  a  L-methionine  of L-methionine  on  s u b s t i t u t i o n s or d e l e t i o n s i n  the s u l p h u r or m e t h y l moiety were a l l o w a b l e .  DISCUSSION A survey o f L-methionine analogues f u r t h e r emphasized  the  specificity  of the e f f e c t o f L - m e t h i o n i n e on P503. A change i n c o n f i g u r a t i o n from the L - t o D-form, replacement o f the m e t h y l group w i t h e t h y l , o x i d a t i o n of the s u l p h u r moiety t o s u l f o n e o r s u l p h o x i d e , o m i s s i o n o f the s u l p h u r and a d d i t i o n of m e t h y l , s u b s t i t u t i o n o f s u l p h u r w i t h s e l e n i u m - a l l r e s u l t e d i n analogues l a c k i n g  the a b i l i t y p o s s e s s e d by L-methionine t o e l i c i t  a P503  peak immediately f o l l o w i n g i t s a d d i t i o n to the c e l l s . C o n s e q u e n t l y , n e i t h e r the s u l p h u r n o r the methyl group p e r se were the s o l e r e q u i r e m e n t s ; r a t h e r , the m o l e c u l e as a whole seemed t o be n e c e s s a r y . The appearance before  the cytochromes  upon a d d i t i o n o f L - e t h i o n i n e f o l l o w e d by  of the P503 peak L-methionine  53  Table IX. E f f e c t of L-methionine analogues on production of the 503nm peak i n wild-type E. c o l i B (The same format i s used as i n Table VII)  Amino acid or analogue  Height of 503nm peak (units)  L-methionine  32.9  D-methionine  0.0  L-ethionine  29.2  DL-norleucine  29.2  DL-methionine  sulfone  first  simultaneous  7.8  L-methionine sulfoxide  10.1  DL-selenomethionine  18.3  N-acetyl L-methionine  **L-ethionine; then  Order o f appearance o f 503nm peak w i t h r e s p e c t t o cytochrome peaks  - (no spectrum) 46.7  first  E-methionine  See Table VII. D-methionine reduced the cytochromes a f t e r a 12 minute l a g , but not the 503nm pigment. L-ethionine reduced both the 503nm pigment and the cytochromes a f t e r a 10 minute l a g . L-ethionine, followed by L-methionine, gave the same r e s u l t as L-methionine alone. N-acetyl L-methionine produced no spectrum at a l l .  54  indicated that t h i s analogue was not e f f e c t i v e as a competitor. If two compounds share a common transport system, then the addition of one compound to the medium can increase the loss of a second compound previously accumulated (Wilbrandt and Rosenberg, 1961). S t r u c t u r a l analogues can be useful i n ascertaining the c h a r a c t e r i s t i c s and d i v e r s i t y of transport systems present, by t h e i r a b i l i t y to p a r t i c i p a t e i n t h i s type of "countertransport". With the exception of the alanine-glycine-serine transport system, the other three systems for neutral amino acid uptake were found to be highly s t e r e o s p e c i f i c (Piperno and Oxender, 1968). When E_. c o l i were preloaded with  cells  -L-methionine then washed with unlabelled L-methio-  nine, 30% of the r a d i o a c t i v i t y was l o s t . This was not the s i t u a t i o n , however, when the c e l l s were washed with L-norleucine; 96% of the l a b e l remained within the c e l l s . Likewise, the D-isomer of methionine and L-ethionine were tested and found to be transported by systems d i f f e r e n t from that f o r L-methionine. In Table IX then, the addition of L-methionine • a f t e r L-ethionine would not have resulted i n loss of any L-ethionine which had already been accumulated by the c e l l s . As the r e s u l t s indicated, the presence of both L-methionine and i t s analogue did not a l t e r the e f f e c t of the amino acid alone on P503. This point w i l l be discussed further i n the next section concerning growth of c e l l s i n the presence of both L-methionine and DL-ethionine (or DL-norleucine). Properties of the various s t r u c t u r a l analogues of L-methionine and the recent research published i n t h i s area i s b r i e f l y reviewed i n the DISCUSSION section under E f f e c t of L-methionine and i t s analogues as growth supplement  on P503 formation (synthesis).  55  4.  E f f e c t of other compounds on production of the 503nm peak  RESULTS The four compounds ascorbic acid, f o l i c acid, betaine and 2-deoxyD-glucose were tested f o r t h e i r a b i l i t y to reduce the 503nm pigment by addition of 0.1 ml of a 0.2% solution to the top cuvette (as described under METHODS V, i ) . Ascorbic acid (sodium s a l t ; Calbiochem) as reductant resulted i n the simultaneous appearance of a small 503nm peak, the cytochrome bands and f l a v i n trough. F o l i c acid (Sigma Chemical Co.) was also tested as a hydrogen donor but f a i l e d to produce a 503nm peak at a l l . I t s metabolism caused the f l a v i n and cytochrome b to become reduced a f t e r 10 minutes. The i d e n t i c a l r e s u l t (as f o l i c acid) was obtained upon addition of betaine anhydrous powder to the upper cuvette. With 2-deoxyD-glucose (20% aqueous solution from Sigma Chemical Co.) the entire spectrum was produced r a p i d l y , s i m i l a r to the metabolism of glucose except f o r a markedly smaller P503 band. As f a r as production of the 503nm peak was concerned then, these additional four compounds were not  effective. DISCUSSION The a b i l i t y of ascorbic acid to undergo reversible oxidation to the  dehydro-form i s w e l l known; dehydroascorbic acid can i n turn be reduced by glutathione. The vitamin i s also known to donate electrons v i a N,N,N',N'-tetramethyl-p-phenylenediamine  (Howland, 1963; Wilson and  Brooks, 1970) or d i r e c t l y (Klingenberg, 1968) to cytochrome c i n mitochondrial systems. The photoreduction of porphyrins was studied by Mauzerall (1962) . The second stage of reduction involves the formation of  tetrahydroporphyrin (porphomethene), having an absorption maximum  56  at 500nm. Ascorbic acid (pH 7.0)  caused reduction of the porphyrin  beyond the second stage to form hexahydroporphyrin or porphyrinogen (a colourless compound). This suggests that ascorbate may produce a more highly reduced (leuco) form of P503 than does glucose. Nosoh (1964) reported that reduction by ascorbate, cysteine, glutathione, potassium cyanide and d i t h i o n i t e could f l a t t e n out the 503nm peak. His difference spectra of substrate-treated c e l l s minus nontreated growing c e l l s showed a trough at 503nm with potassium cyanide, cysteine, and sodium d i t h i o n i t e (Na2S£0 ). Since a 503nm trough was 4  also observed by Nosoh i n the difference spectrum of "growing  cells  upon standing at 30* f o r 4 hours minus growing c e l l s before standing", h i s r e s u l t s might be interpreted i n the following manner: addition of cysteine, cyanide, and d i t h i o n i t e to the top cuvette resulted i n reduction of P503 to i t s colourless form. The "growing c e l l s " i n the bottom cuvette were reduced so that a 503nm band was present, r e s u l t i n g i n a 503nm trough i n the difference spectrum. S i m i l a r l y , the c e l l s (in the top cuvette) l e f t standing f o r 4 hours probably became depleted of endogenous substrate, while those c e l l s ( i n the bottom cuvette) used immediately without standing s t i l l had a supply of substrate. Since the tetrahydro-derivative of f o l i c acid i s a coenzyme involved i n methylation reactions, the vitamin was used to study the possible involvement of methyl transfer with appearance of the P503 band. If the e f f e c t of L-methionine were due to the donation of i t s methyl group, then a compound such as betaine (possessing a l a b i l e methyl group and known to transmethylate homocysteine  to form methio-  nine i n animals) might also produce a peak at 503nm. However, the  57  r e s u l t s with f o l i c acid and betaine were both negative. The evidence appeared  to.be against p a r t i c i p a t i o n of methyl transfer i n production  of the 503nm peak. 2-Deoxy-D-glucose was previously thought  to undergo phosphorylation  at carbon #6 (forming 2-deoxyglucose 6-phosphate at the expense of ATP) without being metabolized further. I f the immediate e f f e c t of L-methionine were to u t i l i z e ATP i n forming S-adenosylmethionine,  thereby causing  a decrease i n the l e v e l of ATP and, i n turn, stimulating the P503 pathway as a compensatory mechanism, one should observe the rapid appearance of a P503 peak upon addition of 2-deoxyglucose as w e l l . This was not the case, however, and a trace of P503 appeared  simultaneously with the f l a v i n  and cytochrome b. Contrary to expectations, 2-deoxyglucose was being metabolized. These rather confusing results were explained when i t was noted that Dietz and Heppel (1971) reported that E_. c o l i c e l l s might be capable of metabolizing 2-deoxyglucose.  These workers found that 2-deoxy-  glucose i n h i b i t e d growth of E_. c o l i and also caused temporary growth s t a s i s when g l y c e r o l or succinate was the carbon source. In contrast, c e l l s grown i n the presence of gluconate or pyruvate resisted these e f f e c t s of 2-deoxyglucose.  This compound was unable to compete with  glucose f o r entry into the c e l l s , and consequently had no e f f e c t on growth when glucose was the source of carbon. I t was also reported that 2-deoxyglucose i n h i b i t e d phosphorylation and fermentation of glucose i n lysozyme lysates (Dietz and Heppel, 1971) of E_. c o l i B, but not i n intact c e l l s . In an e a r l i e r p u b l i c a t i o n , Pogell, Maity, Frumkin and Shapiro (1966) studied the fate of 3 2 p _ i b i i d 2-deoxyglucose a  e  e  6-phosphate a f t e r i t was taken up by E_. c o l i c e l l s . Since only 50% of the compound chromatographed s i m i l a r l y to unchanged  2-deoxyglucose-  58  6-phosphate, they concluded that the nature of the l a b e l gave no information on whether the 2-deoxyglucose  was metabolized or not. Since  phosphorylation by ATP was obviously not the sole reaction of 2-deoxyglucose, this compound d i d not provide a test of the hypothesis that appearance of the 503nm peak, alone (as observed with methionine) might occur as a r e s u l t of a change i n the energy  charge.  59  III.  Formation  (Synthesis) of P503  I.  E f f e c t of 2,4-dinitrophenol on formation of P503 RESULTS 2,4-Dinitrophenol (DNP) has been used extensively i n studies of  the uncoupling of oxidative phosphorylation i n mitochondria since the f i r s t experimental observation of i t s action (Loomis and Lipman, 1948). Ideas regarding mechanism of action, s t i l l being investigated by numerous l a b o r a t o r i e s , appear to d i f f e r depending on which hypothesis chemiosmotic hypothesis ( M i t c h e l l , 1961; (Boyer, 1968)  1967)  (Mitchell's  or the chemical hypothesis  implicating a phosphorylated high-energy  protein intermediate  or "coupling factor") i s favoured for oxidative phosphorylation. This topic w i l l not be dealt with i n this thesis, since another possible reaction of DNP  (namely, the reduction of i t s n i t r o group(s)) provides a more s a t i s -  factory explanation of the r e s u l t s . Stockdale and Selwyn (1971) reported that phenols have two e f f e c t s - one on the coupling a c t i v i t y and the other on i n h i b i t i o n of r e s p i r a t i o n , the two phenomena depending on d i f f e r e n t properties of the phenol. Varied e f f e c t s of DNP  i n E_. c o l i have been reported, depending on i t s  concentration and the growth conditions. Thus, 1.3x10"^ M to 2.4x10"^ M DNP was  found to i n h i b i t r e s p i r a t i o n by 40-85% (Packer and Perry, 1961).  A lower concentration, 10"^ M to 5xl0~5 M DNP r e s p i r a t i o n by 130%  resulted i n stimulation of  (Bovell, Packer and Helgerson, 1963). DNP  has also  been reported to i n h i b i t energy-linked membrane processes such as uptake of substrate (Pavlasova and Harold, 1969; Kay and Kornberg, 1971;  Pogell,  Maity, Frumkin and Shapiro, 1966) . When wild-type E. c o l i B was  grown aerobically on minimal s a l t s -  60  I  Figure 7.  I  Growth of wild-type E_. c o l i B  on minimal s a l t s - l i m i t i n g glucose medium supplemented with various concentrations of 2,4-dinitrophenol: • °  °, 100 uM; •  • , none (control);  250 uM;  , 500 uM.  61  l i m i t i n g glucose medium supplemented with DNT  (Figure 7 ) , the growth  rate and y i e l d of c e l l protein decreased with increasing concentration (100 JIM to 500 uM) of the supplement. The increase i n t u r b i d i t y of c e l l cultures was  followed at 600nm so that the yellow colour of the  DNP-supplemented medium would not contribute to the absorbancy  readings.  Table X shows the decrease i n e f f i c i e n c y from the control value, ranging from 4.1% at 10.0 uM DNP  to 30.9%  at 500 uM DNP.  An increase i n doubling  time accompanied this decrease i n growth e f f i c i e n c y . There was no growth of c e l l s when DNP was present at 1000.0 uM. At a concentration of 500 DNP,  the e f f i c i e n c y of growth was  uM  s i m i l a r to that found for Sm-dependent  E. c o l i B (Table 1). In addition, the doubling time of 87.5 minutes was i n agreement with the value of 85 minutes obtained by Coukell (1969) f o r the Sm-dependent mutant. In order to characterize t h i s e f f e c t of DNP e f f i c i e n c y was  further, the growth  determined under anaerobic conditions (Table XI). The  decrease i n e f f i c i e n c y (from 0.314  to 0.2 51) observed when c e l l s were  grown aerobically was not seen when c e l l s were grown under a nitrogen atmosphere. For both control and DNP-grown c e l l s , the anaerobic e f f i c i e n c y was  approximately 0.100. DNP was  therefore affecting an aerobic (and  not anaerobic) energy-yielding route. A difference i n the e f f e c t of DNP under aerobic versus anaerobic conditions was  also reported by Pavlasova  and Harold (1969); they found that anaerobic generation of ATP i n general was not i n h i b i t e d by uncouplers, but that u t i l i z a t i o n of metabolic  energy  for the active transport of galactosides was prevented. A p o s s i b i l i t y was  that DNP  increased the permeability of the membrane to protons, thereby  abolishing the proton gradient across the membrane, a condition perhaps  62  Table X.  E f f e c t of various concentrations of supplement on the e f f i c i e n c y  (E) of growth f o r wild-type E. c o l i B  supplement  Concentration of supplement i n the medium (uM)  DNP (2 ,4-dinitrophenol)  TBP (1,3,5-tribromophenol)  CM (chloramphenicol)  NH 0H.HC1 (hydroxylaminehydrochloride) 2  Efficiency (ug/ml protein increase per ug/ml glucose consumed  Decrease in E  0.0 10.0 100.0 250.0 500.0 1000.0  0.314 0.301 0.251 0.244 0.217  control 4.1 20.0 22.3 30.9  54.6 56.2 57.5 68.2 87.5  0.0 50.0 75.0 100.0 250.0 350.0  0.314 0.221 0.217 0.206 0.158  control 29.6 30.9 34.4 49.7  54.6 72.8 74.5 93.9 161.0  0.0 1.0 2.5 5.0 10.0  0.314 0.310 0.259 0.199 -  control 1.3 17.5 36.6 -  54.6 74.3 135.7 281.2 -  0.0 10.0 100.0 500.0  0.314 0.294 0.230  control 6.4 26.8  54.6 61.2 115.8  Doubling time (minutes)  * Per cent decrease i n E was calculated using the control value as reference i n each case. - C e l l s d i d not grow at these supplement concentrations. C e l l s were grown on glucose (0.2%)-salts medium f o r 4 hours i n a shaking water bath, harvested, washed once i n phosphate buffer, then r e suspended i n minimal glucose-salts medium supplemented with various concentrations of compounds as indicated. I n i t i a l and f i n a l samples were removed f o r glucose and protein analyses as described i n METHODS.  63  Table XI.  E f f e c t on e f f i c i e n c y (E) during aerobic and anaerobic growth  on 2,4-dinitrophenol (DNP)-supplemented medium f o r wild-type E_. c o l i B  Supplement Aerobic  Anaerobic  None (control)  0.314  0.111  DNP (100 uM)  0.251  0.100  E i s the e f f i c i e n c y of growth defined as ug/ml increase i n protein per ug/ml glucose consumed,  Exponential c e l l s starved f o r glucose were prepared as i n METHODS. The washed c e l l s were resuspended i n minimal salts-glucose (650ug/ml) medium (+DNP supplement) and grown a e r o b i c a l l y . When the Agng reached 0.20, the culture was transferred to anaerobic conditions as described under METHODS I I I , ( i i i ) . Portions of the culture were removed f o r glucose and protein determinations throughout the entire aerobic and anaerobic growth period.  64  e s s e n t i a l i n the maintenance of an energized membrane conformation. An i n v e s t i g a t i o n of the reduced/oxidized difference  spectrum  (Figure 8) indicated that compared with control c e l l s ( s o l i d l i n e ) , growth with 10 uM DNP  decreased the height of P503 by one-half, and  growth with 250 uM DNP  eliminated the 503nm peak completely.  