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Oxidative assimilation of glucose by aerobic bacteria Tomlinson, Geraldine Ann 1964

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OXIDATIVE A S S I M I L A T I O N OF GLUCOSE BY AEROBIC B A C T E R I A by GERALD INE ANN TOMLINSON  B.S.A., T h e U n i v e r s i t y M.A.  (Biochemistry),  of British  Columbia,  The U n i v e r s i t y o f C a l i f o r n i a  1957.  (Berkeley),  A T H E S I S SUBMITTED IN P A R T I A L FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN AGRICULTURAL MICROBIOLOGY in t h e D i v i s i o n o f Animal  We a c c e p t t h i s required  thesis  Science  as c o n f o r m i n g  to the  standard  THE U N I V E R S I T Y OF B R I T I S H COLUMBIA April,  1964.  I960.  In presenting this thesis i n p a r t i a l fulfilment of the requirements for an advanced degree at the University of • B r i t i s h Columbia, I agree that the Library shall make i t freely available for reference and study» . I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives.  It i s understood that:copying or publi-  cation of this thesis for financial gain shall not be allowed without my written permission*.  Department, of  /$?w>'c^/A+,«S  The University.of B r i t i s h Columbia, Vancouver 8, Canada Date  /^^y./  /^'c^U^/^t^  The University of British Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of GERALDINE ANN TOMLINSON B.S.A., The University of British Columbia, 1957 M.A. (Biochemistry), The University of California (Berkeley), 1960 FRIDAY, MAY 1, 1964, IN ROOM 0,  at  2:00 P.M.  AGRICULTURE BUILDING  COMMITTEE IN CHARGE Chairman: F.H. Soward J . J . R . Campbell B.A. Eagles D.P. Ormrod  W.J. Polglase J . J . Stock S.H. Zbarsky  External Examiner: C.E. Clifton Stanford University California  OXIDATIVE ASSIMILATION OF GLUCOSE BY AEROBIC BACTERIA ABSTRACT Oxidative assimilation of glucose-U-Cl^ by several aerobic bacteria was found to involve the assimilation of radioactivity into nitrogenous c e l l components, principally proteinaceous, in. conjunction with the reincorporation of endogenously produced ammonia. In one of these bacteria, Pseudomonas aeruginosa, i f the cells were starved or treated with chloramphenicol/ prior to glucose-C^ the amount of assimilation, especially into protein, was decreased. The incorporation into nucleic acids and lipids was increased by the antibiotic, but was only slightly affected by starvation. A determination of the cytological sites of the assimilated material showed that, in control cell extracts, the soluble proteins of the cytoplasm contained most of the C^. Starved or antibiotic treated c e l l fractions had substantially less of the label in these proteins, whereas the radioactivity incorporated into the ribosomal ribonucleic acid and the "membrane" lipids was greater. A study of the aminoacyl-soluble ribonucleic acid synthetases in P_. aeruginosa revealed that these enzymes were present only in the cytoplasm. Starving the cells resulted in decreased activity of the synthetases, but they were rapidly reactivated during oxidative assimilation. The large amount of heterologous reactions between bacterial soluble ribonucleic acids and synthetases indicated that l i t t l e species specif i c i t y existed. However, cross reactions between the systems in bakers' yeast and the bacteria were poor, showing that some degree of species specificity was present in these instances. Preliminary experiments on the route of assimilation of ammonia in P. aeruginosa and in P. fluorescens gave no evidence for the direct amination of pyruvate by alanine dehydrogenase, but did demonstrate a requirement for concurrent substrate oxidation while ammonia was being incorporated. In contrast, several lines of evidence indicated that ammonia was assimilated via etketoglutarate in P. aeruginosa.  GRADUATE STUDIES Field of Study:  Agricultural Microbiology  Molecular Structure and Biological Function (Proteins and Carbohydrates) Endocrinology Graduate Seminar Chemistry of Proteins Chemistry of Nucleic Acids Enzyme Chemistry Laboratory Techniques in Enzymology Microbial Metabolism Seminars in Biochemistry  W.J. Polglase -•• G.M. Tener G.I. Drummond . P.D. Bragg R.H. Pearce P.H. Jellinck V . J . O'Donnell J . J . R . Campbell R.D, Cole C A . Dekker ,A.B... Pardee J . C . Rabinowitz H.A. Barker M. Dbudoroff R.Y.; Stariier W.T. Jenkins H.A. Barker  Other Studies; Advanced Organic Chemistry  G.G.S.. Dutton D.E. McGreer  PUBLICATIONS Rothstein, M . , and G.A. Tomlinson. 1961. Biosynthesis of amino acids by the nematode Caenorhabditis briggsae. Biochim. Biophys. Acta 49: 625-627. Tomlinson, G.A., and M . Rothstein. 1962. Nematode Biochemistry. I. Culture methods. Biochim. Biophys. Acta 63: 465-470. Rothstein M . , and Tomlinson, G.A. 1962. Nematode Biochemistry. II. Biosynthesis of amino acids. Biochim. Biophys. Acta 63: 471-480. Tomlinson G.A., and J.J.R. Campbell. 1963. Patterns of oxidative assimilation in strains of Pseudomonas and Achromobacter. J . B a c t e r i d . 84: 434-444. Tomlinson, G.A., and J.J.R. Campbell. 1963. Patterns of oxidative assimilation in strains of Acetobacter and Azotobacter. J . Bacteriol. 86: 1165-1172.  ii  Abstract Oxidative assimilation of glucose-U-C^ by Pseudomonas aeruginosa, P. fluorescens, Achromobacter B81, A. viscosus, Azotobacter a g i l i s , A. v i n e l a n d i i , and Acetobacter xylinum was found to involve the a s s i m i l a tion of r a d i o a c t i v i t y  into nitrogenous c e l l components, p r i n c i p a l l y prot-  einaceous in nature in conjunction with the reincorporation of endogenously produced ammonia.  Acetobacter aceti did not exhibit oxidative assimilation  under these circumstances. Further investigation of oxidative assimilation in P. aeruginosa revealed that if the c e l l s were starved or treated with chloramphenicol p r i o r to glucose-c'^ oxidation, the amount of assimilation was decreased. The incorporation of r a d i o a c t i v i t y both treatments.  into protein was severely r e s t r i c t e d by  The amount of l a b e l l i n g of both l i p i d s and nucleic acids  was increased in the presence of the a n t i b i o t i c , but was only s l i g h t l y fected by s t a r v a t i o n .  af-  A determination of the cytological s i t e s of the a s -  similated material showed that,  in control c e l l extracts, the soluble  proteins of the cytoplasm contained most of the c'**.  Starved or a n t i b i o t i c  treated c e l l fractions exhibited a profound decrease in the label of these proteins, whereas the amount of incorporation into the ribosomal r i b o n u c l eic acid and the "membrane" l i p i d s was higher. A study was made of the aminoacy 1-s-RNA synthetases  in f_. aerug-  inosa, and these enzymes were shown to be present only in the cytoplasm, even when the c e l l extracts were prepared and fractionated in a medium of high ionic strength.  Starving the c e l l s resulted in a decrease in the act-  i v i t y of the aminoacyl-s-RNA synthetases, but they were rapidly during oxidative assimilation of glucose.  