At this point, i t should be emphasized that since the c e l l s were grown i n medium containing DNP,  the l a t t e r affected the formation  (synthesis), and not the functioning, of the 503nm pigment. Experiments, i n which c e l l s grown without added DNP were subsequently treated with DNP  j u s t p r i o r to s p e c t r a l a n a l y s i s , indicated no e f f e c t upon reduction  of P503 by glucose. P503 synthesis was the presence of DNP.  therefore i n h i b i t e d by growth i n  In some cases, c e l l s were grown on 250 uM DNP  prior  to the actual experiment. When control c e l l s treated i n t h i s manner were subsequently grown without DNP  supplementation, the 503nm peak re-appeared,  i n d i c a t i n g that the e f f e c t of DNP was not only r e v e r s i b l e , but also exerted only during i t s presence i n the medium. It was possible, then, by growing wild-type E_. c o l i on 250-500 uM DNP,  to eliminate the 503nm  pigment and to decrease the aerobic y i e l d of c e l l protein simultaneously. The marked e f f e c t of DNP  on the 503nm pigment prompted an i n v e s t i g a -  tion of other compounds, i n order to ascertain whether DNP was  specific  i n i t s elimination of P503 or whether a general e f f e c t on energy metabolism was  involved. A l l compounds which behave l i k e DNP  e f f e c t have been shown to possess a phenolic -OH thyroxine and halogenophenols  i n t h e i r uncoupling group; f o r example,  (Parker, 1958; Wilson, Ting and Koppelman,  1971). The degree of d i s s o c i a t i o n of a p a r t i c u l a r uncoupler was  correlated  to i t s potency as an i n h i b i t o r of oxidative phosphorylation, highly  65  T  I  1  !  !  I  400  450  500  550  600  ;  I  650  :  I  700  wavelength (nm) Figure .8.  Reduced/oxidized  difference spectra a f t e r addition of  glucose to air-oxidized suspensions of wild-type E_. c o l i B grown in medium supplemented with 2,4-dinitrophenol: , 10 uM; * ' *', 100 uM; — ' — , 250 uM.  , none (control);  66  dissociated phenols possessing  greater b i o l o g i c a l a c t i v i t y . Hempfling  (1970a) found that 300 uM 2,4-dibromophenol (DBP) abolished the e s t e r i f i c a t i o n of P i i n i n t a c t E_. c o l i c e l l s when a l l three s i t e s of oxidative phosphorylation  were f u n c t i o n a l , but d i d not affect the oxidation of NADH.  It was desirable to test this analogue i n place of DNP to determine whether the n i t r o groups of the l a t t e r compound could be p a r t i c i p a t i n g i n a reaction other than the w e l l known e f f e c t as an uncoupler of o x i dative phosphorylation. However, DBP was not available from the usual chemical  companies. The tribromo-derivative was therefore used i n place  of DBP. In view of the potency of a compound (as uncoupler) depending upon the i o n i z a t i o n of the hydroxyl group, one would assume that TBP, possessing  an extra Br- constituent, would have a greater tendency to  withdraw electrons from the aromatic ring than the dibromo-derivative, consequently r e s u l t i n g i n a greater p o t e n t i a l toward i o n i z a t i o n . Stockdale and Selwyn (1971), i n v e s t i g a t i n g the e f f e c t s of ring substituents on the a c t i v i t y of phenols as i n h i b i t o r s and uncouplers of mitochondrial r e s p i r a t i o n , compared the compounds DNP, DBP, and TBP. They concluded that i n addition to the mediation of proton transport across the membrane, these phenols affected one or more components of the electron transfer system, an e f f e c t not involved i n uncoupling. As seen i n Table X, growth of c e l l s on minimal s a l t s - l i m i t i n g  glucose  supplemented with 50-250 uM TBP resulted i n a gradual decline i n e f f i c i e n c y values, accompanied by an increase i n doubling time. Similar experiments with chloramphenicol  (CM) as supplement indicated the same trend at a  concentration 100-fold l e s s than i n the case of DNP or TBP. Hydroxylaminehydrochloride  (NHoOH.HCl)  also i n h i b i t e d growth of the c e l l s . Corresponding  67  measurements o f the h e i g h t of the 503nm peak f o r each indicated  a d i f f e r e n c e i n the e f f e c t s e x e r t e d by  X I I ) . Hydroxylamine-hydrochloride contrast  c a s e , however,  the i n h i b i t o r s  (Table  had no e f f e c t on P503 h e i g h t ( i n  to the f i n d i n g by Kepes (1964) t h a t P503 r e a c t s w i t h h y d r o x y l a -  mine t o form a c o l o u r l e s s compound), so t h a t i t s e f f e c t must be e x e r t e d on some c e l l u l a r p r o c e s s o t h e r than the P503 pathway o f energy bolism.  (In m i t o c h o n d r i a , Yoshikawa and O r i i  (1970) and  Sekuzu and Okuni (1960) have shown hydroxylamine chrome o x i d a s e r e a c t i o n at 3 . 1 x l 0 ^ M and 1 0 ^ -  -  Takemori,  to i n h i b i t  a g r a d u a l e f f e c t . These two  the c y t o -  M respectively).  e l i m i n a t e d P503 even a t i t s l o w e s t c o n c e n t r a t i o n , w h i l e had  in this  Thus f a r , w i l d - t y p e E_. c o l i B grown on 500  TBP  chloramphenicol  l a t t e r compounds were t e s t e d  and the r e s u l t s w i l l be d i s c u s s e d l a t e r  meta-  further,  section. uM DNP  was  seen t o  "mimic" the Sm-dependent phenotype w i t h r e s p e c t t o a d e c r e a s e d a e r o b i c energy y i e l d  (~30%) and l a c k o r d e c r e a s e o f the 503nm pigment. C o u k e l l  and P o l g l a s e (1969) found t h a t Sm-dependent E_. c o l i B had l e v e l s of c a t a b o l i t e r e p r e s s i b l e enzymes. For purposes  of  de-repressed comparison,  t h e s e v a l u e s were i n c l u d e d i n T a b l e X I I I . When the enzyme l e v e l s were checked  f o r d e - r e p r e s s i o n i n the non-dependent r e v e r t a n t (SBr4) , s i m i l a r  t r e n d s were n o t e d . W i l d - t y p e  c e l l s grown w i t h excess g l u c o s e i n s a l t s  medium c o n t a i n i n g 100-500 uM DNP enzyme l e v e l s . At 500  uM DNP,  showed p r o g r e s s i v e l y g r e a t e r d e r e p r e s s e d  the s p e c i f i c a c t i v i t i e s of fumarase and  a c o n i t a s e were i n c r e a s e d over f i v e - f o l d . G l u c o k i n a s e and dehydrogenase a c t i v i t i e s were not a f f e c t e d by DNP,  isocitrate  i n agreement w i t h  v a l u e s f o r Sm-dependent E_. c o l i B ( C o u k e l l and P o l g l a s e , 1969). An unexpected  r e s u l t was  the f o u r - f o l d i n c r e a s e i n g l u c o s e 6-phos-  68  Table XII.  E f f e c t of various concentrations of supplement on the height  of P503 i n wild-type E. c o l i B  Supplement  DNP (2,4-dinitrophenol)  TBP (1,3,5-tribromophenol)  CM (chloramphenicol)  NH 0H.HC1 (hydroxylaminehydrochloride) 2  Concentration of supplement i n the medium (uM)  Height of P503*  0.0 10.0 100.0 250.0 500.0 1000.0  1.00 (control) 0.48 0.10 0.00 0.00  0.0 50.0 75.0 100.0 250.0 350.0  1.00 (control) 0.00 0.00 0.00 0.00  0.0 1.0 2.5 5.0 10.0  1.00 (control) 0.44 0.12 0.00  0.0 10.0 100.0 500.0  1.00 (control) 0.92 0.92  -  -  -  —  *  Height of P503 i s expressed as a f r a c t i o n of the control value,  designated as 1.00. - C e l l s did not grow at these supplement concentrations.  Experimental conditions were as described i n Table X.  Table XIII.  Specific a c t i v i t i e s of enzymes from wild-type (SB), wild-type supplemented with 2,4-dinitro-  phenol (SB-DNP), Sm-dependent (DB), and non-dependent revertant (SBr4) strains of E_. c o l i B  Ratio of enzyme a c t i v i t i e s Specific a c t i v i t y  (units/mg of protein) DB SB  SBr4 SB  5.35  5.30  3.90  577  5.10  6.98  4.80  452  3.88  1.18  0.72  Strain Supplement...  SB None  DB None  SBr4 None  SB-DNP 2 ,4-dinitrophenol (IOOUM) (250uM) (500uM)  fumarase  481  2545  1872  622  2400  2569  aconitase  113  789  543  246  547  glucose 6-phosphate dehydrogenase  116  137  84  169  403  SB-DNP (500uM) SB  Results f o r Sm-dependent E. c o l i B were taken from Coukell (1969) . The minimal salts medium contained glucose at an i n i t i a l concentration of 0.2%. Enzymes were assayed in u l t r a s o n i c a l l y - t r e a t e d extracts of exponential c e l l s prepared as described i n METHODS. S p e c i f i c a c t i v i t i e s (units of enzyme / mg of protein) are averages from several determinations.  phate dehydrogenase a c t i v i t y , an enzyme not known to be s e n s i t i v e t o c a t a b o l i t e r e p r e s s i o n . From t h e r a t i o s g i v e n i n the l a s t of Table X I I I , the a c t i v i t y  two columns  of t h i s enzyme i n c o n t r o l c e l l s  i s seen  to be s i m i l a r t o t h a t i n the Sm-dependent mutant (DB/SB = 1.18) and s l i g h t l y h i g h e r than t h a t i n t h e non-dependent r e v e r t a n t (SBr4/SB = 0.72). The  similarity  chloramphenicol efficiency  i n the e f f e c t o f 1,3,5-tribromophenol  (TBP)  and  (CM) t o 2 , 4 - d i n i t r o p h e n o l (DNP), b o t h i n decreased  o f growth and i n e l i m i n a t i o n of P503, suggested  n a t i o n o f enzyme l e v e l s . From T a b l e XIV, i t can be seen (at a l l c o n c e n t r a t i o n s used) had v i r t u a l l y no e f f e c t  an exami-  t h a t TBP  on fumarase o r  g l u c o s e 6-phosphate dehydrogenase. CM, on t h e o t h e r hand, a t l / 1 0 0 t h the c o n c e n t r a t i o n o f t h e o t h e r compounds, caused of  a general  p r o t e i n s y n t h e s i s , d e c r e a s i n g t h e l e v e l s o f both  inhibition  enzymes.  When DNP, CM, TBP and Nl^OH.HCl were compared w i t h r e s p e c t to the t h r e e c r i t e r i a o f e f f i c i e n c y , P503 f o r m a t i o n , and d e - r e p r e s s e d enzyme l e v e l s , t h e e f f e c t s of each compound were d i s t i n c t l y  different  DISCUSSION The unique,  o v e r a l l e f f e c t o f 2 , 4 - d i n i t r o p h e n o l (DNP) as supplement was and i n v o l v e d t h e f o l l o w i n g f o u r a s p e c t s i n common w i t h  dependent mutants: (1) slower growth r a t e ; yield  (2) lower  cell  protein  ( 3 0 % ) ; (3) i n h i b i t i o n o r impairment o f P503 f o r m a t i o n ;  r e p r e s s i o n o f c a t a b o l i t e r e p r e s s i b l e enzymes. The o n l y  Sm-  (4)  de-  difference  between Sm-dependent and w i l d - t y p e c e l l s grown w i t h DNP was de-repres s i o n by DNP, as w e l l , o f g l u c o s e 6-phosphate dehydrogenase. I t i s c o n c e i v a b l e t h a t NADPH i s a c o - r e p r e s s o r of g l u c o s e 6-phos  Table XIV.  Specific a c t i v i t i e s of enzymes from wild-type E_. c o l i B grown on glucose supplemented  with 1,3,5-tribromophenol  or chloramphenicol  Specific a c t i v i t y (units/mg of protein)  Supplement...  None  1,3,5-tribromophenol (50uM) (lOOuM) (250uM)  chloramphenicol (l.OuM) (2.5uM) (5.0JUM)  fumarase  481  453  440  423  588  244  139  glucose 6-phosphate dehydrogenase  116  132  130  137  101  75  91  Cultures were grown on 0.2% glucose-salts medium supplemented as indicated. Extracts were prepared f o r enzyme assays as i n Table XIII.  72  phate dehydrogenase. I f t h i s i s t h e c a s e , d e - r e p r e s s i o n o f t h i s enzyme could result its  from r a p i d removal o f i t s p r o d u c t , NADPH, which may t r a n s f e r  hydrogen t o 2 , 4 - d i n i t r o p h e n o l  (an e f f e c t o f DNP d i f f e r e n t  from i t s  r o l e as an u n c o u p l e r o f o x i d a t i v e p h o s p h o r y l a t i o n ) . The medium c o n t a i n i n g DNP d i d undergo a change i n c o l o u r from y e l l o w t o brown when w i l d - t y p e E_.  coli  the  c e l l s were grown i n i t - an i n d i c a t i o n o f p o s s i b l e r e d u c t i o n o f  n i t r o group(s) to t h e amino form. The amount o f DNP a v a i l a b l e f o r NADPH o x i d a t i o n was c a l c u l a t e d f o r  1 litre  o f medium. An i n i t i a l  c o n c e n t r a t i o n o f 0.04% o r 0.40/180 = 2.2x10  moles o f g l u c o s e was p r e s e n t . S e v e r a l s t u d i e s have e s t i m a t e d that 24-30% of  t h e g l u c o s e i s m e t a b o l i z e d by the hexosemonophosphate  shunt  and R i t t e n b e r g , 1969; Model and R i t t e n b e r g , 1967; Wang e t a l . , If  30% i s t a k e n as t h e maximum u t i l i z a t i o n  or  13.2xl0  + H the  +  - 4  moles of NADPH + H  +  (Caprioli 1958).  of the pathway, then 2(6.6x10"  would be formed. S i n c e 3 moles o f NADPH  a r e r e q u i r e d f o r the r e d u c t i o n o f a n i t r o group to t h e amine, then amount of DNP (5.0xl0~4 moles) which e l i m i n a t e d P503 exceeds  that  r e q u i r e d t o r e a c t w i t h a l l of t h e NADPH which might be produced. That is, of  i n the r e d u c t i o n o f one n i t r o group p e r DNP m o l e c u l e , 5.0x10"^ moles DNP would r e a c t w i t h 15.0x10""^ moles of NADPH whereas n o t more than  13.2xl0~4 moles o f NADPH would be e x p e c t e d t o be formed. Thus,  enough  DNP was p r e s e n t i n t h e medium t o remove t h e NADPH which c o u l d be formed by t h e HMP s h u n t . I t was found (Tables I I I and IV) t h a t w i l d - t y p e c e l l s were more efficient  than Sm-dependent mutants  o n l y when grown a e r o b i c a l l y . DNP  l i k e w i s e a f f e c t e d o n l y a e r o b i c e f f i c i e n c y . S i n c e i n b o t h cases t h e impairment i n energy metabolism was accompanied by the d i s a p p e a r a n c e o r e l i m i n a t i o n of P503, one would c o n c l u d e t h a t P503 i s i n v o l v e d i n an  73  aerobic route of energy metabolism. This leads to the general hypothesis: an oxidative pathway i s present i n wild-type E_. c o l i which generates ATP from NADPH v i a an intermediate pigment, P503 (Figure 12). This pathway accounts f o r 25% to 35% of the t o t a l energy or 50% of the solely aerobic energy produced by c e l l s grown on a minimal s a l t s medium with glucose as carbon source. In Sm-dependent mutants, the mutation results i n a P503 d e f i ciency. In DNP-grown wild-type c e l l s , the P503 deficiency was produced in some way by the DNP. In either case, the proposed NADPH-*P503—»ATP pathway was absent. Results which correlate with those obtained i n the present work have been reported by several investigators and can be understood on the basis of the general hypothesis c i t e d above. Mandelstam (1961) found DNP to r e l i e v e catabolite repression i n non-growing E_. c o l i to d i f f e r e n t degrees, depending on the carbon source. Succinate, l a c t a t e , and pyruvate, which are metabolized a e r o b i c a l l y , produced  incomplete  catabolite repression; DNP was able to reverse this repression. Complete repression by gluconate was only p a t i a l l y released by DNP, and glucose repression was not reversible by DNP. I f one postulates that the e f f e c t of DNP was indeed to oxidize NADPH, a possible candidate f o r catabolite co-repressor, then removal of NADPH by DNP explains a l l of Mandelstam's r e s u l t s with non-growing E_. c o l i . The substrates succinate, l a c t a t e , and pyruvate would not generate much NADPH during t h e i r metabolism, so that repression would be incomplete; any NADPH formed, however, could be removed by DNP. Gluconate, generating 1 molecule of NADPH per molecule of gluconate metabolized, would provide enough NADPH to cause complete  74  repression, but DNP could p a r t i a l l y remove the e f f e c t o r . Glucose, producing twice as much NADPH, would also cause complete repression; but enough NADPH would probably remain even with addition of DNP, so that the l a t t e r compound would be i n e f f e c t i v e i n releasing this repression. In a report on the oxidative pathway of carbohydrate metabolism, not known to us u n t i l a f t e r the publication of our preliminary results and speculations  (Kamitakahara and Polglase, 1970), Scott (1956b)  found a f o u r - f o l d de-repression  of glucose 6-phosphate dehydrogenase  when E_. c o l i was grown i n medium containing DNP. I t i s i n t e r e s t i n g at this time to review her observations  and to determine whether or  not they are i n agreement with our hypothesis:  (1) Scott found that  2x10"'* M DNP as supplement affected the y i e l d of c e l l s , but had no e f f e c t on the rate of removal of glucose from the medium. This observation can be r e a d i l y explained i f DNP were accepting hydrogen and re-oxidizing NADPH. Glucose would s t i l l be taken up from the medium since a product (NADPH) i s removed, but the amount of NADPH l e f t for reducing power i n biosynthetic reactions would be l e s s , thereby a f f e c t i n g the o v e r a l l y i e l d of c e l l s ; (2) When c e l l s grown on DNP were removed, washed, and re-grown without DNP, the glucose 6-phosphate dehydrogenase a c t i v i t y was again decreased to the normal l e v e l (Scott, 1956b). Our r e s u l t s were i d e n t i c a l , suggesting  that the e f f e c t of DNP was exerted  only during i t s presence i n the medium; (3) The e f f e c t of DNP on growth and enzyme a c t i v i t y were determined when the carbon sources for growth were arabinose,  l a c t a t e and gluconate (Scott, 1956b).Sfye reported  that  i f arabinose or l a c t a t e were supplied to the c e l l s , no s i g n i f i c a n t e f f e c t of DNP on growth or extractable enzyme a c t i v i t y was perceptible.  75  Gluconate as carbon source de-repressed glucose 6-phosphate dehydrogenase but was less e f f e c t i v e than glucose. These results are consistent with the hypothesis that DNP causes de-repression of glucose 6-phosphate dehydrogenase by re-oxidizing the NADPH formed i n the hexosemonophosphate pathway; (4) In order to determine whether 6-phosphogluconate could be s p l i t without oxidation of NADPH, Scott (1956b) determined the a c t i v i t i e s of the phosphogluconate degrading enzyme (Entner-Doudoroff pathway) versus 6-phosphogluconate dehydrogenase (HMP shunt) during growth on phosphogluconate.She (Scott, 1956b) found that the most active extract had a c t i v i t i e s of 0.012 units and 0.16 units r e s p e c t i v e l y . Although these results indicated that the rate of the phosphogluconate degrading enzyme was less than l/10th that of the 6-phosphogluconate dehydrogenase, since the products of the reactions were not determined, Scott could not estimate the importance of the Entner-Doudoroff pathway i n the metabolism of gluconate; (5) When c e l l s were grown on glucose with DNP, the a c t i v i t y of the phosphogluconate degrading enzyme (EntnerDoudorof f path) was seen to increase, the e f f e c t being greater with prolonged time of exposure to the drug. In addition, the increase obtained in glucose 6-phosphate dehydrogenase a c t i v i t y with DNP as supplement (when glucose or gluconate was the carbon source) was never accompanied by a simultaneous increase i n 6-phosphogluconate dehydrogenase, the f i r s t enzyme s p e c i f i c to the HMP shunt. Our findings were s i m i l a r ; growth of c e l l s i n the presence of 0-500 uM DNP had no e f f e c t on this enzyme. These two results suggest that the accumulation of 6-phosphogluconate r e s u l t s i n the induction of the Entner-Doudoroff enzymes f o r rapid removal of this product. (Eisenberg and Dobrogosz (1967)  76  reported an inducible Entner-Doudoroff pathway i n gluconate-grown E_. c o l i c e l l s ) . Since this pathway i s not known to generate energy during cleavage of 6-phosphogluconate to pyruvate plus t r i o s e phosphate, a lower e f f i c i e n c y would be expected, and this was to be the case;  found  (6) Ascorbic acid or oxidized glutathione with  cysteine were found by Scott to increase NADPH oxidase  activity,  r e s u l t i n g i n an increase i n the rate of re-oxidation of NADPH. In view of the previously mentioned report by Nosoh (1964) that reduction of the c e l l s by ascorbate, cysteine, glutathione, and d i t h i o n i t e resulted i n the f l a t t e n i n g out of the 503nm peak, an explanation f o r both Nosoh and Scott's observations might be that ascorbate,  and oxidized glutathione with cysteine convert the pigment  to a non-absorbing colourless form. The mechanism might be analogous to that operative with sodium d i t h i o n i t e which can e f f e c t the complete reduction of the pigment past the 503nm absorbing form; (7) DNP  was  stage to the leuco  shown by Scott to affect the c e l l s anaerobically  as w e l l . This r e s u l t was  i n contrast to our f i n d i n g s . No d e t a i l s were  given i n Scott's a r t i c l e as to the manner i n which anaerobic  conditions  were produced or maintained. This i s a c r u c i a l point, since i t was discovered i n preliminary experiments that nitrogen gas of 95% purity proved to be unsatisfactory i n eliminating aerobic growth. Scott's explanation for hfctf observed increase i n glucose 6-phosphate dehydrogenase with DNP  was  that the drug blocked other pathways  of metabolism of glucose 6-phosphate (G6-P), r e s u l t i n g i n a pile-up of the substrate so that more enzyme was increased G6-P  formed i n response to the  concentration. A l t e r n a t i v e l y , our explanation i s that  77  the reduced pyridine nucleotide, NADPH, a product and possible corepressor of G6-P of DNP  dehydrogenase, would be removed by i t s reduction  (perhaps to monoamino-mononitrophenol or even further to d i -  aminophenol),  thereby releasing the repression of the enzyme by i t s  product. Previously (point 5, Scott, 1956b), a possible i n t e r p r e t a t i o n was presented concerning the shunting o f f of NADPH and 6-phosphogluconate v i a the inducible Entner-Doudoroff  pathway when DNP  was  present i n the growth medium. A s i m i l a r i n t e r p r e t a t i o n might account for observations of Coukell (1969) which could not be explained at that time. Coukell reported an enhancement i n growth rate (that i s , a decrease i n doubling time from 91 to 74 minutes) i n Sm-dependent c e l l s when gluconate replaced glucose as the carbon source, accompanied by greater repression of the c a t a b o l i t e repressible enzyme, acetohydroxyacid synthetase (that i s , a decrease i n s p e c i f i c a c t i v i t y from 14.1  to 7.85). The l a t t e r observation was unexpected, since h i s  r e s u l t s with glucose-grown c e l l s indicated the Sm-dependent mutant to be de-repressed with respect to the glucose-sensitive enzymes compared to wild-type c e l l s . (Also, the wild-type s t r a i n had the same growth rate on glucose and gluconate). These two observations of enhanced growth rate and increased catabolite repression of acetohydroxyacid synthetase would tend to suggest that gluconate metabolism by the mutant was more e f f i c i e n t than glucose metabolism; however, determination of c e l l y i e l d on the two sugars indicated that the mutant was  38% less e f f i c i e n t than the wild-type s t r a i n , whether grown on  glucose or gluconate (pg dry c e l l s formed / umole carbon source consumed was 59.7  and 59.3 r e s p e c t i v e l y ) . Coukell concluded that the f i r s t  two  78  observations were therefore not a result of increased y i e l d of energy from gluconate. An examination of the metabolic pathways (Figure 12) indicates that gluconate as carbon source v i a the HMP shunt results i n the formation of one less NADPH molecule per molecule of carbon source consumed, than i n the case of glucose. I f gluconate i s d i s s i m i l a t e d v i a the Entner-Doudoroff pathway, then no NADPH i s formed. The chief source of energy would be ATP formed i n the TCA cycle from the pyruvate generated i n two steps i n the Entner-Doudoroff pathway. Although the growth rate i s enhanced, since there are no energy-generating steps for the Sm-dependent s t r a i n v i a the Entner-Doudoroff path or v i a the postulated ATP-generating path of NADPH—*P503, the o v e r a l l e f f i c i e n c y should be the same on glucose and gluconate. It was postulated previously that ATP i s generated from NADPH during oxidation of the l a t t e r compound v i a the P503 pathway. I t should be emphasized  that t h i s pathway i s assumed to be operative as a result  of i t s induction by NADPH. The wild-type organism may then form ATP from NADPH while the Sm-dependent mutant lacks this source of ATP. Indeed, the rate of ATP formation was found to p a r a l l e l the growth rate i n the Sm-dependent organism  (Coukell, 1969) whereas the rate  of ATP production i n the wild-type s t r a i n exceeded the rate of growth. Studies on the reduction of n i t r i t e  (NC^) and n i t r o a r y l  groups  by n i t r i t e reductase have been previously reported i n non-denitrifying organisms.  ( D e n i t r i f y i n g b a c t e r i a such as Pseudomonas aeruginosa are  f a c u l t a t i v e , and can u t i l i z e n i t r a t e or n i t r i t e  ( i n place of oxygen)  as a hydrogen acceptor i n energy-yielding oxidative reactions). L a z z a r i n i  79  and Atkinson (1961) characterized a cyanide-sensitive NADPH-specific n i t r i t e reductase from E_. c o l i which was not stimulated by FMN, FAD, or a variety  of metal ions. Three moles of NADPH were consumed per  mole of n i t r i t e reduced, the reduction product being ammonia. P u r i f i e d n i t r i t e reductase contained three additional a c t i v i t i e s :  cytochrome c  reductase, hydroxylamine reductase, and sulphite reductase, a l l s p e c i f i c for NADPH. In deep-standing (anaerobic)  cultues of E_. c o l i , two other  n i t r i t e reductases were detected, one i n which NADH served as the electron donor, and one in which FMNH^ was the donor. Mager (1960) reported that NADPH-specific sulphite reductase and NADPH-specific hydroxylamine reductase (both FAD dependent) of E_. c o l i , perhaps catalyzed by the same enzyme, were feed-back repressed by methionine, cysteine, or c y s t i n e . Since the Km for sulphite was 100-fold less than for  hydroxyla-  mine, he concluded that sulphite was the "true" physiological substrate and that N^OH reduction was an i n c i d e n t a l capacity of the same enzyme. This point w i l l be considered further i n the discussion of our e x p e r i ments with L-methionine. Taniguchi, Sato and Egami (1956) stated that n i t r a t e with terminal n i t r a t e reductase could serve as a c e l l u l a r  oxidant  instead of oxygen plus the terminal respiratory oxidase, which are the normal oxidants under aerobic conditions. Although they found n i t r a t e metabolism to occur anaerobically as well as a e r o b i c a l l y , the anaerobic process did not proceed beyond the n i t r i t e stage unless oxygen was' added. Thus E_. c o l i was found to possess two d i f f e r e n t types of n i t r a t e metabolism: one being the anaerobic reduction of n i t r a t e to n i t r i t e ,  and the other  being the aerobic reduction of n i t r a t e to n i t r i t e and further to ammonia. They found, however,  that stoichiometric quantities of the expected  intermediates such as hydroxylamine were not obtained; some u n i d e n t i f i e d  80  intermediary product(s) or compound(s) derived from n i t r i t e were therefore assumed to be accumulated. S i l v e r and br^Elroy (1954) showed that wild-type Neurospora mycelia could catalyze the reduction of m-dinitrobenzene to nitrophenylhydroxylamine and f i n a l l y to n i t r o a n i l i n e . The c u l t u r e medium turned a deep amber during growth. As e a r l y as 1935, G r e v i l l e and Stern i d e n t i f i e d the reduction product of 2,4-dinitrophenol i n E_. c o l i c e l l s as 4-nitro-2-aminophenol, the reduction r e q u i r i n g 6 hydrogen equivalents per molecule of DNP reduced. No diaminophenol was detected at t h i s time. I t i s not known whether the metabolism of n i t r i t e proceeds i n microorganisms v i a an inorganic or organic pathway. De l a Haba (1950) postulated that n i t r a t e or n i t r i t e may f i r s t be bound i n an organic form (R-NO-j or R-NO^) p r i o r to i t s reduction to an amino compound (R-Nl^) which then t r a n s f e r s the amino group by transamination, thereby e f f e c t i n g the synthesis of amino a c i d s . In organisms such as E_. c o l i , more than one pathway has been found to e x i s t f o r the d i s s i m i l a t i o n of n i t r a t e (Taniguchi, Sato and Egami, 1956). Although the p h y s i o l o g i c a l s i g n i f i c a n c e of the reduction of n i t r o - a r o m a t i c compounds by microorganisms i s s t i l l obscure, one might suggest that such a process would provide a means of d e t o x i c a t i o n by removal of the nitro-compound. The corresponding amino-form has been shown to be non-toxic (Cain, 1958). The p a r t i c i p a t i o n of one p a r t i c u l a r enzyme system over another would then depend upon the environmental conditions surrounding the organism- any subsequent change i n the medium (for example, aerobic to anaerobic) causing an a l t e r n a t e pathway to come i n t o play. In studies on c a t a b o l i t e r e p r e s s i o n , Prevost and Moses (1967) found that glucose and gluconate were the only sugars whose a d d i t i o n to  81  E_. c o l i growing exponentially on g l y c e r o l resulted i n severe i n h i b i t i o n of ^-galactosidase synthesis. Sixty minutes after the addition of the glucose or gluconate, the c e l l s recovered and synthesis of the enzyme resumed at the normal rate. These workers suggested that the e f f e c t o r exerting this catabolite repression might be either d i r e c t l y produced metabolically from glucose, or i t s concentration might be changed when glucose i s added to the c e l l s , even though the e f f e c t o r i s derived from another source. When the l e v e l s of intermediates of glucose metabolism were followed during the experiments, an increase was observed i n the pool sizes of glucose 6-phosphate, 6-phosphogluconate,  fructose 1,6-  diphosphate, and NADPH immediately upon the addition of glucose. Sixty minutes a f t e r the addition of glucose, the pool sizes of a l l four compounds decreased. On the contrary, they found no change i n the concentrations of ATP, NADH, and several other phosphorylated intermediates. From these r e s u l t s , they concluded that the a c t i v i t y of the pentose phosphate  cycle  was of central importance i n determining the s e n s i t i v i t y of the c e l l s to catabolite repression by glucose. The rapid production of NADPH was presumed to exceed both the c e l l s ' requirement for the reduced coenzyme, as well as the c e l l s ' a b i l i t y to oxidize NADPH i n terminal r e s p i r a t i o n . An accumulat i o n then resulted i n a metabolic imbalance which persisted u n t i l the c e l l s were able to achieve a new steady metabolic state i n the changed environment  caused by adding glucose. They stated, furthermore, that under  anaerobic conditions, the a c t i v i t y of the pentose phosphate  cycle would  be n e g l i g i b l e . In view of the general hypothesis that ATP i s readily  formed  from NADPH v i a P503, i t i s equally p l a u s i b l e that catabolite repression r e s u l t s from the maintenance of a high energy charge v i a this pathway.  82  2.  E f f e c t of a n a e r o b i c growth on f o r m a t i o n of P503  RESULTS The 503nm pigment has p r e v i o u s l y been found i n a n a e r o b i c a l l y grown c e l l s o f Saccharomyces  cerevisiae  mayer and Smith, 1964; Nosoh, when w i l d - t y p e E_. c o l i  (Lindenmayer, 1959; L i n d e n -  1964). S i m i l a r r e s u l t s were o b t a i n e d  B was grown i n i t i a l l y under a e r o b i c c o n d i t i o n s ,  then changed t o an a n a e r o b i c n i t r o g e n atmosphere of  f o r the remainder  the growth p e r i o d ( F i g u r e 9 ) . P503 and the cytochromes were p r e s e n t  b u t i n an o x i d i z e d s t a t e a f t e r a n a e r o b i c growth, i n d i c a t i n g they were not f u n c t i o n a l and d i d n o t a c c e p t e l e c t r o n s i n the absence o f oxygen.  DISCUSSION C e l l s were grown a e r o b i c a l l y  f o r a short period i n i t i a l l y  so t h a t  a s u f f i c i e n t amount o f energy would have accumulated t o c a r r y them over t h e change t o a n a e r o b i o s i s . When a p p r o x i m a t e l y h a l f t h e amount of  P503 was formed under these c o n d i t i o n s , i t was s u s p e c t e d  that  perhaps P503 was b e i n g formed d u r i n g the a e r o b i c phase o f t h e e x p e r i ment and b e i n g d i l u t e d out d u r i n g a n a e r o b i c growth. T h i s  possibility  was d i s m i s s e d , however, when c e l l s were grown i n DNP-supplemented medium (known t o e l i m i n a t e P503) p r i o r t o a n a e r o b i c growth. Chloramp h e n i c o l was added at t h e end o f the experiment ( t o i n h i b i t p r o t e i n s y n t h e s i s ) and the c e l l s were c h i l l e d in  t h e a n a e r o b i c atmosphere p r i o r  i n an i c e - s a l t  further bath  to h a r v e s t i n g . The r e s u l t s were  i d e n t i c a l t o those o b t a i n e d by the f i r s t method o f a n a e r o b i c  growth.  P503 and the cytochromes were formed under a n a e r o b i c c o n d i t i o n s b u t remained i n an o x i d i z e d s t a t e .  83  400  450  500  550  wavelength  600  650  (nm)  Figure 9. Reduced/oxidized difference spectra after addition of glucose to wild-type E_. c o l i B grown anaerobically under a nitrogen (N ) atmosphere. Cells were f i r s t grown a e r o b i c a l l y from A^2o 0.10 to 0.40 i n minimal s a l t s medium containing 650 pg glucose/ml, then transferred to anaerobic conditions as described under METHODS. At the end of growth, the 37* water bath was replaced by an i c e - s a l t bath, and N flow continued f o r 1 hr. The c e l l s were then harvested, washed, and analyzed immediately. Since the cytochromes and P503 were s t i l l oxidized under these conditions, glucose was added to produce the difference spectra. , aerobically grown control c e l l s ; • • • «, anaerobically grown c e l l s . 2  2  700  84  The  formation of these pigments anaerobically i s not s u r p r i s i n g  in view of several observations. An adaptive change i n the type and amount of cytochrome content from anaerobiosis to aerobiosis was  first  established by Ephrussi and Slonimski (1950), and Chin (1950) i n yeast c e l l s . These workers reported the absence of the "normal" cytochrome system i n anaerobically grown c e l l s of Saccharomyces c e r e v i s i a e , and the appearance of these enzymes upon exposure of the c e l l s to oxygen under non-growing conditions. This r e s u l t implied that the inducer of the active enzymes was molecular oxygen, and that the oxygen-binding compound was  synthesized i n the absence of oxygen.  More recently, Chen and Charalampous (1969), Tuppy and Birkmayer (1969), and Henson, Perlman, Weber and Mahler (1968) found that under anaerobic conditions, yeast c e l l s possess cytochrome oxidase apoenzymes which are converted to active enzymes during de-repression and addition of oxygen i n the absence of c e l l d i v i s i o n . In eukaryotes, of the interdependence  of mitochondria  (Chen and Charalampous, 1969; machinery of the cytoplasm was  several cases  and cytoplasm have been reported  Birkmayer, 1971). The protein-synthesizing responsible for synthesis of the apoenzymes,  and only when aerobic conditions were met  did the mitochondria  synthesize  the active oxidase. In t h i s case, oxygen served not only as an inducer i n i t i a t i n g the cytochrome oxidase formation, but also as a cosubstrate i n the biosynthesis of heme a. Perhaps a s i m i l a r but simpler s i t u a t i o n prevails i n E_. c o l i  cells.  