reactivated  There was found to be l i t t l e  i ii  species s p e c i f i c i t y P.  between the s-RNA's  and s y n t h e t a s e s  f l u o r e s c e n s , Achromobacter B81, and E.  col i,  were o b t a i n e d , but t h e h e t e r o l o g o u s r e a c t i o n s t h e b a c t e r i a were p o o r , except coli  of £_. a e r u g i n o s a ,  s i n c e good c r o s s  between b a k e r s '  in t h e c a s e o f t h e yeast  reactions  yeast  and  s-RNA and t h e  E.  enzyme. Preliminary  experiments on t h e r o u t e o f ammonia a s s i m i l a t i o n  P_. a e r u g i n o s a and in P.  f l u o r e s c e n s gave no e v i d e n c e f o r t h e d i r e c t  in  ami n a -  t i o n o f p y r u v a t e by a l a n i n e dehydrogenase, but d i d demonstrate a r e q u i r e ment f o r c o n c u r r e n t s u b s t r a t e o x i d a t i o n w h i l e ammonia was b e i n g a s s i m i l ated.  In c o n t r a s t ,  several  lines of evidence  a s s i m i l a t e d v i a 9(*-ketoglutarate  in P.  i n d i c a t e d that  ammonia was  aeruginosa.  J . J . R . Campbell  xi  ACKNOWLEDGEMENT  I wish to express my appreciation to Dr. J.J.R. Campbell, for his continued interest and encouragement throughout my years of study and r e search under his d i r e c t i o n , and also to my husband, without whose help, understanding, and forebearance, the completion of this thesis would not have been p o s s i b l e .  In addition, acknowledgement is due to my f e l -  low students, for t h e i r helpful discussions and assistance during the course of this research.  iv  TABLE OF CONTENTS Page INTRODUCTION  1  LITERATURE REVIEW  3  I. II. III.  Oxidative Assimilation Nitrogen Assimilation  3 13  Species S p e c i f i c i t y of s-RNA's and Aminoacyl-s-RNA Synthetases  19  MATERIALS AND METHODS I.  Oxidative Assimilation into Whole Cells of Aerobic Bacteria A.  Bacteriological methods  26  B.  Assimilation studies  27  C.  Starved c e l l experiments  28  1.  Starvation procedure  28  2.  Assimilation experiments  29  D.  E. II. III.  26  Analytical methods  29  1.  Analysis of residual fractions  29  2.  Analysis of cold t r i c h l o r o a c e t i c acid fractions  30  3.  Chemical methods  31  k.  Paper chromatography and electrophoresis  31  Isotopic methods  32  Inorganic Nitrogen Assimilation by Pseudomonas aeruginosa and Pseudomonas fluorescens Oxidative Assimilation into the Cytological Fractions of Normal, Starved, or Chloramphenicol Treated C e l l s of Pseudomonas aeruginosa ATCC 9027 A. Assimilation studies B.  Preparation of c e l l fractions  32  32 33 33  Page C.  Analysis  of  cytological  D.  Chemical  fractionation  E.  Analysis  of  chemical  1.  "Membrane"  2.  Cytoplasmic cold  residual  Preparation  G.  Assay procedure f o r  H. IV.  fractions  fraction  34  trichloroacetic  acid  soluble  P_.  a e r u g i n o s a s-RNA  35  incorporation of  c'4  amino 36  methods  Specificity  of  37 s-RNA's  and  Aminoacy1-s-RNA  Synthetases  37  A.  Preparation  of  s-RNA's  37  B.  Preparation  of  enzymes  38  C.  Preparation  of  aminoacyl-s-RNA's  38  D.  Paper  chromatography  39  RESULTS AND DISCUSSION I.  34 34  i n t o s-RNA  Isotopic  Species  the c y t o l o g i c a l  35  F.  acids  of  34  fractions  fractions  of  fractions  kO  Oxidative Assimilation Bacteria A.  Pseudotnonas  2.  3.  Aerobic  oxidative  assimilation  and A c h r o m o b a c t e r  into strains  of kO  M a n o m e t r i c o b s e r v a t i o n s , ammonia p r o d u c t i o n and u p t a k e , and e x c r e t i o n o f radioactive products into the supernatant f l u i d s during glucose-U-C^4 o x i d a t i o n I n c o r p o r a t i o n o f C^4 tive assimilation Assimilation soluble  4.  of  40  Patterns of 1.  i n t o Whole C e l l s  of  in c o l d  A s s i m i l a t i o n of insoluble  c'4  into c e l l s  oxida50  ; to n  the  cell  trichloroacetic C^4  during  kO  into the  fraction  acid  cell  in c o l d t r i c h l o r o a c e t i c  55  fractions  acid  56  vi  Page B.  Patterns of oxidative assimilation o f A c e t o b a c t e r and A z o t o b a c t e r 1.  2.  3.  4.  C.  A.  B. II.  60  60  I n c o r p o r a t i o n o f c'^ i n t o c e l l s oxidative assimilation  69  during  I n c o r p o r a t i o n o f c'^ i n t o t h e c e l l f r a c t i o n soluble in cold t r i c h l o r o a c e t i c acid  69  Incorporation of insoluble in cold  74  into the c e l l fractions t r i c h l o r o a c e t i c acid by s t a r v e d  cells  o f P.  76  1.  Manometric o b s e r v a t i o n s  76  2.  Ammonia  77  3.  Excretion of radioactive supernatant f l u i d s  excretion  and u p t a k e  4.  Distribution of C ^  5.  Analysis of the cold soluble fractions  1  The i n f l u e n c e assimilation  products  into the 81  in the cells  81  trichloroacetic acid 83  of vitamin  Bg o n  oxidative 86  Inorganic Nitrogen A s s i m i l a t i o n and  strains  M a n o m e t r i c o b s e r v a t i o n s , ammonia p r o d u c t i o n , and e x c r e t i o n o f r a d i o a c t i v e p r o d u c t s i n t o the supernatant f l u i d s during glucose-U-Cl^ oxidation  Oxidative assimilation a e r u g i n o s a ATCC 9027  6.  II.  into  by P s e u d o m o n a s  aeruginosa  Pseudomonas f l u o r e s c e n s  87  9027  Experiments with  f_. a e r u g i n o s a ATCC  1.  Assimilation  o f ammonia d u r i n g t h e o x i d a t i o n  2.  of keto acids A s s i m i l a t i o n o f ammonia inhibitors  Experiments with  88 88  in the presence of  P. f l u o r e s c e n s  92 A 3.12  Oxidative Assimilation into the Cytological Fractions Normal, Chloramphenicol Treated, o r Starved C e l l s o f Pseudomonas a e r u g i n o s a ATCC 9027  93 of  97  vi i Page A.  Chemical composition of cytological fractions  97  B.  Incorporation of glucose-U-C^4 into cytological fractions  98  C.  1.  "Membrane" fractions  100  2.  Ribosomal fractions  105  3.  Cytoplasmic fractions  108  Experiments with the cytoplasmic proteins 1.  2. IV.  114  Amount of r a d i o a c t i v i t y contained in the "pH 5 enzyme"  114  Effect of starvation on the a c t i v i t y of the aminoacyl-s-RNA synthetases  115  Species S p e c i f i c i t y of s-RNA's and Aminoacyl-s-RNA Synthetases A.  124  Cytological location of aminoacyl-s-RNA synthetases in f_. aeruginosa  124  B.  Interspecific reactions between s-RNA's and aminoacyl -s-RNA synthetases  127  C.  Patterns of amino acid incorporation into homologous systems  133  D.  Patterns of amino acid incorporation into h e t e r o l ogous systems  137  GENERAL DISCUSSION  153  BIBLIOGRAPHY  158  FIGURES  Oxygen uptake w i t h 5 Mmoles of s u b s t r a t e and d i s a p p e a r a n c e of g l u c o s e and c'^ from supernatant f l u i d s w i t h washed c e l l s u s p e n s i o n s of Pseudomonas a e r u g i n o s a . Time c o u r s e of N H 3 and keto a c i d p r o d u c t i o n and incorp o r a t i o n d u r i n g o x i d a t i o n o f 5 Mmoles of g l u c o s e - U - C ^ by washed c e l l suspensions of Pseudomonas a e r u g i n o s a . Oxygen uptake w i t h 5 H of s u b s t r a t e and d i s a p p e a r a n c e of g l u c o s e and fJ4 from supernatant f l u i d s w i t h washed c e l l s u s p e n s i o n s of Pseudomonas f l u o r e s c e n s . m o l e s  Time c o u r s e of N H 3 and keto a c i d p r o d u c t i o n and incorporation during oxidation of 5 M ° l e s of g l u c o s e - U - C b y washed c e l l suspensions o f Pseudomonas f l u o r e s c e n s . m  Oxygen uptake w i t h 5 Mmoles of s u b s t r a t e and d i s a p p e a r a n c e of g l u c o s e and C ^ from supernatant f l u i d s w i t h washed c e l l suspensions of Achromobacter B81. Time c o u r s e of N H 3 and keto a c i d p r o d u c t i o n and incorp o r a t i o n d u r i n g o x i d a t i o n o f 5 Mmoles o f g l u c o s e - U - C ^ by washed c e l l s u s p e n s i o n s o f Achromobacter B81. Oxygen uptake w i t h 5 Mmoles o f s u b s t r a t e and d i s a p p e a r a n c e o f g l u c o s e and from supernatant f l u i d s w i t h washed c e l l s u s p e n s i o n s o f Achromobacter v i s c o s u s . Time c o u r s e ~of N H 3 and keto a c i d p r o d u c t i o n and c ' 4 i n c o r poration during oxidation of 5 M " ! of g l u c o s e - U - C ' 4 by washed eel 1 suspensions of Achromobacter v i s c o s u s . 