In contrast to yeast, reports on the oxygen-mediated regulation of r e s p i r a tory a c t i v i t y or the content of r e s p i r a t o r y enzymes i n b a c t e r i a are scarce and c o n f l i c t i n g . Hino and Maeda (1966) found that the r e s p i r a t o r y a c t i v i t y  85  o f a n a e r o b i c a l l y grown E_. c o l i cells  i n the absence o f c e l l p r o l i f e r a t i o n . S i n c e  nitrogen  remained c o n s t a n t ,  of proteins for  c o u l d be i n c r e a s e d by a e r a t i n g the t h e amount o f c e l l  they c o n c l u d e d t h a t t h e g e n e r a l  synthesis  d i d n o t o c c u r ; however, s e v e r a l amino a c i d s were  t h i s development o f r e s p i r a t o r y a c t i v i t y , s u g g e s t i n g  synthesis  of protein(s)  occurred.  The i n c r e a s e  responsible  required  that  specific  f o r t h e r e s p i r a t i o n o f E_.  i n respiratory activity  coli  reached the l e v e l o f  a e r o b i c a l l y grown c e l l s when the c u l t u r e was a e r a t e d  i nbuffer  containing  casamino a c i d s , w i t h o u t a concomitant i n c r e a s e i n the c o n t e n t of c y t o chromes bit a-p o r a . They i n f e r r e d from these r e s u l t s t h a t 2  were n o t the l i m i t i n g and  t h a t other Little  f a c t o r f o r o v e r a l l r a t e o f r e s p i r a t i o n i n E_.  coli,  t e r m i n a l o x i d a s e systems might be p r e s e n t .  i s known about the s t r u c t u r a l and f u n c t i o n a l changes i n t h e  membrane system o f E_. c o l i sis.  cytochromes  E_. c o l i  and o t h e r  during  t h e change from a n a e r o b i o s i s  f a c u l t a t i v e b a c t e r i a do n o t p o s s e s s  to aerobio-  extensive  membrane systems (Gray, Wimpenny, Hughes and Mossman, 1966).  Anaerobically  grown c e l l s were found by these workers t o r e t a i n many of t h e membranebound components that a r e c h a r a c t e r i s t i c o f aerobes and  (cytochromes o x i d a s e  b-^) , b u t t o form, i n a d d i t i o n , s o l u b l e enzymes s i m i l a r t o those  t y p i c a l o f anaerobes ( s o l u b l e cytochrome c ) . I n 1966, Mossman r e p o r t e d  that  Gray, Wimpenny and  the amount o f NADH o x i d a s e was d e c r e a s e d  during  a n a e r o b i c growth, w h i l e adequate amounts o f cytochromes a-^, a , and b-^ 2  were s t i l l  present.  They s t a t e d t h a t t h i s decrease i n NADH o x i d a s e was  p r o b a b l y due t o a d e f i c i e n c y i n some e l e c t r o n c a r r i e r o t h e r cytochromes. I n a d d i t i o n , C a v a r i , A v i - D o r , and Grossowicz  than these  (1968) observed  a r e l a t i o n s h i p between t h e development o f t h e r e s p i r a t o r y system and t h e  86  energy-conserving  phosphorylative mechanism of E_. c o l i during the  t r a n s i t i o n from anaerobic to aerobic growth. Upon exposure to oxygen, the NADH oxidase a c t i v i t y of anaerobically grown c e l l s increased to the aerobic l e v e l i n 10 minutes although  the cytochrome content was  re-established a f t e r 60 minutes. This development of NADH oxidase a c t i v i t y was p a r a l l e l e d by the synthesis of protein and r i b o n u c l e i c acid (but not deoxyribonucleic a c i d ) . The implication of t h e i r r e s u l t s was  that the respiratory chain between NADH and the cytochromes was  linked only when oxygen was present. Our r e s u l t s were s i m i l a r to those of Gray, Wimpenny, Hughes and Mossman (1966) i n that the cytochrome spectra were obtained under both aerobic and anaerobic compartmentalization  conditions. Since prokaryotes  do not possess  of various functions and have only one type of  protein-synthesizing apparatus,  the cytochromes and P503 may be produced  under anaerobic conditions, but remain non-functional u n t i l the system i s exposed to oxygen. Such a mechanism would prepare the c e l l s f o r immediate u t i l i z a t i o n of high energy aerobic routes i n preference to the lower energy-yielding anaerobic pathways whenever oxygen i s made available to them.  87  3.  N u t r i t i o n a l effects on formation of P503 RESULTS Since the height of the 503nm peak and i t s order of appearance  with respect to the cytochrome bands i n the difference spectra were found to be dependent upon the carbon source used as reductant  (Table  VII) , wild-type c e l l s of E_. c o l i B were grown on a v a r i e t y of sugars and acids to determine t h e i r e f f e c t on formation  (synthesis) of P503  (Table XV). The presence of P503 was tested by glucose reduction of a i r - o x i d i z e d suspensions,  since this method was shown previously to  produce the maximum height of the P503 peak. Glucose-grown, glucose-reduced  c e l l s showed a very large 503nm  peak and were used as the c o n t r o l . Gluconate as carbon source yielded a s l i g h t l y smaller peak. Growth on g l y c e r o l , succinate, and l a c t a t e resulted i n a smaller but s t i l l noticeable 503nm pigment. The cytochromes and f l a v i n were not affected by changes i n carbon source. Supplementation of succinate medium with iron ( f e r r i c chloride) did not a f f e c t the r e s u l t s , i n d i c a t i n g that iron was not l i m i t i n g under the growth conditions u t i l i z e d . Monitoring of pH throughout the f i n a l growth on succinate showed no deviat i o n from pH 7.0. When c e l l s were grown on 0.04% glucose plus 0.04% 2-deoxy-D-glucose, glucose reduction produced one-half the amount of P503 of c o n t r o l c e l l s (without added 2-deoxyglucose). A correspondingly  lower  growth rate was observed. In general, P503 was formed i n variable amounts on a l l carbon sources tested. When the concentration of glucose or of gluconate i n the medium was increased, an e f f e c t was observed- on the amount of P503 formed (Table XVI). With glucose, the height of the P503 peak decreased  from 38.9 units  88  Table XV.  E f f e c t of various carbon sources as growth supplement  on P503 formation (synthesis) i n wild-type E. c o l i B  Sugar or acid supplement  Concentration of supplement i n the medium  Per cent o formed  (%)  glucose  0.04  . 100.0  gluconate  0.04  74.3  glucose + gluconate  0.02 0.02  77.0  succinate  0.07 0.53  59.8 30.9  glycerol  0.05 2.00  32.4 6.4  lactate  0.20  30.2  The height of the 503nm peak was taken as an i n d i c a t i o n of the amount of P503 formed. The per cent of P503 formed was  determined  r e l a t i v e to the control (0.04% glucose) value of 100.0%. The cytochromes and f l a v i n spectra were not affected. C e l l s were grown on minimal s a l t s medium supplemented with various carbon sources as indicated. In a l l cases, the presence of P503 was tested by glucose reduction of a i r - o x i d i z e d For experimental d e t a i l s , see METHODS.  suspensions.  89  Table XVI.  E f f e c t of concentration of carbon source i n medium (glucose  or gluconate) on P503 formation (synthesis) i n wild-type E_. c o l i B  Supplement  Concentration of supplement i n the medium  Height of P503 (units)  (%) glucose  gluconate  Height of cytochrome b** (units)  0.05 0.25 0.75 1.25 1.75 2.00  38.9 31.3 27.1 14.7 13.0 0.0  29.2 •i  0.05 0.16 0.20 0.50 2.00  22.8 13.3 10.2 6.8 0.0  29.2 ti  Ratio of he: height cytoi  II II  ti it  II  it ti  1.36 1.07 0.93 0.52  0.45 0.00 0.78  0.46 0.35 0.23 0.00  The height of the 503nm peak was taken as an i n d i c a t i o n of the amount of P503 formed. The height of cytochrome b was normalized to 29.2 units i n each case (see Table V). C e l l s were grown on 0.2% glucose (0.2% gluconate), harvested during log phase, washed once i n phosphate buffer, then resuspended  i n minimal  s a l t s medium supplemented with the concentrations of glucose or gluconate as indicated. During l o g phase (see METHODS), c e l l s were harvested, washed, and resuspended  i n 0.01 M phosphate buffer. The reduced/oxidized difference  spectra were obtained by addition of hydrogen peroxide to the bottom cavette.  90  (at 0.05% glucose) to 0.0 units (at 2.00% glucose); cytochrome b remained constant i n height. Gluconate was e f f e c t i v e i n decreasing P503 synthesis at lower concentrations. Gluconate at a concentration of 0.16% or glucose at 1.75% gave the same r a t i o of 0.45 f o r height of P503 / height of cytochrome b. The rates of growth with excess carbon source were i d e n t i c a l throughout  the range of concentrations tested.  DISCUSSION In microorganisms, a change i n the n u t r i t i o n a l environment i s accompanied by an adaptive change i n the metabolic route u t i l i z e d f o r d i s s i m i l a t i o n of the nutrient (Romberg, 1961; Sanwal, 1970). Since prokaryotes lack the physical compartmentation found i n higher organisms (eukaryotes), they have evolved precise regulatory mechanisms to channel metabolic intermediates into the appropriate anabolic, c a t a b o l i c , or amphibolic pathway i n response to t h e i r energy and growth requirements. The degree to which these pathways are linked with P503 may be r e f l e c t e d in the height of P503 obtained. Generally, the P503 peak was found to decrease i n height (but not be eliminated) with carbon sources whose metabolism does not involve NADPH production at an early stage. During the process of glucose and gluconate oxidation, NADPH i s synthesized v i a the hexosemonophosphate (HMP) shunt. Numerous workers have followed the break-down of r a d i o a c t i v e l y l a b e l l e d glucose i n an attempt to determine the extent to which various pathways are u t i l i z e d . S p e c i f i c y i e l d s of -^O-labelled CO2 produced from E_. c o l i grown i n minimal medium on (1-^0) glucose, (6--^0) glucose, and ( l - ^ o ) g l u c o n a t e indicated that 25% of the sugar was channelled through the HMP shunt  (Model and  Rittenberg, 1967). In s i m i l a r studies, C a p r i o l i and Rittenberg (1969)  91 reported a value of 30% and Wang et a l . (1958) 28%. The remainder the carbon source (up to 70%) was  of  found to be metabolized v i a the Embden-  Meyerhof (E-M) path of g l y c o l y s i s . The Entner-Doudoroff  (E-D) path  was  of l i t t l e s i g n i f i c a n c e under these conditions (Wang et a l . , 1958; Scott and Cohen, 1953;  Scott, 1956a). Only when the HMP  pathway i s blocked  or not required f o r ribose and NADPH production does the E-D  route come  into play. This inducible pathway provides a means of producing pyruvate and glyceraldehyde 3-phosphate more d i r e c t l y than v i a g l y c o l y s i s , but i s not known to r e s u l t i n any form of energy production p r i o r to the TCA cycle. C o n f l i c t i n g evidence was (1967), who  reported by Eisenberg and Dobrogosz  favoured the E-D path as the primary route of gluconate  metabolism i n E, c o l i . However, since they found no s i g n i f i c a n t differences i n the l e v e l s of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase i n c e l l - f r e e extracts of glucose-grown and gluconategrown c e l l s , one can assume that the u t i l i z a t i o n of the HMP  shunt remained  constant; induction of the E-D pathway allowed rapid production of pyruvate for a more d i r e c t entrance into the TCA cycle. Nevertheless, NADPH w i l l be produced even though the main route of gluconate metabolism may the E-D  be  path.  Glycerol metabolism involves the induction of g l y c e r o l kinase and ^-glycerophosphate  dehydrogenase (Koch, Hayashi and L i n , 1964); i t s  conversion to dihydroxyacetone phosphate and subsequent entrance into the g l y c o l y t i c or glucogenic pathway does not r e s u l t i n NADPH production. A smaller amount of P503 was  detected i n the reduced/oxidized difference spectra  of glycerol-grown c e l l s when compared to the control case  (glucose-grown).  92  Growth of E_. c o l i B on lactate gave a doubling time of 120 minutes (Scott, 1956a). D i s s i m i l a t i o n of the C^ compound occurs v i a the induction of phosphoenolpyruvate  (PEP)-synthase, an enzyme catalyzing the conversion  of pyruvate to PEP (Romberg and Smith, 1967; Cooper and Romberg, 1967). Lactate, therefore, does not p a r t i c i p a t e i n NADPH generation during i t s metabolism; a lower amount of P503 was observed. When succinate serves as a carbon source, the glucogenic enzyme PEP carboxykinase i s induced, catalyzing the step from oxalacetate to PEP (Sanwal, 1970). The immediate reactions of succinate involve i t s oxidation to fumarate ( v i a the f l a v i n - l i n k e d dehydrogenase) and further to malate. At this stage, either of two routes may be followed: (1) malate oxalacetate (involving NADH) or (2) malate  ^  >• pyruvate (generating  NADPH). Ratsuki, Takeo, Rameda and Tanaka (1967) reported that the NADP +  linked malic enzyme was operative i n E_. c o l i c e l l s grown on minimal medium whereas the NAD -linked malic enzyme was active when an enriched medium +  was a v a i l a b l e . The p a r t i c i p a t i o n of these enzymes i s therefore dictated by the energy state and biosynthetic requirements of the c e l l . I t follows then that E_. c o l i grown under our conditions of minimal salts-succinate would possess de-repressed l e v e l s of the NADP -specific malic enzyme. +  It i s therefore expected that succinate-grown. c e l l s would eventually accumulate enough NADPH to e l i c i t a P503 band. The 503nm pigment could be detected i n succinate-grown c e l l s . Less P503 was formed at high than at low glucose or gluconate concentrations suggesting a l i n k between P503 and catabolite repression. These sugars have been reported to produce severe repression singly (Prevost and Moses, 1967; Mandelstam, 1961). But the simultaneous presence of both  93  glucose and gluconate should be most e f f e c t i v e i n causing catabolite repression (Hsie, Rickenberg, Schulz and Kirsch, 1969; Perlman, de Crombrugghe and Pastan, 1969; Moses and Sharp, 1970). Since P503 was  as large i n this case (glucose and gluconate together) as i n  the control, i t s r e l a t i o n s h i p to catabolite repression i s neither simple nor straight forward. NADPH i s an a t t r a c t i v e candidate for catabolite co-repressor. Repression has been found to depend on the rapid metabolism of the carbon source, r e s u l t i n g i n a metabolic imbalance  of some compound. Model and Rittenberg (1967) reported the  HMP shunt to function maximally  during log phase when reducing power  i s needed f o r biosynthetic purposes. By stationary phase, as b a c t e r i a l growth ceases, the pathway would no longer be needed as a source for NADPH or pentose so that the HMP route decreases i n a c t i v i t y . Similar observations have been found by several independent  groups. A l l e n and  Powelson (1958) and Scott and Cohen (1953) observed that on l i m i t i n g carbon source, rapid growth resulted i n the d i s s i m i l a t i o n of glucose v i a the HMP shunt; at the onset of stationary phase, a s h i f t occurred from the HMP pathway to the E-M route. Concentrations of the nicotinamide adenine dinucleotide coenzymes were reported to fluctuate between b a c t e r i a of d i f f e r e n t p h y s i o l o g i c a l types (obligate anaerobes, anaerobes,  facultative  and obligate aerobes) and with the n u t r i t i o n a l and c u l t u r a l  nature of the medium surrounding microorganisms  (London and Knight, 1966).  E_. c o l i grown aerobically on minimal medium and l i m i t i n g glucose possessed the highest concentration of the NADP-coenzyme (0.84 umoles / gm dry wt); a change to anaerobiosis, excess glucose, or completely defined medium decreased t h i s value to 0.58, 0.24, and 0.48 respectively. The NAD-coenzyme  94  remained f a i r l y constant under a l l conditions at ~2.5 jumoles / gm dry wt. It i s i n t e r e s t i n g that Sanwal (1970) reported the l e v e l of NADH (opposite to that of NADPH) to be high i n early log phase (5.5 umoles / gm dry wt) , decrease during mid log (3.0) and increase again by stationary phase (4.5). In keeping with the a c t i v i t y of the HMP  shunt, Hempfling  (1970b) found  that E_. c o l i B grown on glucose possessed a lower e f f i c i e n c y of phosphoryl a t i o n when harvested during the log period (due to the presence of glucose) than c e l l s harvested i n the stationary phase of growth. These r e s u l t s suggested to him that part of the enzymatic or s t r u c t u r a l apparatus of oxidative phosphorylation was  subject to catabolite repression.  