1 0  6 5  Oxygen uptake w i t h 5 Mmoles of s u b s t r a t e and d i s a p p e a r a n c e of g l u c o s e and C.14 from supernatant f l u i d s w i t h washed c e l l s u s p e n s i o n s o f A c e t o b a c t e r acet i . Time c o u r s e o f N H 3 and keto a c i d p r o d u c t i o n and incorp o r a t i o n d u r i n g o x i d a t i o n of 5 Mmoles o f g l u c o s e - U - C l 4 by washed c e l l suspensions o f A c e t o b a c t e r a c e t i . Oxygen uptake w i t h 5 Mmoles of s u b s t r a t e and d i s a p p e a r a n c e of g l u c o s e and CJ4 from supernatant f l u i d s w i t h washed c e l l s u s p e n s i o n s of A c e t o b a c t e r x y l i n u m . Time c o u r s e o f N H 3 and keto a c i d p r o d u c t i o n and incorp o r a t i o n d u r i n g o x i d a t i o n of 5 Mmoles o f g l u c o s e - U - C ^ by washed c e l l s u s p e n s i o n s of A c e t o b a c t e r x y l i n u m .  ix' Figure  Page Oxygen uptake with 5 Mmoles of substrate and disappearance of glucose and c ' ^ from supernatant f l u i d s with washed c e l l suspensions of Azotobacter v i n e l a n d i i .  66  Time course of NH3 and keto acid production, and C ^ incorporation during oxidation of 5 Mmoles of glucose-U-C'4 by washed c e l l suspensions of Azotobacter v i n e l a n d i i .  66  Oxygen uptake with 5 Rmoles of substrate and disappearance of glucose and from supernatant f l u i d s with washed c e l l suspensions of Azotobacter a g i l i s .  68  Time course of NH3 and keto acid production, and incorporation during the oxidation of 5 Mmoles of glucose-U-c'^ by washed c e l l suspensions of Azotobacter a g i l i s .  68  9  Oxygen uptake during oxidation of pyruvate and glucose by washed control and starved c e l l s of Pseudomonas aeruginosa.  79  10  Production and uptake of NH3 by washed, starved c e l l pensions of Pseudomonas aeruginosa.  80  11  Disappearance of and glucose from, and excretion of keto acids into, supernatant f l u i d s during oxidation of glucoseU-Cl4 by washed, starved c e l l s of Pseudomonas aeruginosa.  80  Incorporation of during oxidation of 5 Mmoles of glucose-U-cJ^ by washed c o n t r o l , c h l o r a m p h e n i c o l ( C h l o r o m y c e t i n ) treated, or starved c e l l s of Pseudomonas aeruginosa.  85  Uptake of oxygen and NH3 by washed c e l l s of Pseudomonas f l u o rescens during the oxidation of 10 M ° l e s of pyruvate with and without 50 Mmoles of fluoroacetate.  95  7A  7B  8A  8B  12  13  1  sus-  m  14  Incorporation of into the protein residue and the l i p i d of the "membrane" fractions during oxidation of glucose-U-C'4 by washed c o n t r o l , chloramphenicol ( C h l o r o m y c e t i n ) treated, or starved c e l l s of Pseudomonas aeruginosa. 103  15  Incorporation of C l ^ into the ribosomal fractions during oxidation of glucose-U-Cl4 by washed c o n t r o l , chloramphenicol (Chloromycetin) treated, or starved c e l l s of Pseudomonas aeruginosa.  16  Incorporationoof C'^ into the RNA and protein residue of the ribosomes during oxidation of glucose-U-C'4 by washed control,  chloramphenicol  (Chloromycetin)  c e l l s of Pseudomonas aeruginosa. 17  107  Incorporation of  treated,  or  jnto the cold t r i c h l o r o a c e t i c  starved  acid  109  s o l u b l e p o o l and t h e p r o t e i n r e s i d u e o f t h e c y t o p l a s m i c f r a c t i o n s d u r i n g o x i d a t i o n o f g l u c o s e - U - C ^ by w a s h e d c o n t r o l , c h l o r a m p h e n i c o l (Chloromycetin) treated c e l l s o f Pseudomonas a e r u g i n o s a . Incorporation mic f r a c t i o n s  o f C l 4 a m i n o a c i d s i n t o s-RNA by o f Pseudomonas aeruginosa.  cytoplas-  1.  INTRODUCTION  " O x i d a t i v e a s s i m i l a t i o n " is the poration  of carbon  i n t o c e l l u l a r components  tion of part  in the  Energy f o r t h i s  a b s e n c e o f added n i t r o g e n ,  of the substrate,  w h i l e the  remainder  t h e a s s i m i l a t i o n o c c u r s w i t h o u t added n i t r o g e n , the  designated  "primary products",  t o be t r u e f o r a number o f m i c r o o r g a n i s m s . evident  that,  ety of c e l l  components,  b e i n g d e r i v e d from t h e endogenous  in t h i s  l a b o r a t o r y h a v e shown t h a t  not  indeed, t h i s  has  R e c e n t l y , however,  including nitrogenous  o f Pseudomonas  aeruginosa.  this  Since that  i n non n i t r o g e n o u s  i n some m i c r o o r g a n i s m s , a s s i m i l a t i o n t a k e s p l a c e  process  cells  i s p r o v i d e d by o x i d a -  i t m i g h t be e x p e c t e d  and  ones;  the  it  has  that  found  in  found become  into a  nitrogen for  is the s i t u a t i o n  cell  been  varithis  r e s p i r a t i o n of the c e l l s .  and m o r e o v e r ,  sub-  process,  is a s s i m i l a t e d .  i n c o r p o r a t e d c a r b o n would be f o u n d e x c l u s i v e l y  constituents,  incor-  during the o x i d a t i o n of a  (100).  s t r a t e by w a s h e d c e l l s o f m i c r o o r g a n i s m s which takes p l a c e  term used t o d e s c r i b e t h e  Studies resting  a primary product  is  formed d u r i n g o x i d a t i v e a s s i m i l a t i o n o f g l u c o s e ( 5 5 ) . This thesis  similation  in aerobic b a c t e r i a .  Duncan and C a m p b e l l aerobic bacteria, a special  is concerned w i t h several  (55) w i t h  to determine  reserve product  components  determined, assimilation  the  experimental  i f , under  approach used  these c o n d i t i o n s , they would  s u c h as c a r b o h y d r a t e o r  extent  asby  P . a e r u g i n o s a was a p p l i e d t o a number o f  w o u l d be s y n t h e s i z e d .  a e r u g i n o s a c e l l s on t h e  sites  Firstly,  aspects of o x i d a t i v e  Secondly, the  lipid,  effect  o r whether  form  all  cell  o f s t a r v a t i o n o f P_.  and p a t t e r n s o f o x i d a t i v e a s s i m i l a t i o n was  and an a t t e m p t was made t o e l u c i d a t e t h e m e c h a n i s m o f ammonia in P. aeruginosa  and P . f l u o r e s c e n s .  o f o x i d a t i v e a s s i m i l a t i o n were  investigated  Thirdly,  the  cytological  in P. aeruginosa,  and  the  2. influence of starvation or was s t u d i e d .  