Whether P503 i s s e n s i t i v e to, or whether i t a f f e c t s catabolite repression i s not known. Indeed, the two may not even be connected. In view of our hypothesis r e l a t i n g P503 with NADPH, the observed results may  reflect  the action of the reduced coenzyme as opposed to the pigment i t s e l f . If the amount of P503 present i n growing c e l l s r e f l e c t e d the energy  state  and r e l a t i v e proportion of metabolic routes u t i l i z e d , then one would expect the height of P503 to vary also. Labbe et a l . , (1967) and Nosoh (1964), using a synchronous culture of yeast c e l l s , found the 503nm peak was highest during log growth. When the culture was stationary phase, the 503nm band was  continued to the  lowered appreciably. Depending upon  the time of harvesting, therefore, one might obtain the same r e s u l t s seen i n Table XVI. Again the d i s t i n c t i o n between limited versus excess  carbon  source f o r growth must be emphasized. The constancy i n growth rate even with increasing concentrations of glucose (or gluconate), i n contrast to the decline i n P503, may be i n d i c a t i v e of the c e l l s ' need f o r the u t i l i zation of the P503 pathway. Only under l i m i t i n g conditions, when a given  95  substrate i s broken down and oxidized f u l l y , i s a high energy-yielding path needed, and therefore used. Under enriched conditions (such as an excess of substrate), other routes such as glycogen synthesis are also u t i l i z e d and the path of P503 i s no longer needed immediately, nor i s i t used. Labbe, Volland and Chaix (1967) noted the lack of a P503 peak when c e l l s were grown on enriched media such as nutrient agar or peptone broth. Similar findings were reported by Nosoh and Itoh (1965). E_. c o l i and B a c i l l u s s u b t i l i s grown i n complex medium (8 g nutrient broth and 40 g glucose per l i t r e ) showed no P503 band. Absence of t h i s pigment  was  confirmed by difference spectrophotometry. Since Nosoh (1964) had previously observed that r e s p i r a t i o n seemed to be e s s e n t i a l to obtain the 503nm band, the respiratory a c t i v i t y was obtained  determined on complex medium. The high values  (1965) l e d to t h e i r conclusion that P503 i s absent i n b a c t e r i a l  c e l l s under favourable conditions. The a c t i v i t y of the HMP  shunt  was  reported by Model and Rittenberg (1967) to be high during logarithmic growth on glucose i n minimal medium and s i g n i f i c a n t l y less as c e l l s entered the stationary phase. The i d e n t i c a l behavioural pattern of the appearance of the P503 band and the a c t i v i t y of the HMP  shunt i s remarkable.  This group also reported the shunt a c t i v i t y to be less when the minimal medium was  enriched with casein hydrolysate or yeast extract- again  p a r a l l e l i n g observations made with P503. Since the amino acids and other compounds necessary  for growth and d i v i s i o n of the c e l l s are  already present i n the medium, the c e l l s need not b u i l d up stores of NADPH for reducing power i n biosynthetic reactions. Consequently, P503 would not be induced and a d d i t i o n a l pathways producing high energy ATP would not be required.  96  4.  E f f e c t of L-methionine and i t s analogues as growth  supplement  on P503 formation RESULTS Olden and Hempfling (1970) mentioned i n a b r i e f abstract that E_. c o l i grown i n the presence of 1 mg/ml methionine had no P503. I t was of i n t e r e s t to study t h i s observation i n more d e t a i l , especially since previous results (Tables VIII and IX) indicated a s p e c i f i c e f f e c t of L-methionine on e l i c i t i n g a P503 band i n non-growing  c e l l s . Table XVII summarizes the e f f e c t on P503  synthesis when JE. c o l i B i s grown on minimal s a l t s - l i m i t e d glucose med:m supplemented with L-methionine and/or i t s analogues (Figure 10). In the case of ethionine, norleucine and selenomethionine, a large amount of P503 was formed; no e f f e c t of concentration was noticeable. Growth on L-methionine, at. a concentration of 0.01%, repressed P503. In the presence of both L-methionine and an analogue (ethionine or norleucine), P503 formation was markedly decreased when compared to the e f f e c t of the analogue alone. This decrease was due to L-methionine, even though i t was present at a concent r a t i o n l/10th that of the analogue. Other i n d i v i d u a l amino acids such as L-cysteine and glycine as growth supplement had no e f f e c t on P503 formation (L-cysteine, however, adversely affected the growth r a t e ) . Thus, a more complete mixture was  tested. Casein  hydrolysate proved to be just as e f f e c t i v e as L-methionine i n i t s a b i l i t y to abolish P503 synthesis. When a l l the amino acids of casein hydrolysate were added to the growth medium i n the appropriate proportions (West and Todd, 1955) including L-methionine, then the P503 band did not appear. However, when L-methionine was excluded from the mixture, then a peak at 503nm was once again produced. From these r e s u l t s , i t would appear that  97  Table XVII. E f f e c t of L-methionine and i t s analogues as growth supplement on P503 formation (synthesis) i n wild-type E_. c o l i B (The same format i s used as i n Table XVI) Supplement  Concentration of supplement i n the medium  Height of P503 (units)  (%)  0.01  73.0  ti  0.10  71.7  DL-norleucine  0.01  94.2  II  0.10  98.7  0.01  50.5  0.05  52.7  0.01  0.0  0.10  0.0  DL-ethionine  DL-selenomethionine II  L-methionine II  L-methionine  + DL-ethionine  0.01 + 0.10  12.8  L-methionine  + DL-norleucine  0.01 + 0.10  29.2  0.05  0.0  0.10  0.0  0.10  0.0  0.10  61.7  casein hydrolysate  it 18 aa's plus  L-methionine  ** 18 aa's without  L-methionine  ^ee Table XVI. The amino acids (aa's) i ^-methionine were used i n the proportions given f o r casein hydrolysate (West and Todd, 1955) ( i . e . , the 21 aa's minus tryptophan, glutamine and asparagine). The cytochrome and f l a v i n spectra were not affected by the supplements. C e l l s were grown on 0.2% glucose, harvested, washed i n phosphate buffer, a i r - o x i d i z e d , then resuspended i n minimal salts-glucose (0.04%) medium supplemented with L-methionine or i t s analogues at the concentrations indicated. At the end of l o g phase, the c e l l s were harvested, washed, and air-oxidized f o r 1 hr. The reduced/oxidized difference spectra were obtained by addition of glucose i n each case.  methionine  CH.  (CH ) , H—C—NHCOOH selenomethionine  norleucine  CH.  CH-  Se  CH.  ethionine  methionine sulfone  methionine sulfoxide  CH.  CH.  CH,  0«-S-K)  N-acetyl methionine  CH.  S=0  I (CH-) CH, — C-  I  7  -N—C—H  il I I  0 Figure  H  COOH  10. Structural analogues of methionine. The f i r s t 5 analogues have the same structure as methionine  in the portion below the horizontal l i n e . The l a s t analogue d i f f e r s from methionine only by the addition of an acetyl group attached to the nitrogen.  99  the lack of P503 formation when c e l l s were grown on casein hydrolysate was i n fact due to the presence of L-methionine. The presence of L-methionine and/or a s t r u c t u r a l analogue i n the growth medium was found to influence the rate of growth (Table XVIII) with respect to control conditions (glucose alone). In a l l cases, the semilog plot of increase i n absorbance at 420nm versus time indicated that exponential c e l l growth was maintained when an analogue was  included  i n the medium. The doubling time decreased s l i g h t l y with L-methionine or casein hydrolysate supplementation. When a l l the amino acids of casein hydrolysate excluding L-methionine were added to the medium, the doubling time of 51.8 minutes was s i m i l a r to the control (glucose-salts) value of 53.8 minutes. The i n c l u s i o n of L-methionine i n the mixture resulted i n a marked decrease i n generation time to 39.0 minutesequivalent to an increase i n growth rate of 27.4%. Growth i n medium supplemented with DL-ethionine or DL-norleucine was s l i g h t l y slower than the control when these compounds were present at a concentration of 0.01%, and much less at 0.10%. The addition of 0.01% L-methionine as w e l l , to the medium containing 0.10% DL-ethionine or DL-norleucine countered the e f f e c t of the analogue alone, such that the new growth rate was s i m i l a r to the control value. DL-selenomethionine exhibited a s i m i l a r pattern of slowing down the growth rate, i t s e f f e c t being enhanced at the higher concentration (0.05%). Of the analogues tested, the amount of decrease i n growth rate was maximal i n the case of selenomethionine. The increase i n growth rate with 0.01% L-methionine was not  accompanied  by a simultaneous increase i n growth e f f i c i e n c y . In f a c t , 0.01% L-methionine as supplement  or 0.10%  i n limiting' glucose-salts medium produced the  Table XVIII. E f f e c t of L-methionine and i t s analogues as growth supplement on rate of growth (doubling time) of wild-type E_. c o l i B Supplement  Concentration Doubling time of supplement (minutes) i n the medium  none (control)  -  53.8  L-methionine  0.01  49.3  casein hydrolysate  0.10  46.1  18 aa's plus L-methionine  0.10  39.0  0.10  51.8  0.01  58.7  0.10  76.2  0.10 + 0.01  58.7  0.01  58.8  0.10  80.0  0.10 + 0.01  53.8  0.01  68.5  0.05  85.8  *18 aa's without L-methionine DL-ethionine " DL-ethionine + L-methionine DL-norleucine  DL-norleucine + L-methionine DL-selenomethionine "  Control c e l l s were grown on 0.04% glucose. See Table XVII. Semilog plots were prepared of increase i n absorbance^n  w  i  t  n  time f o r the experiments described i n Table XVII, and the doubling times (minutes) calculated i n each case.  101  same e f f i c i e n c y as the control (0.04% glucose alone). The unique e f f e c t of L-methionine  supplementation  i n causing the  elimination of P503 i n wild-type E_. c o l i B prompted an investigation of a methionine-requiring (met ) auxotroph, E_. c o l i K12W-6. In order -  to ascertain generally i f an optimal concentration of  L-methionine  existed which resulted i n the highest e f f i c i e n c y of growth, c e l l s were grown on minimal s a l t s - l i m i t i n g (0.04%) glucose medium supplemented with L-methionine  from 0.001% to 0.10%. Although the growth rate for  this mutant was e s s e n t i a l l y the same i n a l l cases, L-methionine concentration of 0.02%  appeared  at a  to r e s u l t i n a s l i g h t l y higher e f f i c i e n c y  in c e l l y i e l d . A peculiar observation was  the biphasic nature of the  arithmetic plot of e f f i c i e n c y (pg/ml increase i n protein versus ug/ml glucose consumed). Two  slopes were obtained i n a l l cases, the i n i t i a l  slope (0.270) being less than the second for wild-type K12 was  (0.380). Although the e f f i c i e n c y  found to be 0.289, this s t r a i n was not the parent  of the met" mutant, so that the two organisms could not be compared. This unexplainable biphasic property was thought at f i r s t to be an a r t i f a c t ; however, i t s occurrence i n a l l experiments  involving this  mutant would tend to suggest otherwise. In f a c t , semilog plots of growth rate (absorbance^o versus time) and rate of glucose consumption (ug/ml glucose consumed versus time) also were biphasic. Although the i n i t i a l rates were less than the f i n a l for growth and e f f i c i e n c y , glucose consumption showed the opposite e f f e c t ; namely, consumption was more rapid at the beginning of growth. Replacement of L-methionine by DL-ethionine resulted i n a decreased growth rate and lower e f f i c i e n c y but the biphasic nature of the three plots persisted (Figure 11) .  102  Figure 11.  Biphasic nature of growth, glucose consumption,  and protein y i e l d from glucose f o r the met  -  mutant, E_. c o l i  K12W-6. Slope changes at broken l i n e s . Cells were grown on minimal salts-0.04% glucose medium supplemented with L-methionine.  (a) semilog plot of increase i n  0.02%  absorbance^o  versus time (hours); (b) semilog plot of decrease i n glucose (ug/ml) versus time (hours); (c) arithmetic plot of protein y i e l d (ug/ml) versus glucose consumed  (ug/ml).  103  104  The reduced/oxidized difference spectra of the met  -  mutant  indicated no P503 peak with L-methionine supplementation; however, s u b s t i t u t i o n of L-methionine by DL-ethionine i n the medium produced the  503nm band. Since these studies with the met  -  mutant d i d not  further elucidate the e f f e c t of L-methionine on P503, our focus was s h i f t e d once again to wild-type E_. c o l i B. For was  several reasons (explained i n the DISCUSSION) L-methionine  thought to be a modulator of enzyme a c t i v i t y , the target enzyme  being either glucose 6-phosphate dehydrogenase or  6-phosphogluconate  dehydrogenase. B r i e f experiments were therefore c a r r i e d out to determine whether t h i s amino acid exerted an i n h i b i t o r y e f f e c t on either enzyme. The substrate concentration (glucose 6-phosphate or 6-phosphogluconate) was varied i n the reaction mixture from 1/20  to 1/100  the  normal amount, and controls (minus L-methionine) were run to e s t a b l i s h the  minimum substrate concentration allowable before i t became l i m i t i n g ,  thereby r e s u l t i n g i n a decrease i n enzyme a c t i v i t y . L-methionine (from equimolar amounts to lOx the concentration of the substrate) was then added to c e l l extracts p r i o r to addition of the reaction mixture to eliminate the p o s s i b i l i t y of NADP or glucose 6-phosphate (or 6-phospho+  gluconate) protecting the active or a l l o s t e r i c s i t e and/or preventing any binding of L-methionine to the enzyme. The results were negative for both enzymes; neither enzyme a c t i v i t y was affected by the presence of L-methionine. The p o s s i b i l i t y that S-adenosylmethionine was the active compound responsible f o r i n h i b i t i o n was also tested; however, no e f f e c t was observed on either enzyme. The other substrate, NADP , was varied i n a s i m i l a r +  manner, keeping glucose 6-phosphate (or 6-phosphogluconate)  constant at  105  1/25  (or 1/50)  the normal concentration. The results were again negative.  It should be emphasized  that these investigations on the e f f e c t of  L-methionine or S-adenosylmethionine on enzyme function were not carried out  i n d e t a i l , nor were they repeated. Their sole purpose was to deter-  mine i f a general i n h i b i t i o n could be obtained. If the r e s u l t s had indeed indicated a possible i n h i b i t o r y e f f e c t , then further detailed enzyme studies would have been i n order. Only a q u a l i t a t i v e statement can be made, therefore, that L-methionine alone does not i n h i b i t either glucose 6-phosphate dehydrogenase or 6-phosphogluconate  dehydrogenase,  so that the c o l l e c t i v e e f f e c t s of L-methionine on P503 cannot be explained on the basis of an e f f e c t of the amino acid on these HMP  enzymes.  DISCUSSION The u t i l i z a t i o n of s t r u c t u r a l analogues of L-methionine by E_. c o l i has been investigated by numerous laboratories in recent years. L-selenomethionine i s most l i k e l y the closest analogue resembling L-methionine, replacement of the sulphur ( i n methionine) by selenium causing l i t t l e disturbance of the molecular conformation. In f a c t , the analogue has been shown to substitute for L-methionine i n diverse reactions. Nisman and Hirsch (1958) studied the a c t i v a t i o n ( f o r protein synthesis) of L-methionine and i t s analogues by enzymatic fractions of E_. c o l i . whereas D-methionine showed no a c t i v a t i o n , and DL-ethionine and norleucine stimulated PP-^2 exchange only to 30 and 15% of the methionine rate, DL-selenomethionine proved to be a better substrate than L-methionine i t s e l f . The same authors reported the formation of Se-adenosylselenomethionine catalyzed by S-adenosylmethionine synthetase. Se-adenosylselenomethionine can serve as a methyl donor f o r b a c t e r i a l RNA and  DNA  106  methylations  (Wu and Wachsman, 1971), as well as f o r the biosynthesis  of creatine and choline (Mudd and Cantoni, 1957). Furthermore, i t has been shown to be an excellent substrate for spermidine biosynthesis (Pegg, 1969)  although another analogue, S-adenosylethionine,  proved  to be a poor s u b s t i t u t e . The question then arises whether selenium can replace a l l the sulphur i n E_. c o l i . Cowie and Cohen (1957) found no selenoglutathione formed, jj-Galactosidase (containing 80 cystine and 150 methionine residues per mole protein) was  i s o l a t e d and  analyzed  from E. c o l i grown on Na selenate (Huber and C r i d d l e , 1967). Since none of the cystine or cysteine moieties of the enzyme were replaced by t h e i r selenium analogues while 80 out of 150 of the methionine residues had been replaced by the corresponding selenium compound, these workers concluded  that the use of selenium i n amino acids was  formation of selenomethionine.  r e s t r i c t e d to the  (Perhaps s u b s t i t u t i o n by seleno- cystine  and cysteine would prove to have a d r a s t i c e f f e c t on the chemical prop e r t i e s i n contrast to selenomethionine). The newly-formed |3-galactosidase was  unchanged i n i t s c a t a l y t i c properties (Km and Vmax) and  pH  optimum. However, properties depending on the t e r t i a r y structure of the enzyme were found to be a f f e c t e d , the Se-enzyme being less stable to heat and urea denaturation than the S-enzyme. Cowie and Cohen (1957) showed selenomethionine  could replace methionine completely  i n proteins of  E. c o l i and s t i l l allow exponential growth and active enzyme synthesis; however, the concentration of the analogue i n the growth medium was found to influence the growth rate. While a concentration of 10 ^ -  selenomethionine Greene, 1971;  M  gave the same rate of growth as the control (Coch and  Cowie and Cohen, 1957), increasing amounts (10"^ to 10~  2  M)  107  caused a progressive Our  decrease i n the f i n a l rate and l e v e l of growth.  