Finally,  inhibition  i n v e s t i g a t i o n was made o f t h e  acid  (aminoacyl-s-RNA)  on t h i s  s i n c e p r o t e i n was o n e o f t h e m a i n p r o d u c t s  t i v e a s s i m i l a t i o n i n P_. a e r u g i n o s a an  of protein synthesis  (as w e l l  as  in the other  process of  aerobes  oxidastudied),  formation o f aminoacyl s o l u b l e r i b o n u c l e i c  in P. aeruginosa,  m i c r o o r g a n i s m was c o m p a r e d t o t h o s e  and t h e s y s t e m p r e s e n t  in other  bacteria,  and  in  in  this  yeast.  3.  LITERATURE REVIEW I.  Oxidative Assimilation H. A . Barker  (11)  first  used t h e term " o x i d a t i v e a s s i m i l a t i o n "  t o d e s c r i b e t h e c o n v e r s i o n , by t h e c o l o r l e s s a l g a P r o t o t h e c a zopf i i , p a r t o f an o r g a n i c m o l e c u l e i n t o c e l l u l a r m a t e r i a l , w h i l e t h e was o x i d i z e d .  of  remainder  T h i s p r o c e s s was m a n i f e s t e d by an oxygen uptake which was  l e s s than e x p e c t e d , a l t h o u g h t h e s u b s t r a t e had c o m p l e t e l y d i s a p p e a r e d . addition,  the dry weights  of t h e microorganisms had r i s e n somewhat.  s i m i l a r phenomenon had been noted p r e v i o u s l y and Stephenson  (41),  but no reason f o r  in E s c h e r i c h i a c o l i  In  A  by Cook  i t s o c c u r r e n c e had been p o s t u l a t e d .  From t h e m o l e c u l a r q u a n t i t i e s o f t h e r e a c t a n t s ,  and t h e f a c t  t h a t one o f  t h e p r o d u c t s was carbon d i o x i d e , Barker was a b l e t o w r i t e balanced e q u a t i o n s f o r the o x i d a t i o n of several  substrates.  o x i d a t i o n o f a c e t a t e was w r i t t e n as CH3COOH + 0  Thus, the equation f o r  the  follows: )  2  C0  2  + H 0 + (CH 0) 2  2  The f o r m u l a f o r t h e p r o d u c t o f a s s i m i l a t i o n o f s e v e r a l  o r g a n i c compounds  c o u l d a l s o be w r i t t e n as C H 0 .  S i n c e P.  and s t o r e g l y c o g e n , Barker  concluded t h a t t h i s was t h e primary p r o -  2  (11)  duct o f o x i d a t i v e a s s i m i l a t i o n t h e s o u r c e o f raw m a t e r i a l  zopf i i was known t o s y n t h e s i z e  in t h e a l g a , and t h a t  for cellular  i t might be used as  s y n t h e s i s d u r i n g p r o l o n g e d incuba-  t ion o r growth. The Warburg manometric t e c h n i q u e was used in t h e subsequent y e a r s by a s u c c e s s i o n o f authors t o study o x i d a t i v e a s s i m i l a t i o n , and t h e i r r e s u l t s supported B a r k e r ' s h y p o t h e s i s . calcoacet?  and E.  col?  T h u s , C l i f t o n w i t h Pseudomonas  (32), G i e s b e r g e r u s i n g Spir?11 urn (70),  w i t h Pseudomonas s a c c h a r o p h i l a (52).  all  and Doudoroff  p o s t u l a t e d primary p r o d u c t s o f  assimilation s i m i l a r to that suggested by Barker.  These results were  based on the amount of oxygen consumed, and the carbon dioxide l i b e r a t e d , at the time of the f i r s t sharp break in the oxygen curve for the substrate in question.  That substrate assimilation was not confined to respiring  systems was suggested by the experiments of Winzler and Baumberger on heat production during alcoholic fermentation.  (174)  From the heats of f o r -  mation, and the heat produced during exogenous r e s p i r a t i o n , they concluded that 70.5% of the glucose was fermented to ethyl alcohol and carbon d i o x ide, and 29.5% stored. were 24.5% and 75.5%.  Corresponding figures for aerobic d i s s i m i l a t i o n Fermentative assimilation was also shown in Candida  albicans by van Niel and Anderson (155), but could not be detected in Streptococcus faecal i s . Siegel and C l i f t o n (134,135) have studied the relationship between the amount of assimilation of several carbohydrates and organic acids and the available free energies of these compounds.  The extent of assimila  tion of arabinose was investigated in both growing and resting c e l l s of E. coli  (134).  From manometry with resting c e l l s , the percent of oxidative  assimilation with each sugar was determined, and found to be 40% for arabin ose, and about 50% for glucose and lactose.  In addition, the free energy  changes for the oxidative reactions were calculated from these data, based on the actual amount of each sugar oxidized.  The value so calculated for  arabinose was found to be twice as high as that for glucose and arabinose, yet from carbon balances done on growing c e l l s , arabinose was assimilated to the greatest extent, glucose l e s s , and lactose the l e a s t .  Previous  studies on the extent of assimilation in resting and growing c e l l s of JE. c o l i had indicated that this occurred to a s i m i l a r measure in both cases (37).  In a second paper, Siegel and C l i f t o n (135)  reported on a s i m i l a r  investigation of the energetics  involved in the assimilation of succinate,  fumarate, lactate, pyruvate and glycerol by short term cultures of E. colI.  The same s i t u a t i o n was found with these compounds as with the sugars  a similar amount of carbon was assimilated from pyruvate as from lactate, and from fumarate as from succinate, although the free energy of oxidative assimilation was found to be less for the f i r s t compound of each p a i r . In these experiments, both the amount of oxygen taken up and the carbon assimilated were determined with growing c e l l s , and therefore should be comparable, in contrast to the data on sugars, where resting c e l l s were used in assessing one value, and growing c e l l s for the other.  When the  amounts of assimilation in resting c e l l s of the acids and the sugars were calculated solely on the basis of oxygen uptake, there appeared to be somewhat less assimilation than in growing c e l l s .  The e f f i c i e n c y of u t i l -  ization of the acids was found to be lower than that of the sugars.  This  was considered to be due in large measure to the circumstance that some of the molecules of the compounds with fewer carbons had to be oxidized completely to provide energy for the assimilation of others, or for the assimilation of the intermediates derived from them.  With substrates  having more carbons, the energy evolving processes generated the mediates needed for synthesis.  inter-  That the same s i t u a t i o n existed in Baci1 -  lus s u b t i l is was suggested by the studies of Wilner and C l i f t o n  (172)  also with organic acids and glucose as substrates for a s s i m i l a t i o n . Again, fumarate and pyruvate were assimilated to a greater degree than would have been predicted by the f r e e energy changes from oxidation of the molecule.  C l i f t o n and his associates have concluded from these i n -  vestigations that the chemical structure of a compound, which determines how it  is metabolized, appears to be more important in determining the  6. extent of its assimilation than the amount of free energy inherent in the molecule. Photosynthetic assimilation was f i r s t studied by Gaffron using resting c e l l s of purple bacteria.  (65)  As a result of these investiga-  t i o n s , he postulated the presence of a primary assimilatory product with the empirical formula (C^HgO^),-,.  Small quantities of a polymeric substance  with this elementary composition were isolated from cultures grown photosynthet ical ly on butyrate.  