r e s u l t s (Table XVIII) confirmed t h e i r observations,  5.1x10"^ M) increasing the doubling and 0.05%  (or 2.6x10  time from 53.8  M) s t i l l further to 85.8  the greatest detrimental  0.01%  to 68.5  (or  minutes,  minutes. Perhaps  e f f e c t exhibited by t h i s analogue (more  than with ethionine or norleucine)  can be attributed to i t s having  the closest resemblance to the natural amino acid (substitution of the sulphur by selenium c o n s t i t u t i n g a more subtle change) , consequently replacing methionine i n more processes than the other analogues. The  other analogues tested, d i f f e r i n g more i n structure  and  chemical properties, would be expected to a l t e r enzyme functions much more d r a s t i c a l l y if_ they are substituted for L-methionine i n proteins. For example, introduction of an extra methylene group into the methionine side chain  (as i n ethionine) would most l i k e l y i n t e r f e r e with  s p a t i a l r e l a t i o n s h i p s necessary f o r the active three dimensional s t r u c ture of the enzyme. Thus Spizek and Janecek (1969) found that increasing l e v e l s of ethionine led to a decrease i n j3-galactosidase  a c t i v i t y , an  enzyme not known to involve methionine i n i t s c a t a l y t i c mechanism (Wallenfels and Malhotra, 1960). Since the changed 6-galactosidase as e f f e c t i v e as the normal enzyme with respect ability  (antigen-antibody  was  to i t s cross-reacting  p r e c i p i t a t i o n ) , the e f f e c t of ethionine as a  replacement must have resulted i n formation of an i n a c t i v e protein. The a b i l i t y of an analogue to decrease the synthesis of methionine i n organisms by feedback repression would be one  i n d i c a t i o n of i t s  s i m i l a r i t y to the natural amino acid (with respect to shape, s i z e , and i o n i c configuration), such that the s t r u c t u r a l requirements of the  108  biosynthetic enzymes would be s a t i s f i e d . Rowbury and Woods (1961) found that growth i n the presence of L-methionirie repressed i t s biosynthesis in E. c o l i ;  the e f f e c t was r e v e r s i b l e , and the c e l l s regained this  a c t i v i t y when incubated i n a growth medium without ^-methionine. An analysis of several analogues showed effectiveness of repression i n the following order: DL-methionine sulphone >  sulphoxide >  DL-norleucine >  DL-methionine  DL-ethionine. The e f f e c t of methionine sulphoxide was  attri-  buted to i t s conversion to methionine, since Sourkes and Trano (1953) showed that the analogue could be reduced by E_. c o l i i n the presence of molecular hydrogen v i a the hydrogenase system. In contrast, the 60% repression exerted by the sulphone was assumed to be legitimate since this compound was not reduced v i a the hydrogen-hydrogenase  system nor  could i t support the growth of methionine auxotrophs. An alternate determinant of the s i m i l a r i t y of an analogue to a natural amino acid i s i t s a b i l i t y to displace the amino acid from i n t e r n a l pools. Richmond (1962) found that L-methionine-C''"^ could be displaced by L-norleucine but not by D-norleucine or D-methionine i n E_. c o l i . These r e s u l t s suggested that s t r u c t u r a l requirements were involved both i n holding an amino acid i n the pool and i n the a b i l i t y of an exogenous compound to displace this amino acid from the pool. Richmond also found that whereas L.-norleucine could displace both valine and methionine from i n t e r n a l pools, neither of the natural amino acids was able to displace the other. Thus norleucine was s u f f i c i e n t l y s i m i l a r to both valine and methionine to be taken up at their s i t e s . In  contrast to the findings of Rowbury and Woods (1961) , Munier and  Cohen (1959) found that norleucine did not i n h i b i t methionine biosynthesis, but rather i n h i b i t e d the incorporation of methionine into proteins. The  109  i n h i b i t i o n of growth v i a norleucine was  therefore a r e s u l t of the  synthesis of abnormal proteins; replacement of i n t e r n a l - and methionine as well as leucine residues was  N-terminal  thought to produce a cata-  l y t i c a l l y i n a c t i v e enzyme. The a b i l i t y of norleucine to replace methionine i n the i n i t i a t i o n of protein synthesis i n E_. c o l i was Trupin, Dickerman, Nirenberg that tRNA^  et  and tRNA^  that norleu-tRNA^  et  et  and Weissbach (1966), who  confirmed by  observed not only  could be acylated with norleucine, but further  could also be converted  to f-norleu-tRNA , m  et  in a  reaction analogous to the formylation of met-tRNA™ . Kerwar and Weisset  bach (1970) found f-norle -tRNA™ u  et  could substitute f o r f-met-tRNA^  i n subsequent reactions leading to the incorporation of the  et  formylated  species into newly synthesized protein; no differences were observed i n binding to ribosomes, puromycin reaction, and deformylation. They noted, however, that norleucine could not substitute for methionine i n reactions involving the metabolism of the amino a c i d . Of the analogues u t i l i z e d i n our methionine studies, the N-acetyl compound appears to be the least investigated; very l i t t l e has been published on i t s properties. B r i e f mention was made by Davis and M i n g i o l i (1950) that this compound could substitute for methionine to support growth of B-^2 auxotrophs (methionine could apparently  the  replace the vitamin  in the growth medium). The unique e f f e c t of L-methionine on prevention of P503 synthesis (even at the low concentration of 0.01%) was stances  unexplainable. Under circum-  i n which a p a r t i c u l a r f u n c t i o n a l group i s responsible for promoting  a given reaction, removal of the required moiety or i t s replacement by an alternate (but s i m i l a r ) compound usually pinpoints the target. In the  110  s i t u a t i o n concerning P503, however, none of the analogues was a successful substitute. Plausible explanations were therefore sought as to what e f f e c t the presence of L-methionine might have on the b a c t e r i a l c e l l s . The r e s u l t s obtained thus f a r with L-methionine and i t s s t r u c t u r a l analogues suggested that L-methionine was behaving l i k e an e f f e c t o r or modulator of enzyme a c t i v i t y , possibly of glucose 6-phosphate- or 6-phosphogluconate dehydrogenase: (1) Addition of L-methionine to glucose-grown c e l l s caused the appearance of a peak at 503nm i n less than 0.9 minutes (the shortest time i t was possible to measure), suggesting an e f f e c t either by i t s presence per se, or by i t s " a c t i v e " form, S-adenosylmethionine; (2) The stereos p e c i f i c nature of the e f f e c t of L-methionine on e l i c i t i n g a P503 peak i n non-growing c e l l s as well as on formation of the pigment would suggest a s t e r e o s p e c i f i c recognition of L-methionine by some a l l o s t e r i c s i t e on an enzyme; (3) The low concentration of L-methionine required (and i t s being e f f e c t i v e against a ten-fold greater concentration of i t s analogue) would imply a low Km value, c a t a l y t i c nature, and p h y s i o l o g i c a l range- a l l involved with enzyme functions; (4) The sulphur or methyl moiety per se were not s i g n i f i c a n t ; rather, the complete structure was e s s e n t i a l , poss i b l y implicating a three-dimensional conformation required i n a recognition process or f o r i t s f i t into a groove i n an enzyme; (5) Presence of L-methionine i n the medium did not increase the e f f i c i e n c y of growth although the rate of growth was enhanced. The p o s s i b i l i t y of a role of L-methionine i n c o n t r o l l i n g an enzyme function prompted  investigations on glucose 6-phosphate-  and 6-phosphogluconate dehydrogenase, the two enzymes responsible f o r formation of NADPH. The increase i n G6-P dehydrogenase a c t i v i t y i n contrast to th  constancy of 6-phosphogluconate dehydrogenase under the influence  Ill  of DNP was interpreted as i n d i c a t i n g the l a t t e r enzyme to be under stringent c o n t r o l . The modern concept of feedback regulation of the f i r s t enzyme i n a biosynthetic pathway also favoured 6-phosphogluconate dehydrogenase  (the f i r s t enzyme s p e c i f i c to the hexosemonophosphate  shunt) as the more l i k e l y candidate. The limited experiments testing t h i s p o s s i b i l i t y were a l l negative, and i t appeared that methionine or S-adenosylmethionine did not regulate either of the dehydrogenases. On the other hand, i f t h e i r e f f e c t depended on the presence of other factors such as coenzymes or a p a r t i c u l a r c e l l energy charge i n the i n h i b i t o r y mechanism, the lack of appropriate conditions may have been responsible f o r the negative r e s u l t s obtained. The influence of methionine versus i t s analogues on the growth rate of E_. c o l i was yet another property peculiar to methionine. The dramatic e f f e c t observed when the medium was supplemented with 18 amino acids * methionine emphasized  this point even further. The increase i n growth  rate without a concomitant increase i n growth e f f i c i e n c y suggested an e f f e c t of methionine due to i t s presence per se. This phenomenon was mentioned e a r l i e r by Davis and M i n g i o l i (1950) but was not discussed by them. They found that growth of wild-type E_. c o l i i n minimal medium was accelerated by the addition of either methionine or B12; other amino acids and vitamins did not s i n g l y produce this acceleration. In view of the s p e c i f i c i t y i n the relationship between P503 and methionine, an explanation may arise only a f t e r the complete story of P503 i s elucidated. The l i m i t e d studies on the methionine auxotroph, E_. c o l i K12W-6, indicated an optimal concentration of methionine (0.02%) which resulted in the highest e f f i c i e n c y of growth. Various laboratories u t i l i z i n g a  112  met" mutant i n their investigations have reported supplement concentrations ranging from 0.002% to 0.20%. Mansouri, Decter and S i l b e r (1972) studied the regulation of one-carbon metabolism i n E_. c o l i . They found that the interconversion of serine and glycine, mediated by serine hydroxymethyltransferase, was  controlled by the l e v e l of methionine i n  the medium. Thus low l e v e l s of methionine  (1.3xl0 ^ M) enhanced the -  s p e c i f i c a c t i v i t y , whereas higher l e v e l s (1.3x10"^ M) repressed enzyme a c t i v i t y . Since the addition of methionine  or S-adenosylmethionine  to the  serine hydroxymethyltransferase assay mixture had no e f f e c t on s p e c i f i c a c t i v i t y , regulation by methionine was  considered to be not v i a feedback  i n h i b i t i o n ; rather, the l e v e l of the amino acid i n the growth medium was instrumental i n causing the repression-derepression of the enzyme. It i s not therefore s u r p r i s i n g that an optimal concentration of methionine does e x i s t . The semilog plots of growth rate and glucose consumption as w e l l as the arithmetic plot of e f f i c i e n c y were biphasic. Whereas the f i n a l slope i n the two cases, growth rate and e f f i c i e n c y , was  greater than  that during the i n i t i a l phase, glucose consumption was  observed to be  more rapid at the beginning of growth. A possible explanation for this difference might be that the c e l l s were low i n energy i n i t i a l l y rapid metabolism of glucose occurred to b u i l d up a supply of ATP  and and  NADPH for b i o s y n t h e t i c reactions. Once the higher steady state l e v e l was  reached, less glucose was needed, and protein synthesis increased,  accounting f o r greater e f f i c i e n c y and a faster growth rate.  113  Growth of the methionine auxotroph on ethionine decreased both the growth rate and the e f f i c i e n c y . Since the mutant can use neither inorganic sulphate nor cysteine as a source of methionine, i t follows that ethionine must replace a l l the methionine residues i n proteins. As discussed prev i o u s l y , ethionine i s not s u f f i c i e n t l y s i m i l a r to methionine i n structure that i t can replace the amino acid without causing adverse e f f e c t s . Furthermore, Beaud and Hayes (1971) found that ribosomes of an auxotroph grown on ethionine i n place of methionine were submethylated, r e s u l t i n g i n the formation of 30S and 50S subunits defective i n the capacity to associate at a high Mg  concentration (10 mM)  to form 70S p a r t i c l e s .  The lack of P503 i n wild-type E_. c o l i grown i n the presence of L-methionine was observed i n the met  -  mutant. Since the pigment was  formed when  ethionine was used, one can conclude that the mutation to methionine dependence did not a f f e c t the P503 system. The question s t i l l remains as to the mechanism underlying the a b i l i t y of methionine to prevent formation of P503. I t was previously mentioned that L a z z a r i n i and Atkinson (1961) reported a NADPH-specific n i t r i t e reductase i n E_. c o l i . The p u r i f i e d enzyme was found to contain three additional a c t i v i t i e s : cytochrome c reductase, hydroxylamine reductase, and s u l f i t e reductase, a l l s p e c i f i c for NADPH. In addition, Mager (1960) reported the NADPH-specific s u l f i t e reductase and NADPH-specific hydroxylamine reductase (perhaps the same enzyme) to be feedback repressed by cysteine or cystine and to a lesser extent by methionine. Lampen, Roepke and Jones (1947) showed that the second (and not the f i r s t ) step of s u l f i t e reductase (HSO^ — > -  ^S)  was catalyzed by an enzyme s p e c i f i c a l l y linked  to NADPH. The i n i t i a l stages of s u l f i t e assimilation p r i o r to attachment  114  of the sulphur atom to the carbon skeleton of serine (to form cysteine) requires much energy i n the form of ATP as well as NADPH. Perhaps then the energy-sparing e f f e c t and/or a s p e c i f i c repression by methionine of the enzyme catalyzing the step NADPH  > P503 (a j u s t i f i e d p o s s i b i l i t y )  i s responsible f o r the lack of P503 formation. With regard to the "energy-sparing" e f f e c t of L-methionine, one might argue a s i m i l a r r e s u l t should then be observed v i a supplementation with cysteine or cystine. Thus cysteine, an intermediate i n the biosynthesis of methionine, should accelerate methionine synthesis and consequently f a c i l i t a t e growth. This was not observed; contrary to expectations, the presence of L-cysteine i n the medium increased the doubling time from 53.8 (control value) to 146 minutes. The severe repression of cysteine on s u l f i t e reductase might affect the synthesis of some other e s s e n t i a l sulphur compound. On the other hand, a completely separate process altogether  _3 may be affected. Schachet and Squire (1971) reported that 7.5x10  M  cysteine caused very rapid i n a c t i v a t i o n of bovine adrenal glucose 6-phosphate dehydrogenase.  Although t h i s action of cysteine was not observed i n our  experiments, the difference may have been due to a concentration e f f e c t . The reasons f o r the adverse e f f e c t of cysteine on c e l l growth remains obscure. Another possible s i t e of enzyme control by L-methionine may be between NADPH  >P503. The p o s s i b i l i t y of methionine repressing the NADPH  »P503 step i s not only a t t r a c t i v e , but also quite f e a s i b l e . The previously mentioned studies of Mansouri, Decter and S i l b e r (1972) showed methionine to control the a c t i v i t y of serine hydroxymethyltransferase. Furthermore, methionine has been shown to regulate other folate-dependent  115  reactions including methyl transferase (catalyzing f o l a t e + homocysteine  5-methyltetrahydro-  > methionine) and reductase (catalyzing 5,10-  methylenetetrahydrofolate—>5-methyltetrahydrofolate). Indeed the d i v e r s i t y of reactions already found to be controlled by methionine supports the p o s s i b i l i t y of i t s regulatory action over yet another enzyme. 5.  E f f e c t of other compounds as growth supplement on P503 formation  RESULTS Growth of wild-type E_. c o l i B on l i m i t i n g glucose supplemented with 0.01 to 0.05% ascorbic acid, 0.01 to 0.10% f o l i c acid, or 0.04% 2-deoxyglucose had no e f f e c t on e i t h e r growth rate or P503 formation. In a l l cases, a large amount of P503 was synthesized.  