However, further investigations of this unique  polymeric product was discouraged by van N i e l ' s report  (154) that the gross  composition of purple bacteria corresponded closely to the composition of Gaffron's proposed product of a s s i m i l a t i o n . The use of manometric techniques introduced several  limitations  which, despite the considerable volume of work which was undertaken, p r e vented the i d e n t i f i c a t i o n of the nature of the assimilated material.  One  d i f f i c u l t y was the uncertainty of the actual oxygen uptake, since it was not known whether endogenous respiration continued unabated, was s t i m u l a t ed, or was inhibited by, the addition of oxidizable substrate.  Qualita-  t i v e tests were usually carried out on supernatant f l u i d s for predetermined typical products or intermediates  in the oxidation of the substrate, and  if these were negative, then no other tests were done.  However,  in ex-  periments where carbon balances were performed, discrepancies were sometimes found, which were due to the presence of unidentified products. S t i l l another question was whether the substrates were assimilated  intact,  or whether they were cleaved to smaller fragments p r i o r to the assimilatory process.  Some of these questions were answered by later experiments with  radioisotopes, which showed that the amount of oxidative assimilation was often much less than that indicated by manometric measurements.  This  7. technique also made possible the separation and characterization of the products of a s s i m i l a t i o n . Since some bacteria appeared to assimilate carbon as e f f i c i e n t l y in the absence of nitrogen as in its presence, it seemed evident that a s s i m i l a t i o n was not necessarily coupled with general c e l l synthesis.  One  could, therefore, conclude that the assimilated material would be found in a limited number of c e l l constituents, or primary products. organisms this was found to be the case.  In many micro-  The assimilation of carbon by P.  zopfi i into a product having the empirical formula of glycogen has been discussed.  This material, or one with the same empirical formula, was  also suggested by the findings of other workers with a number of b a c t e r i a . Polysaccaride  is the primary product of assimilation by many  b a c t e r i a , e s p e c i a l l y the enteric group, which have been found to contain levels of glycogen as high as 48% of the dry weight of the c e l l s  (103),  present as a reserve product within the c e l l , or as structural components, especially capsules (170).  Holme and Palmstierna (85) have made detailed  studies of an a l k a l i stable polyglucose, apparently identical to glycogen in its p r o p e r t i e s , formed from glucose by growing or resting c e l l s of E_. c o l i during nitrogen s t a r v a t i o n .  If the polysaccharide containing c e l l s  were transferred to a medium containing inorganic nitrogen, but no carbon, the newly formed polyglucose was broken down, and used for protein s y n thesis. The oxidative assimilation of glucose, and other substrates to Sajxlna Jutea was investigated by Binnie, Dawes and Holms (21).  in-  When  the c e l l s were grown on peptone, carbohydrate made up 10% of t h e i r dry weight, and after oxidation of glucose by freshly harvested or l y o p h i l ized endogenous diminished c e l l s , this rose to 28%.  The assimilated  8 material ing  was u t i l i z e d  i n 3.5  hours,  d u r i n g endogenous  the  r e s t more s l o w l y .  to assimilate uniformly bulk of the yielded  r a d i o a c t i v i t y was f o u n d  the  authors  When c e l l s w h i c h had b e e n  l a b e l l e d glucose were f r a c t i o n a t e d  g l u c o s e on h y d r o l y s i s .  glucose,  in the  alcohol  concluded that  the  result  i n an  bacteria  aid of  increase  into both  the assimilated r a d i o a c t i v i t y into the  lipid  30% o f t h e d r y w e i g h t o f t h e c e l l s , w h i c h made up l e s s  i t has  than  s i t e of assimilation.  lipid  mines t h e p r o d u c t o f a s s i m i l a t i o n . acetate tion,  increased the formation of  whereas  lipid,  found  an  increase  ized a c e t a t e .  the form o f  in the  This  incorporated  that  lipid  into  (115).  i t was  the substrate  and d e c r e a s e d  The  the  deter-  the presence  of  glycogen forma-  (171).  g l u c o s e had t h e o p p o s i t e e f f e c t  takes  accounted  8% o f t h e c e l l m a t e r i a l  o f a number o f s t u d i e s  and t h e r e seems t o b e some d i s a g r e e m e n t  ated m a t e r i a l  carbohydrate.  i n c o r p o r a t e d 37% o f  T h u s , w i t h JE. c o l ? ,  Y e a s t s have been t h e s u b j e c t tion,  and  52% was  W i t h some b a c t e r i a ,  cells,  b e e n shown t h a t  f r a c t i o n , which  whereas  sub-  intermediates.  higher, s p e c i f i c a c t i v i t y of the carbohydrate suggested initial  i t was o f  in the cold t r i c h l o r o -  metabolic  labelled substrates,  are able to a s s i m i l a t e carbon  carbohydrate,  that  in the carbohydrate of the  N o c a r d i a c o r a l U n a , when o x i d i z i n g p r o p i o n a t e - 3 - C ^  for  poly-  The u s e o f a c e t a t e o r p y r u v a t e as  acid soluble f r a c t i o n s , with other With the  and  g l u c o s e had b e e n a s s i m i l a t e d d i r e c t l y .  r a d i o a c t i v i t y f r o m t h e s e compounds b e i n g f o u n d  acetic  chemically, the  From t h e s p e c i f i c a c t i v i t y o f t h e  low m o l e c u l a r w e i g h t .  s t r a t e s d i d not  allowed  soluble material,  The c h r o m a t o g r a p h i c b e h a v i o u r o f t h e p o l y m e r s u g g e s t e d relatively  50% d i s a p p e a r -  r e s p i r a t i o n , about  as t o w h e t h e r  or carbohydrate.  reducing sugar content  of yeast  i n c r e a s e was e n o u g h t o a c c o u n t  on a s s i m i l a -  the  incorpor-  Winzler  (173)  w h i c h had  f o r 80% o f t h e  oxidmaterial  9. t h e o r e t i c a l l y assimilated.  On the other hand, McLeod and Smedly-Mclean  (111) found that l i p i d was synthesized from acetate, without the mediate formation of carbohydrate.  These  inter-  experiments were generally of  a longer duration, and were carried out in the presence of higher concentrations of acetate than employed by Winzler. earlier  Perhaps an analysis made  in the course of the oxidation would have revealed the primary  synthesis of carbohydrate.  Pickett and C l i f t o n (125) established that  carbohydrate was the primary product of assimilation by Saccharomyces cerevisiae during glucose oxidation, since the increase in readily hydrolysable carbohydrate was equal to the increase in dry weight of the c e l l s . When a second yeast, Torulopsis u t i l i s . was allowed to oxidize sucrose plus acetate-C^ j  n  a nitrogen free medium, the r a d i o a c t i v i t y was incor-  porated into both carbohydrate and l i p i d (92).  if acetate-c'^ were used  alone, however, nearly 50% of the assimilated material was c a l c u l a t e d , by d i f f e r e n c e , to be " p r o t e i n , " although there was no net increase of protein in the c e l l s during the experiment.  