116  GENERAL DISCUSSION AND CONCLUSIONS  A study of four Escherichia c o l l s t r a i n s (B, K12, UL and CRX) indicated a consistent pattern of 25-35% decreased aerobic e f f i c i e n c y i n the Smdependent mutant compared to the wild-type revertant  parent. A non-dependent  (SBr4) , derived from the Sm-dependent mutant of E_. c o l i B,  showed a s i m i l a r decreased aerobic e f f i c i e n c y . The anaerobic protein y i e l d proved to be i d e n t i c a l i n a l l cases although lower than under aerobic conditions. Subsequent c a l c u l a t i o n of the s o l e l y aerobic portion of growth revealed that wild-type  c e l l s produced double the amount of  energy that could be ascribable to aerobic metabolism, as that produced by dependent c e l l s . In view of previous  findings regarding  the l e s s e f f i c i e n t , uncontrolled  nature of metabolism peculiar to this mutant (namely, excretion of v a l i n e when grown on glucose (Bragg and Polglase, 1962; Tirunarayanan, Vischer and Renner, 1962) as well as de-repressed l e v e l s of catabolite repressible enzymes (Coukell, 1969)), investigations were i n i t i a t e d on hydrogen metabolism i n an attempt to elucidate the mechanism(s) responsible f o r this decrease i n energy y i e l d . The only difference noticeable i n the reduced/ oxidized difference spectra of a e r o b i c a l l y grown c e l l s was that the large, transient, symmetrical peak at 503nm i n wild-type  E_. c o l i was absent or  markedly decreased i n Sm-dependent mutants (and the non-dependent revertant) ; the cytochromes and f l a v i n were i d e n t i c a l . Consequently, the remainder of the thesis was focussed  on characterizing the properties  of t h i s 503nm pigment (P503) i n wild-type  E_, c o l i B.  Numerous and varied attempts to obtain a peak at 503nm i n permeabil i z e d c e l l s or i n crude extracts were f u t i l e ; the pigment proved to be quite l a b i l e under a l l conditions and could not be detected.  Further  117  investigations were therefore c a r r i e d out on whole c e l l s . Subsequent studies involved characterizing either (1) reduction of the  pigment i n non-growing  c e l l s or (2) i t s formation (synthesis) during  growth. When the effects of several types of compounds on production of the 503nm peak were compared, a high l e v e l of s p e c i f i c i t y was found. In the case of  sugars and acids, whereas reduction by gluconate produced only a substan-  t i a l P503 peak i n the f i r s t few minutes p r i o r to the steady-state levels of the cytochrome and f l a v i n bands, succinate as substrate resulted i n the i n i t i a l appearance of f l a v i n followed by cytochrome b with only a trace of P503. Glucose (as control) e l i c i t e d the entire spectrum immediately. Other substrates such as g l y c e r o l , l a c t a t e , and acetate showed an absence or n e g l i g i b l e amount of P503 although cytochrome b and f l a v i n were constant. For  a l l cases i n which l i t t l e (or no) P503 peak was obtained, the addition  of glucose a f t e r a 10-15 minute l a g caused the rapid appearance of the 503nm peak, i n d i c a t i n g that the c e l l s d i d possess P503 but that the substrates added i n i t i a l l y were unable to e l i c i t  i t s appearance. These r e s u l t s i m p l i -  cated a r e l a t i o n s h i p between P503 and NADPH, such that the reduced coenzyme might transfer i t s hydrogen to the 503nm pigment. A s i m i l a r survey of L-amino acids as reductants singled out the e f f e c t of methionine. Addition of L-methionine or casein hydrolysate (but no other amino acids) produced only a P503 band within 0.9 minutes; a f t e r a l a g of 10-14 minutes, the cytochromes and f l a v i n were reduced. Structural analogues of L-methionine were then studied to test both the s t e r e o s p e c i f i c i t y of action as well as the various functional substituents. The results indicated that no changes whatsoever were allowable, further emphasizing the uniqueness of  the e f f e c t of L-methionine on P503. Among other compounds tested ascorbic  118  acid, f o l i c acid, betaine, and 2-deoxy-D-glucose were also i n e f f e c t i v e i n e l i c i t i n g a large P503 peak i n non-growing c e l l s . Experiments concerning the e f f e c t of growth conditions and  supple-  ments furnished information regarding the formation or synthesis of the 503nm pigment during growth. Anaerobically grown wild-type E_. c o l i B possessed P503, the cytochromes, and f l a v i n ; however, since these were present in an oxidized s t a t e , i t was  concluded that they were nonfunctional  anaerobically and did not accept electrons i n the absence of oxygen. Inclusion of increasing concentrations of 2,4-dinitrophenol  (DNP)  in the minimal s a l t s - l i m i t i n g glucose medium resulted i n a progressive decrease i n aerobic growth rate, y i e l d of c e l l protein, and height of the 503nm peak. At 500 uM DNP,  wild-type c e l l s were found to resemble  the Sm-dependent mutant and non-dependent revertant i n the following ways: (1) decreased (87.5 minutes),  aerobic e f f i c i e n c y (30%), (2) enhanced doubling time  (3) lack of P503 and (4) de-repressed  l e v e l s of the  catabolite r e p r e s s i b l e enzymes fumarase and aconitase. An unexpected r e s u l t was  the f o u r - f o l d increase i n glucose 6-phosphate dehydrogenase  as w e l l - an enzyme not known to be s e n s i t i v e to c a t a b o l i t e repression. No e f f e c t of DNP was phenol was  observed under anaerobic conditions or when the  added to control c e l l s just p r i o r to analysis of the difference  spectrum. Furthermore, i t s e f f e c t was grown c e l l s i n the absence of DNP  reversible and re-growth of  DNP-  resulted i n synthesis of the 503nm  pigment once again. Other i n h i b i t o r s including 50.0-250.0 uM 1,3,5tribromophenol,  1.0-5.0 uM chloramphenicol,  mine-hydrochloride  and 10.0-  100.0  did not have the same e f f e c t as DNP,  pll hydroxyla-  emphasizing the  rather unique quality of the last compound. The c o l l e c t i v e results  119  obtained with DNP were compatible with the following i n t e r p r e t a t i o n : DNP might undergo reduction by oxidizing NADPH. Removal of the NADPH, a possible co-repressor of glucose 6-phosphate dehydrogenase,  would  then displace the equilibrium of the enzyme, thereby increasing i t s a c t i v i t y . In addition, removal of the reduced coenzyme would prevent induction of the 503nm pigment, previously suggested as receiving hydrogen  from NADPH. The decreased growth e f f i c i e n c y under the  influence of DNP  observed only i n aerobiosis (and not anaerobiosis)  which accompanied the elimination of P503 could implicate P503 i n a major aerobic pathway of energy  metabolism.  The following general hypothesis was subsequently put f o r t h : an oxidative pathway which generates ATP from NADPH v i a an intermediate pigment, P503, and which accounts f o r 25-35% of the t o t a l energy or 50% of the solely aerobic energy with glucose as carbon source, i s present i n wild-type E_. c o l i c e l l s grown on a minimal s a l t s medium (Figure 12). In Sm-dependent c e l l s and DNP-grown wild-type c e l l s , a deficiency i n P503 (caused by mutation i n the former and some e f f e c t of the drug i n the l a t t e r case) resulted i n elimination of the NADPH >P503  > ATP pathway.  A survey of compounds (analogous to that conducted on the reduction of the 503nm pigment) further elucidated the nature of P503 synthesis. Glucose and gluconate as carbon source resulted i n the formation of a s i g n i f i c a n t peak at 503nm. In contrast, succinate, g l y c e r o l , or lactate as sole carbon and energy source produced a smaller P503 peak. Glucose and gluconate metabolism involve the synthesis of NADPH during the process of t h e i r d i s s i m i l a t i o n v i a the hexosemonophosphate shunt,  Figure 12. Pathways of carbohydrate metabolism i n Escherichia c o l i :>ATP  P503 glucose  HEXOSEMONOPHOSPHATE SHUNT  NADP  glucose 6-phosphate  NADP  NADPH  NADPH  'y —-^6-phosphogluconate-  I I  —-^pentoses  I I  EMBDEN-MEYERHOF PATHWAY  %  \  glycerol-  ENTNER-DOUDOROFF PATHWAY  !  triose-phosphate s >' — ^ pyruvate  lactate  | succinate  ATP  TRICARBOXYLIC ACID CYCLE I I !  NADH  ^ cytochromes  postulated new energy pathway  ->ATP  121  whereas the l a t t e r three compounds (succinate, g l y c e r o l , and lactate) do not generate NADPH at an early step i n t h e i r metabolism. A concent r a t i o n e f f e c t of glucose and gluconate on P503 formation was also observed, such that increasing concentrations of either sugar caused a progressive decrease i n height of the 503nm peak. Although a poss i b l e relationship between P503 and catabolite repression was suggested, the connection was obscure and no conclusions could be made i n this regard. The previously discovered uniqueness of L-methionine on reduction of the 503nm pigment i n non-growing c e l l s was extended to the e f f e c t of t h i s amino acid as supplement on P503 synthesis i n growing c e l l s . No other L-amino acid s i n g l y , nor a mixture of the 18 amino acids of casein hydrolysate (minus methionine), possessed the a b i l i t y of methionine to prevent the formation of P503; indeed, no s t r u c t u r a l analogue of L-methionine, even at a ten-fold higher concentration, was able to duplicate the e f f e c t of this amino acid, implicating s t e r e o s p e c i f i c i t y and chemical i n t e g r i t y as being r e q u i s i t e i n both reduction and format i o n (synthesis) processes. Another d i s t i n c t i o n of L-methionine was the enhanced growth rate of the c e l l s during i t s presence i n the medium without a concomitant increase i n growth e f f i c i e n c y ( y i e l d of p r o t e i n ) . Further studies to elucidate the explanations underlying these observations indicated that L-methionine (or i t s active form, S-adenosylmethionine) alone was not exerting i t s e f f e c t by i n h i b i t i n g either glucose 6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase. Other cofactors or the energy state of the c e l l s might be involved i n the mechanism of inhibition.  122  At this point, i t should be c l e a r l y stated that methionine holds a singular p o s i t i o n in the realm of amino acids. As more biochemical knowledge i s accumulated, the d i v e r s i t y of reactions in which methionine p a r t i c i p a t e s i s being extended. This amino acid i s required not  only  for incorporation into proteins, but also for various methylation reactions, synthesis of polyamines, and i n i t i a t i o n of protein  synthesis.  Although i t s r e l a t i o n s h i p to P503 i s s t i l l obscure, a consideration  of  the t o t a l influence of this amino acid supports i t s role as an e f f e c t o r i n the control of an enzyme function, possibly between NADPH—»P503. The  f i n a l proof that L-methionine i s a modulator of such a reaction,  however, must await a demonstration of i t s e f f e c t i n a p u r i f i e d c e l l free system, a seemingly d i f f i c u l t task i n view of the extreme l a b i l i t y of the 503nm pigment. The  chemical i d e n t i t y of the 503nm pigment has not yet been esta-  b l i s h e d . Although such a study would perhaps e n t a i l i t s p r i o r i s o l a t i o n , one might evaluate the o v e r a l l c h a r a c t e r i s t i c s observed i n support of or i n contrast to those structures already proposed i n the l i t e r a t u r e . The  likeness of P503 to a cytochrome- or flavin-type compound has been  favoured by several i n v e s t i g a t o r s , while rejected by others. Nosoh (1964) reported  that the appearance of the 503nm peak was  related to  the disappearance of a 519nm peak and vice versa; i n f a c t , he found the change i n height i n various  of the 503nm band to be accompanied always by a change  cytochrome bands. Olden and Hempfling (1970) stated that  the  pigment absorbing at 503nm could not be attributed to a cytochrome, but that i t appeared to be i n k i n e t i c equilibrium with f l a v i n . On the other hand, Lindenmayer (1959) and Lindenmayer and Smith (1964) found no  123  other absorption band to appear p a r a l l e l i n g the change i n P503. Two r e s u l t s obtained from our work would tend to support the d i s s i m i l a r i t y of P503 with respect to both the cytochromes and f l a v i n : (1) Whereas the height of the 503nm peak was seen to increase, decrease, and eventually disappear upon reduction by glucose, the peaks of the cytochromes and f l a v i n trough were constant; families of curves recorded from 700-400nm during the progressive decline of the 503nm peak showed p e r f e c t l y superimposed bands for the other pigments; (2) The time lapse between the appearance of the steady-state l e v e l of the 503nm peak versus that of the cytochromes (and f l a v i n ) upon reduction by gluconate and L-methionine  (approximately 3 and  10-14  minutes respectively) suggests that the 503nm pigment has a lower (more negative) redox p o t e n t i a l than the respiratory pigments. Other properties of P503, namely the s i n g l e absorption band as well as the r e a c t i v i t y , were i n agreement with observations reported by Lindenmayer and Smith (1964). In view of the i n s t a b i l i t y and formation anaerobically of P503, i t i s u n l i k e l y that P503 i s the semiquinone of ubiquinone. The  absorp-  t i o n c h a r a c t e r i s t i c of the semiquinone form of free f l a v i n or enzymebound f l a v i n i s also d i s s i m i l a r to that obtained for the 503nm pigment. One  f i n a l proposal was  the protoporphomethene (or tetrahydropor-  phyrin) nature of P503 (Labbe, Volland and Chaix, 1967; Hempfling,  Olden and  1970). The a b i l i t y of porphyrins to undergo stepwise reduc-  tion (Mauzerall, 1962)  through two stages to protoporphomethene (500nm),  and further with certain reducing agents to a t h i r d colourless form (porphyrinogen), makes t h i s group of compounds a very favourable candidate. There are several points, however, that must be explained before  124  one can state this structure of P503 with c e r t a i n t y : (1) The (400, 550, 590nm) and dihydroporphyrin  porphyrin  (440, 735nm) forms were not  detected i n any of the s p e c t r a l runs so that a precursor-type  rela-  tionship could not be established; (2) Mauzerall (1962) reported the h a l f - l i f e of the tetrahydroporphyrin (500nm) to be approximately  20  hours at 100*C. In contrast, our experiments with P503 indicated the pigment to be transient, even when c e l l suspensions were heated  to  80* a f t e r the appearance of P503, i n an attempt to denature enzymes and "trap" the structure i n i t s 503nm absorbing state; (3) Mauzerall (1962) found reduction by sodium d i t h i o n i t e (and s u l f i t e ) to give a 500nm peak (tetrahydroporphyrin), but reduction by ascorbic acid to cause reduction beyond the second stage to a colourless compound; (4) Labbe et a l . (1967) and Mauzerall (1962) suggested  the formation  of a complex between s u l f i t e and the methene bridge of tetrahydroporphyrin to produce the colourless structure. Although such a linkage i s f e a s i b l e and could explain the e f f e c t of cysteine, cyanide, and L-methionine as w e l l , the difference with the seleno- analogue of methionine does not s a t i s f y this proposed mechanism. The f i r s t point might be explained i n part by the time f a c t o r the i n a b i l i t y of the instrument  to record the appearance of bands at  several wavelengths simultaneously. Perhaps an analysis using a dual wavelength spectrophotometer set at the absorption maxima of  two  d i f f e r e n t reductive forms might reveal such a stepwise sequence. In addition, the 550 and 440nm bands might be obscured by the cytochrome b and broad f l a v i n absorption pattern; however, one would then expect to see a shoulder i n the peak (or trough) which disappeared with This observation was  not made.  time.  125  The comparatively  long h a l f - l i f e of tetrahydroporphyrin tends to  rule out this compound, since i f P503 had this structure, one would expect the pigment to be found i n c e l l extracts, e s p e c i a l l y i f P503 were obtained f i r s t v i a reduction by glucose and the c e l l s then plunged immediately  into an 80' bath followed by disruption of c e l l s . Several  experiments on s t a b i l i z i n g or "trapping" the pigment i n the 503nm absorbing form were a l l negative. Assuming that "chemical" reduction i s more complete than the substrate or "enzymatic" process  (Beinert, 1956a), i t i s puzzling  that Mauzerall obtained only p a r t i a l reduction with d i t h i o n i t e and complete reduction with ascorbic a c i d , the l a t t e r compound stated by him to be a "mild" reducing agent. Although  the concentration  factor might account f o r the difference i n extent of reduction, the concentrations were not given f o r either of these reductants. Selenomethionine has been a useful substitute f o r methionine i n experiments determining  the importance of the sulphur atom, due to  the subtlety of the change. Selenium and sulphur are s i m i l a r i n t h e i r physical and chemical properties (Jauregui-Addell, 1966; Rosenfeld and Beath, 1964). Since the b i v a l e n t type of linkage i s common to both compounds, one would not expect the replacement of sulphur by selenium to a f f e c t covalent bonding. 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Answer: Coukell  (1969) found that at low substrate  concentrations  ( i . e . l i m i t i n g glucose), the increase i n absorbance at 420nm was  directly  proportional to the ug/ml increase i n dry weight as well as to the ug/ml increase i n t o t a l c e l l u l a r protein, up to an A^Q  of 1.0.  