This f i n d i n g , by Jackson and Johnson,  of assimilation into p r o t e i n , was one of the f i r s t reports of a nitrogenous product of a s s i m i l a t i o n . Although the l i p i d formed by assimilation into yeast c e l l s thought to consist of conventional t r i g l y c e r i d e s  is  (171), the importance of  the l i p i d material poly-jS-hydroxybutyrate as a product of assimilation by bacteria has recently been recognized.  Although it had been known for  years that this l i p i d was found as a major component of Bac i11 us species (101,102), it remained for McRae and Wilkinson in 1958 (108) to show that it functioned as an i n t r a c e l l u l a r reserve of carbon and energy in B_. megaterium and B. cereus.  Glucose, pyruvate or fi -hydroxybutyrate served  as substrates for synthesis of the polymer by washed c e l l suspensions, but  10. acetate could not, although Ft greatly enhanced the formation of poly-^?hydroxybutyrate  in the presence of one of the other substrates.  The wide-  spread occurrence of t h i s polymer was soon demonstrated by Forsyth, Hayward and Roberts  (60),  who found it to be present  in many Gram negative  including Azotobacter species and nonpigmented pseudomonads. of poly-^-hydroxybutyrate  in  bacteria,  The presence  cocci was demonstrated by Sierra and Gibbons  (136), who studied the biosynthesis and oxidation of the polymer in Micrococcus h a l o d e n i t r i f i c a n s .  Doudoroff and Stanier (53)  found that poly- /3 -  hydroxybutyrate was the product of photosynthetic assimilation in Rhodos p i r i l i u m rubrum. thus confirming the much e a r l i e r conclusion of Gaffron (65)  with purple b a c t e r i a .  The role of the polymer as an endogenous carbon  and energy source <ih R. rubrum was also demonstrated. Previous work by Wiame and Doudoroff (167) s i m i l a t i o n of  on the oxidative a s -  labelled substrates by f_. saccharophila had shown that,  in this microorganism, two carbon fragments were the fundamental building blocks of a s s i m i l a t i o n .  When lactate was used as the substrate, the carb-  oxyl carbon was oxidized almost completely, while the remaining two carbons were used for synthetic reactions. cinate,  i.e.,  A s i m i l a r s i t u a t i o n occurred with s u c -  the carboxyl carbons were oxidized, and the methyl carbons  were assimilated, and whereas both carbons of acetate were assimilated, the methyl carbon was favoured.  However, the nature of the assimilated  material was not established, although it was suggested, without any supporting data being given, that "the carbon flowed into many different materials  in the c e l l ,  including p r o t e i n . "  The finding that  poly-/?-  hydroxybutyric acid occurred in some pseudomonads, and that two carbon fragments were assimilated by P. saccharophila. led Doudoroff and Stanier (53)  to reexamine the products.of assimilation in this organism.  They  n. found  that  freshly  harvested  cells,  a s s i m i l a t e d o n l y 21% o f t h e appeared before  added  when  r a d i o a c t i v i t y , and o f t h i s ,  as p o l y - fi - h y d r o x y b u t y r a t e .  the  a g a i n as  experiment,  the polymer.  However,  m o r e t h a n 50% o f t h e  butyrate.  but  net  for  P. saccharoph?la  transfer  in the  the  two-thirds  c e l l s were  g l u c o s e was  starved  assimilated,  formed d u r i n g  D o u d o r o f f and S t a n i e r  p o l y - ^ - h y d r o x y b u t y r a t e c o u l d s e r v e as piration  i f the  T h i s was a l s o t h e m a i n p r o d u c t  oxidation of acetate or  glucose-c'^,  incubated with  reported  the  that  s u b s t r a t e f o r endogenous  res-  a b s e n c e o f an o x i d i z a b l e s u b s t r a t e ,  of polymer carbon to other  cell  constituents  c o u l d not  be  demonstrated. Up t o t h i s cerned tions  the  time,  reports  o n a s s i m i l a t i o n by b a c t e r i a  formation of a primary carbonaceous  that  this  experiments  might not  be a u n i v e r s a l  of Warren, E l l s  and C a m p b e l l  inosa c o n s i s t e n t l y produced c o n s i d e r a b l e endogenous  respiration,  c o s e was a d d e d .  and t h a t  it  as w e l l  as t o (38)  However,  (163), who showed t h a t quantities  (74)  this  extended  this  finding  glucose-c'^  assimilated  by r e s t i n g c e l l  suspensions  of this  most o f t h e  l a b e l was f o u n d  in the  trichloroacetic acid  components,  but  i n t o t h e compounds s o l u b l e and  revealed  that  cold  50% o f t h e  number o f  Clifton  organism  (35).  bacteria,  has  since reported  i n c l u d i n g Bac?1lus megaterium,  later  Initially,  s o l u b l e pool  i n s o l u b l e in  w h i c h w o u l d be p r i m a r i l y n u c l e i c a c i d s  (36)  a  r a d i o a c t i v i t y was  and  F o r m a t i o n o f a p o l y m e r s u c h as p o l y - ^ - h y d r o x y b u t y r a t e d i d  appear t o o c c u r .  glu-  was  t o p r o d u c e ammonia e n d o g e n o u s 1y, and  with  teins.  aerug-  to  Bac? 11 us c e r e u s  S,. c e r e v i s i a e .  experiments  trichloroacetic acid,  P.  the  ammonia when  and S o b e k  hot  indica-  o f ammonia d u r i n g  shown by C l i f t o n  soon passed  con-  s i t u a t i o n w e r e p r o v i d e d by  reincorporated  G r o n l u n d and C a m p b e l l  number o f b a c t e r i a ,  reserve.  had  similar  results  pro-  not for  A z o t o b a c t e r ag?1 i s ,  a  12. B. subti1 i s , and Hydrogenomonas faci1 is  (45).  Almost simultaneously with C l i f t o n ' s report on B_. cereus. Duncan and Campbell (55),  using P. aeruginosa, demonstrated that t h i s microorgan-  ism also formed no primary product during oxidative assimilation of g l u cose, but followed the same route of incorporation of carbon as did B_. cereus, i . e . ,  the label appeared f i r s t  in the cold t r i c h l o r o a c e t i c  acid  soluble p o o l , then passed into compounds soluble and insoluble in hot t r i chloroacetic acid and a l c o h o l .  However,  in contrast to the b a c i l l u s , and  to the conclusions suggested by oxygen uptake data, the pseudomonad assimilated only 16% of the substrate. assimilation  Duncan and Campbell were able to r e l a t e  in P. aeruginosa to the formation of V - k e t o g l u t a r a t e ,  which  was excreted into the medium during the early stages of glucose oxidation, and reincorporated concurrently with ammonia provided by endogenous r e s piration.  Since this microorganism has an active glutamic acid dehydro-  genase, Duncan and Campbell believe that "the p a r t i a l  block of glucose  oxidation at of-ketoglutarate represents a control mechanism, ensuring that the method of entry for nitrogen w i l l be present as soon as any becomes available by d i f f u s i o n , leaching, or from endogenous storage products." Assimilation by P. aeruginosa appeared to be limited by the amount of nitrogen a v a i l a b l e , since the addition of ammonia greatly  increased the  amount of C ^ incorporated, and prevented the accumulation of •( -ketoglutarate  in the suspending f l u i d .  When chloramphenicol was added to  resting c e l l suspensions oxidizing glucose, the amount of r a d i o a c t i v i t y was decreased, and assimilation  incorporated  into the l i p i d , cold t r i -  chloroacetic acid soluble p o o l , and hot t r i c h l o r o a c e t i c acid soluble fractions was increased, at the expense of the residual f r a c t i o n , which is mainly protein.  The reincorporation of endogenously produced ammonia during  13. oxidative assimilation has led to the hypothesis, advanced by Duncan and Campbell (55), and by C l i f t o n (36), that oxidative assimilation may serve, at least  in p a r t , to replenish the nitrogenous endogenous reserves of some  microorgan isms. 11.  Inorganic Nitrogen Assimilation In the previous s e c t i o n , the l i t e r a t u r e on oxidative a s s i m i l a -  tion was reviewed, and it was shown that this process, although by d e f i n i tion occurring in the absence of added nitrogen, often involves protein and nucleic acid biosynthesis.  For this reason, the assimilation of i n -  organic nitrogen becomes important in the overall phenomenon of oxidative ass i m i l a t i o n . The major pathway by which organisms incorporate inorganic nitrogen into organic compounds involves the ammonium ion. 1926, Quastel and Woolf  As early as  (127) reported the formation of aspartate from  fumarate and ammonia by c e l l s of E. c o l ? , and later this was extended to several anaerobes (42).  The equilibrium of this reaction was found to  favour aspartate formation, with a K q of approximately 20. e  Virtanen and  Tarnenen (157) extracted aspartase from c e l l s of Pseudomonas fluorescens. and demonstrated that the reaction occurred in c e l l free extracts.  The  enzyme proved to be fumarate s p e c i f i c , and later work by Ichihara et_ a l . (91)  indicated that f o l i c a c i d , reduced glutathione, and cobalt  required by the p u r i f i e d enzyme.  ions were  This report, however, remains to be  confirmed or extended, and the mechanism of the action of aspartase is s t i l l unknown. The enzyme alanine dehydrogenase was f i r s t described by Wiame and Pierard  (168)  in a mutant of B_. subt ? 1 is which lacked glutamic acid  14  dehydrogenase,  but o x i d i z e d g l u t a m a t e .  tion of glutamic acid with  I t was assumed  that  transamina-  a n o t h e r k e t o a c i d was o c c u r r i n g , and t h a t  the  a m i n o a c i d s o f o r m e d was t h e n o x i d i z e d by an enzyme as y e t  unknown.  ination of c e l l  s p e c i f i c f o r L_-  extracts  revealed  a l a n i n e and n i c o t i n a m i d e a d e n i n e oxidation product,  an a l a n i n e d e h y d r o g e n a s e dinucleotide  (NAD).  and a l a n i n e was s y n t h e s i z e d  in the presence of  nicotinamide adenine d i n u c l e o t i d e  (NADH), p y r u v a t e  enzyme has  bacilli,  been  hydrogenase,  reported  but  in other  been  reported  that  several  a l s o o x i d i z e d (123). a t pH 10 has b e +18.6 ate  been  kcal.  not y e t  postulated  has  (58,86,87).  Although  i t has  the  recently  of the deamination  are  reaction to  of alanine they  but  are  unless  in b a c t e r i a l  (122).  b e e n shown t o o c c u r  been e l u c i d a t e d ,  is favoured,  an e n z y m e - b o u n d  pyruv-  spores,  The r e a c t i o n intermediate  in  mechanhas  been  (71).  in y e a s t ,  bacterial,  primary route for  and a n i m a l s y s t e m s ,  the  in E . c o l i  w h i c h has has  a l w a y s been t h o u g h t  i n c o r p o r a t i o n o f ammonia  (2) c e l l  free  been e x h a u s t i v e l y  preparations,  the  i n two  has  the  In y e a s t been  shown  (NADP) s p e c i f i c , and  to  stages.  L - g l u t a m i c a c i d + NADP  ^  iminoglutaric acid + H 0 2  A t pH 6.5,  reaction  studied  t o be  i n t o amino a c i d s .  t o be n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e p h o s p h a t e occur  de-  (  Glutamic a c i d dehydrogenase,  (3), and  This  1.4xl0~'5 and t h e f r e e e n e r g y c h a n g e  t o be  removed by o x i d a t i o n , as  which the deamination  and ammonium i o n s .  L-alanine,  The e q u i l i b r i u m c o n s t a n t  reported  reduced  a l i p h a t i c , m o n o c a r b o x y l i c amino a c i d s  Thus, the synthesis  and NADH a r e  i s m has  t o be s p e c i f i c f o r other  the  many o f w h i c h l a c k g l u t a m i c  some o f w h i c h h a v e b o t h enzymes  enzyme was f o r m e r l y t h o u g h t  P y r u v a t e was  Exam-  the  free  energy  V  ^  change  i m i n o g l u t a r i c a c i d + NADPH 1  pf-ketoglutaric  i s +17.6  kcal  in the  acid +  NH3  direction of  °f-keto-  15. g l u t a r i c acid formation, and therefore, l i k e alanine dehydrogenase, favours the synthesis of the amino acid (124).  NADP s p e c i f i c glutamate  dehydrogenases have also been found in Neurospora crassa (59), and in many bacteria (2). acceptors.  The animal enzymes can use either NADP or NAD as hydrogen  Like alanine dehydrogenase, glutamic acid dehydrogenase was  thought to be s p e c i f i c for one substrate only.  However, Struck and Sizer  (145) found that, under appropriate conditions, certain other a l i p h a t i c , monocarboxylic acids were also oxidized by the chicken l i v e r enzyme. These other amino acids were oxidized at a much higher pH than is optimal for glutamate, similar to the conditions found to give the maximum rate for alanine dehydrogenase and alanine.  Leucine was the best substrate among  these compounds, and alanine was also oxidized slowly.  Aspartic acid was  not oxidized. Both glutamic acid (124) and alanine dehydrogenases (71,122,168) are inactivated by iodoacetate and p-chloromercuribenzoate, but not by arsenite, suggesting that t h i o l , but not d i t h i o l groups are involved in the enzymatic reaction.  The inhibition of alanine dehydrogenase a c t i v i t y  by the sulfhydral binding reagents can be reversed by L_-cysteine (122). Possibly related to this finding are the reports of the i n h i b i t i o n , by shaking, of alanine biosynthesis, from pyruvate and ammonia, by resting c e l l s of B. subt?1 is (58) and Brucella abortus (7).  However, a recent  report by Freeze and Oosterwyck (63) indicated that alanine dehydrogenase in B. subt?1 is is an inducible enzyme, and that induction is repressed by aeration, probably because of the oxidation of NADH to NAD. Thus, no alanine would be available to act as an inducer.  Since the levels of the  enzyme were low in aerated B. s u b t i l is c e l l s grown in the presence of pyruvate, there must be some mechanism for-assimilation of ammonia in these  16. c e l l s other than the animation of t h i s keto acid to give alanine;  however,  no glutamic acid dehydrogenase was found ih the presence or absence of aeration, nor did L_-glutamate act as an inducer for alanine dehydrogenase. There are several other less important pathways of ammonia i n corporation in microorganisms.  A s i n g l e report exists on the formation of  glycine from glyoxylate, ammonia, and NADH by an enzyme from Mycobacterium tuberculosis var. hominis (39).  The standard free energy of glycine forma-  tion was calculated to be -11 kcal per mole. ducts of nitrogen f i x a t i o n (6),  Amides are known to be p r o -  and the synthesis of amides from ammonia  and either aspartate or glutamate is a major ammonia incorporation reaction of plant t i s s u e (143).  