In our  preli-  minary studies, using 0.04%  glucose as C source, repeated protein  minations of the wild-type,  Sm-dependent, and r e s i s t a n t (JIDHSm) organisms  of each s t r a i n indicated that the increase i n A^Q  was  deter-  also d i r e c t l y  proportional to the ug/ml increase i n protein. In addition, under these experimental conditions  ( i . e . , minimal s a l t s  and l i m i t e d glucose), Coukell reported that a l i n e a r r e l a t i o n s h i p existed i n the p l o t of ug/ml increase i n c e l l weight versus ug/ml glucose consumed. Our plots of ug/ml increase i n protein versus ug/ml glucose consumed showed a s i m i l a r l i n e a r r e l a t i o n s h i p . It follows, then, that the use of protein determinations i n growth y i e l d calculations i s j u s t i f i e d . Since determinations of protein were reproducible than i n the case of c e l l weight, we estimating  and s e n s i t i v e , yet simpler and faster chose to use the former method i n  the e f f i c i e n c y values.  It should also be mentioned that the growth rates ( i . e . doubling  times)  were given for the wild-type and Sm-dependent mutant of E_. c o l i B, the s t r a i n  A-2  used i n most of the experiments. (lb)  Objection: Coliform b a c t e r i a may produce acetate during aerobic glucose metabolism, and i t i s possible that the lower growth y i e l d s for some of the b a c t e r i a l strains are due to incomplete oxidation of glucose. Product assays were not done. The experiments using limited glucose somewhat temper this objection. Answer: The l a s t statement  i t s e l f counters the c r i t i c i s m . The c e l l s  were grown not on an excess, but rather a l i m i t i n g amount of glucose, so that intermediate metabolites would not be expected to accumulate to the extent expected when an excess of substrate i s present. Experiments, i n which absorbance measurements were continued f o r 2 1/2 hours after the glucose had been exhausted, indicated no further increase i n the  A  4 2  Q.  If acetate were an end product of incomplete glucose metabolism, one would then have expected the induction of the glyoxylate pathway a f t e r the exhaustion of glucose, and a resumption  of growth.  In addition, Sm-dependent E_. c o l i B grown aerobically on succinate as C source also resulted i n a decreased c e l l y i e l d compared to the wild-type organism. F i n a l l y , i n preliminary studies, determination of the CO2 produced indicated that the Sm-dependent organism produced twice as much CO2 as the wild-type when grown on l i m i t i n g glucose. This result would imply complete oxidation of glucose. (lc) Objection: Some measure of variance of the growth y i e l d data should be included so that the s i g n i f i c a n c e of the differences of 25-30% can be evaluated. Answer: This c r i t i c i s m would be j u s t i f i e d if_ growth y i e l d were determined on only a few occasions. However, c e l l y i e l d experiments, using c e l l weight, had been carried out by Coukell many times; s i m i l a r l y , the protein y i e l d s had been estimated i n our experiments,  on each s t r a i n , f o r  well over s i x months. In addition, i n every experiment,  the control c e l l s  were checked for growth e f f i c i e n c y as well as the presence of P503. The r e s u l t s were consistent, the value recorded i n the thesis being the average of a l l the determinations. In view of the numerous repetitions by both groups, the difference of 25-35% found i n the growth y i e l d between the Sm-dependent and wild-type organisms i s s i g n i f i c a n t and r e l i a b l e .  (2) Objection: The P503 content may vary with time course of growth on d i f f e r e n t C sources, so that the c e l l u l a r content of P503 should have been monitored during growth. Answer: Kropinski, from our group, d i d monitor the P503 content during growth. He found that the pigment was produced early during log phase and that the steady-state l e v e l remained high throughout  growth.  In our studies using d i f f e r e n t C sources of growth, the experimental conditions were kept constant to determine the s p e c i f i c e f f e c t of the p a r t i c u l a r sugar or acid. Thus, c e l l s were adapted f o r growth on the appropriate compound p r i o r to the actual experiment. After the c e l l s were harvested, the suspensions were a i r - o x i d i z e d f o r 1 hour i n b u f f e r to deplete any endogenous substrates which might obscure the r e s u l t s . In a l l cases, the presence of P503 was tested by the addition of glucose as reductant, since glucose had previously been found to e l i c i t the highest 503nm peak. Therefore, i f any synthesis of the 503nm pigment had been allowed on the p a r t i c u l a r C source used, this method would give consistent results which could be used i n comparison studies.  (3) Objection: The author did not test her hypothesis of NADPH oxidation through P503; therefore, an experiment i s suggested. The growth y i e l d of the wild-type should be measured i n the presence and absence of methionine. If the P503 energy conservation pathway hypothesis i s correct, then the addition of methionine should decrease the growth y i e l d by an appropriate amount. The experiment should be carried out  A-4  with U-C^-methionine to estimate the amount of methionine carbon incorporated which results i n more oxidative metabolism of glucose. In another experiment U-C^-glucose might be used so that an assessment of soluble products could be made. Should the growth y i e l d not change or should i t increase, the hypothesis would be untenable. Answer: From the suggested experiment, Dr. Hempfling reveals, unfortunately,  that he does not f u l l y understand the hypothesis presented  i n the t h e s i s , and has misinterpreted  the implications of i t .  i  F i r s t , the hypothesis r e l a t i n g NADPH oxidation'through  P503 was  developed from studies comparing the growth e f f i c i e n c i e s of the Sm-dependent mutant and the wild-type  organism. When the presence or absence of  P503 was observed to be the only difference between the two c e l l further studies were done using 2,4-dinitrophenol.  types,  This drug was found  to decrease the growth e f f i c i e n c y , eliminate P503, and cause  de-repression  of the c a t a b o l i t e repressible enzymes. Its e f f e c t on glucose 6-phosphate dehydrogenase as well, implicated the reduced pyridine nucleotide, NADPH. The investigations on the s p e c i f i c i t y of the C sources as reductants i n e l i c i t i n g the spectrum of P503 also pointed to NADPH. A d i r e c t study of NADPH as reductant  could be performed only on permeabilized  c e l l s , since  c e l l s are impermeable to this coenzyme. However, a l l attempts to obtain a P503 peak i n disrupted or permeabilized  c e l l s were negative. Due to  the extreme l a b i l i t y of P503, i n d i r e c t evidence for the r e l a t i o n s h i p between NADPH and P503 was necessitated using whole c e l l s . Secondly, as stated i n the thesis (commencing on page 99, l i n e 24), supplementation of the growth medium with 0.01% L-methionine increased the growth rate, but not the growth e f f i c i e n c y . The l a t t e r was found to be i d e n t i c a l to the control condition (0.04% glucose alone). This result implies that the action of methionine was as the intact molecule; i n f a c t , the conclusion was that methionine appeared to be acting as an e f f e c t o r  A-5  of an enzyme function, possibly d i v e r t i n g the metabolic route from the P503 system. Methionine may have a small "sparing" e f f e c t since i t can be incorporated  into proteins, but i t i s added i n such a minute quantity to the  medium compared to the amount of glucose, that i t would probably not be used as a C source. In addition, the s t r u c t u r a l analogues, which have been reported to substitute for L-methionine i n various reactions such as protein synthesis, methylation  of DNA and RNA, and biosynthesis of  choline and spermine, were not e f f e c t i v e i n s u b s t i t u t i n g for L-methionine with regard to the P503 system, suggesting  again that the three-dimen-  sional structure of the complete molecule i s e s s e n t i a l . The l a s t statement of the objection i s i n c o r r e c t . I f the growth y i e l d increases, then the hypothesis would be tenable. The P503 system i s assumed to be operative as a result of i t s induction by the presence of excess NADPH. In the Sm-dependent mutant, a p o s s i b i l i t y for i t s excretion of valine was that the excess NADPH formed might be removed by the synthesis of valine (the l a t t e r amino acid serving as a neutral hydrogen acceptor). If methionine somehow prevents the over-production of NADPH (and therefore the induction of the P503 system), then growth of Sm-dependent mutants i n medium supplemented with methionine should eliminate the wasteful production  of NADPH, so that the intermediate  product of glucose metabolism, pyruvate, would be metabolized v i a the TCA  cycle, and not be used f o r the synthesis of v a l i n e . An increase i n  e f f i c i e n c y should therefore be observed, although perhaps not to the same extent as i n the wild-type  organism. This prediction can be tested  using either the Sm-dependent mutant, or DNP-grown wild-type  c e l l s , since  A-6  b o t h c o n d i t i o n s were shown to be e q u i v a l e n t w i t h r e s p e c t t o the P503 system.  S i n c e the c o m p l e t i o n o f t h i s t h e s i s , such an experiment has  been done by Joyce Boon. W i l d - t y p e c e l l s were grown on medium  supple-  mented w i t h DNP  observed  in  and methionine  the DNP-reduction  s i m u l t a n e o u s l y . A d e c r e a s e was  p r o d u c t s , i m p l y i n g t h a t methionine d i d d i v e r t the  route from the NADPH—>DNP p a t h . A s l i g h t a l s o o b s e r v e d . These experiments  increase i n e f f i c i e n c y  was  a r e s t i l l b e i n g conducted, u s i n g  v a r y i n g c o n c e n t r a t i o n s o f m e t h i o n i n e , so t h a t a d e f i n i t e v a l u e f o r the increase i n e f f i c i e n c y The  cannot be s t a t e d at t h i s  time.  constancy i n the e f f i c i e n c y o f w i l d - t y p e c e l l s  w i t h methionine  (as s t a t e d p r e v i o u s l y ) i s not unexpected,  supplemented since although  the P503 path i s no l o n g e r a v a i l a b l e , t h i s r o u t e i s assumed t o be used o n l y when an excess o f NADPH i s p r e s e n t , and a d d i t i o n o f m e t h i o n i n e p r e v e n t s the w a s t e f u l o v e r - p r o d u c t i o n o f NADPH, thereby c o n s e r v i n g on energy. The g l y c o l y t i c p a t h f o r ATP s y n t h e s i s , as w e l l as the TCA c y c l e , a r e presumed to be the same i n both w i l d - t y p e , and w i l d - t y p e with  methionine.  supplemented  S-1  SUGGESTIONS FOR FUTURE WORK Before the complete story of the 503nm pigment can be elucidated and i t s functional s i g n i f i c a n c e i n c e l l u l a r metabolism established, much more research must be done. Despite the widespread occurrence of this pigment, l i t t l e attention has been given to i t i n the l i t e r a t u r e . It i s the author's hope that the findings reported i n this thesis w i l l contribute i n part to the understanding  of the role of this pigment,  and i n s t i g a t e renewed interest i n this area. Due to the shortage of time, several points relevant to the support or contradiction of the hypothesis were not able to be examined f u l l y . These suggestions are outlined below. (1) The i s o l a t i o n , p u r i f i c a t i o n , i d e n t i f i c a t i o n and quantitative determination of the reduction products of 2,4-dinitrophenol would enable one to ascertain whether the reduction of DNP  does, i n f a c t ,  account f o r a l l the NADPH which i s channelled off and removed p r i o r to the reduction of P503. The results would provide a check on the calculations presented i n the thesis. (2) A comparison of wild-type control c e l l s versus DNP-grown wild-type c e l l s with regard to the a c t i v i t y of a DNP-reductase might be carried out by following NADPH oxidation at 340nm. The following questions might then be asked: (a) Is there a s u f f i c i e n t difference of a c t i v i t y i n the two conditions to account f o r the excess NADPH being oxidized? (b) Is DNP-reductase "induced" during growth of the c e l l s on DNP Since DNP  or i s the enzyme always present?  added to c e l l suspensions just p r i o r to spectral analysis has  S-2  no e f f e c t on P503, the enzyme should be inducible. The k i n e t i c parameters and optimal condition f o r the DNP-reductase could also be investigated. (3) The p o s s i b i l i t y of a connection between P503 and catabolite repression was suggested by the decrease and eventual elimination of P503 when wild-type c e l l s were grown on increasing concentrations of glucose or gluconate. Gluconate was e f f e c t i v e i n causing this decreased synthesis of P503 at a lower concentration than i n the case of glucose. Is the r e l a t i o n s h i p between catabolite repression and (a) P503, (b) NADPH or (c) the energy charge (adenylate r a t i o ) of the c e l l ? (4) The suggestion was made that L-methionine added to the growth medium might control the NADPH—*P503  ^ATP reaction sequence. At what  point i s i t s e f f e c t exerted, and what i s the mechanism involved? (5) A possible explanation f o r the excretion of v a l i n e by Sm-dependent mutants was that the excess NADPH could not be channelled o f f v i a the P503 system (since this pathway was impaired), thereby r e s u l t i n g i n an accumul a t i o n of the reduced coenzyme. The u t i l i z a t i o n of NADPH f o r v a l i n e biosynthesis would o f f - s e t t h i s increase i n NADPH. I f the action of methionine i s , indeed, to i n h i b i t this NADPH—> P503 — * ATP path, or to divert metabolism away from the P503 system, then growth of Sm-dependent (or DNP-wild-type) c e l l s i n the presence of methionine might prevent  excess  accumulation of NADPH. Such an e f f e c t would predict three things: (a) Sm-dependent c e l l s grown with methionine or none at a l l ;  should excrete less valine  (b) since no wasteful NADPH i s made, the e f f i c i e n c y should  increase; (c) less DNP should be reduced. (6) Various attempts to obtain P503 i n disrupted or permeabilized wild-type c e l l s were negative. I f further studies were continued along this l i n e , one might t r y using the Sm-resistant  ( i n d i f f e r e n t ) s t r a i n of  S-3  E_. c o l i B. Although the height of the 503nm peak was not as high i n this mutant as i n the wild-type organism, i t was more persistent (less trans i e n t ) . I f one did succeed i n t h i s regard, a large area of research would be opened for scrutiny, (a) Addition of NADPH versus NADH to permeabilized c e l l s would test the s p e c i f i c i t y of P503 d i r e c t l y , (b) I f the P503 system generates ATP (as stated i n the hypothesis), then t h i s pathway should not be operative when the c e l l has a high enough energy charge. The energy charge explanation, given as a possible e f f e c t of L-methionine i n e l i c i t i n g the 503nm peak r a p i d l y , could then be tested by addition of ATP plus L-methionine simultaneously to the c e l l . The added ATP should help maintain the energy charge of the c e l l , so that the u t i l i z a t i o n of the P503 path would not be needed as a compensatory response.  Alternatively,  one could add S-adenosylmethionine to determine whether the e f f e c t of L-methionine i s exerted v i a i t s " a c t i v e " form, or as the amino acid per se. (c) The cofactor requirements  for the s t a b i l i z a t i o n of the pigment could  be investigated, (d) I f the appropriate conditions were found f o r the s t a b i l i z a t i o n of P503, the p o s s i b i l i t y would then arise of i t s i s o l a t i o n and p u r i f i c a t i o n , (e) The i s o l a t i o n of P503 would, i n turn, enable i t s s t r u c t u r a l determination. (8) The d i s t r i b u t i o n of P503 i n b i o l o g i c a l systems, both i n eukaryotes and prokaryotes, might be determined  to ascertain whether a pattern r e s u l t s  which may contribute to i t s further functional s i g n i f i c a n c e . (9) Mutant studies might provide a test of c e r t a i n explanations which were advanced to support the hypothesis. For example, (a) E_. c o l i mutants which lack 6-phosphogluconate dehydrogenase would most l i k e l y a l l the gluconate provided v i a the Entner-Doudoroff  metabolize  path. Since an excess  of NADPH would not then be expected to accumulate, the P503 system should  S-4  not be induced, (b) E_. c o l i mutants lacking glucose 6-phosphate dehydrogenase would d i s s i m i l a t e glucose v i a the Embden-Meyerhof path, and none should be metabolized v i a the HMP  shunt. One would then  expect to see a decreased l e v e l of the 503nm peak with glucose. (c) E_. c o l i mutants lacking phosphoglucose to convert G6-P  isomerase would be unable  to F6-P i n the Embden-Meyerhof path, thereby forcing  glucose metabolism  to proceed v i a the HMP  greater use of the HMP  shunt and E-D path. The  shunt would then predict a higher l e v e l of P503.  

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