Cell free preparations of Staphylococcus aureus  (57) and Proteus vulgaris  (77),  have been shown to synthesize glutamine  from glutamic a c i d , ammonia, adenosine triphosphate (ATP), and magnesium. Nothing is known of the mechanism of asparagine synthesis in bacteria; however,  in p l a n t s , the cofactor and metal requirements appear to be the  same as for glutamine synthesis  (165).  Mortenson suggests that this  is  because there is a transamidation reaction from glutamine to aspartic a c i d , y i e l d i n g asparagine and glutamic acid  (116).  in the opinion of O'Connor and Halvorson (123), alanine and glutamic acid dehydrogenases are the primary routes for assimilation of ammonia in microorganisms.  Support for the major role of glutamic acid  dehydrogenase is found in the work on nitrogen f i x a t i o n by Azotobacter vinelandi ? (27), Clostridium pasteurianum (180). photosynthetic bacteria (161), algae (169), and soybean nodules (181).. Exposures of growing cultures of these agents to N ^ , or to N ' ^ H ^ , for short periods of time resulted in +  the highest s p e c i f i c a c t i v i t y of the isotope being found in glutamate. concentration of  i  n  the glutamic acid was usually two or more times  The  17. that of the nearest compound.  C. pasteurianum c e l l free extracts were  found to follow the same route of nitrogen f i x a t i o n , and the addition of of-ketoglutarate to the reaction mixture reduced the amount of isotope in the ammonia, probably by formation of glutamic acid via glutamic acid dehydrogenase (116).  Virtanen et aJL  (156) also found that yeast c e l l s ,  exposed to ammonium or n i t r a t e salts after a period of nitrogen starvation, synthesized glutamic acid the e a r l i e s t , and the most actively of a l l  the  amino acids. The synthesis of alanine from pyruvate and ammonia has been demonstrated to occur in several species of bacteria and actinomycetes (7,20,58,71),  in yeast  (95),  in plants  (96), and in animals (15,126),  in  situations where transamination reactions between pyruvate and glutamate have been eliminated.  Altenbern and Housewright (7) demonstrated that the  transamination of pyruvate with glutamate played the largest role in the formation of alanine by resting c e l l s of B_. abortus, but that reductive amination of pyruvate occurred to about one-third the amount of transamination.  Washed c e l l suspensions of B_. subt ? 1 is were able to synthesize  alanine from pyruvate and ammonia much more rapidly than glutamate from oC~ketoglutarate and ammonia, according to Fairhurst et al_. (58).  In  Streptomyces species, the primary reaction for assimilation of ammonia was found to be the amination of pyruvate by ammonia, with that of fumarate being of secondary importance (20). oxalacetate could be demonstrated.  No amination of of-ketoglutarate or Burk and Pateman (26) showed that  mutants of N_. crassa lacked glutamic acid dehydrogenase, but possessed an alanine dehydrogenase s p e c i f i c for NADPH, and were able to synthesize alanine when incubated with pyruvate, ammonia, and NADPH.  There have been  several reports by Halvorson and his associates on the presence and func-  18. tion of alanine dehydrogenase in B_. cereus spores (109,122,123). enzyme was found to be able to act vegetative c e l l s , but it  in the direction of synthesis  The in  is probable that the function of the enzyme in  spores is to deaminate alanine, to y i e l d the pyruvate which has been r e ported to be necessary for spore germination.  Although the enzyme has  been found in Rhizobium species, a recent report by Brouvers (25)  indicates  that alanine dehydrogenase levels in these microorganisms are not related to t h e i r nitrogen f i x i n g e f f i c i e n c y .  To date, however, despite the p r o -  l i f e r a t i o n of reports on the presence of alanine dehydrogenase in a number of bacteria, yeasts, and in actinomycetes, there does not appear to have been a case in which the enzyme has been found in a Pseudomonas species. The necessity for energy u t i l i z a t i o n by microbial c e l l s during the assimilation of nitrogenous compounds has been suggested by a number of studies.  For instance, Winzler et aj_. (175) reported that the a s s i m i l a -  tion of ammonia by yeast was dependent both on the b i o t i n content of the c e l l s , and on g l y c o l y s i s ; requiring the presence of glucose in the environment, and being inhibited by azide.  Possibly an expenditure of energy is  required for active transport across c e l l b a r r i e r s , or for endergonic u t i l i z a t i o n of the compound, or both.  McLean and Fisher (110)  calculated  that, with Serrat ia marcescens. approximately 2.2 rnoles of oxygen were consumed per mole of ammonia assimilated.  S. faecal is was found to be  able to u t i l i z e glutamate only in the presence of glucose, or a s i m i l a r energy source (66,67).  Endogenous uptake of added ammonia occurred in M.  tuberculosis. the oxygen consumption in the presence of added ammonia being greater than in its absence (19).  Heating prevented the assimilation  of the ammonia, although oxygen uptake at the level c h a r a c t e r i s t i c of the absence of ammonia continued.  Following a recovery period after heating,  19. assimilation of ammonia started once more, and was accompanied by an i n crease in oxygen uptake.  Hence, an oxidative process appeared to be  coupled to the endogenous assimilation of ammonia in M. tuberculos i s . Bernheim (18) has reported that both an oxidizable substrate and potassium were necessary for the u t i l i z a t i o n of ammonia by f_. aeruginosa under normal conditions, but in the absence of the metal, assimilation could be restored to a s i g n i f i c a n t degree by surface active agents, such as polymyxin B, or behzalkonium c h l o r i d e . There has so far been no resolution of the path of ammonia uptake in microorganisms possessing both dehydrogenases.  However,  in those lack-  ing glutamic acid dehydrogenase, for example, some b a c i l l i  (168),  it would  seem that alanine dehydrogenase must play the major r o l e .  The primary  function of this enzyme in spores of Baci1lus species has been found to be that of providing a supply of pyruvate, which is a requirement for germination.  McCormick and Halvorson (109) have recently reported that the  alanine dehydrogenase of mature spores of B. cereus was heat  resistant.  The preferential and very active p a r t i c i p a t i o n of glutamate in transamination reactions led Braunstein,  in 1957 (23), to attribute to glutamic acid  dehydrogenase and ° f - k e t o g l u t a r a t e , the key positions in ammonia a s s i m i l a tion.  All  indications s t i l l  are that he was probably correct, with pyruvate  amination by alanine dehydrogenase, and the formation of amides, playing supporting roles in some s i t u a t i o n s . III.  Species S p e c i f i c i t y of s-RNA's and Aminoacyl-s-RNA Synthetases Recently,  it has become evident that the process of oxidative  a s s i m i l a t i o n , rather than y i e l d i n g only a carbonaceous product within c e l l , often results  the  in the synthesis of a number of c e l l constituents, in-  20. eluding proteins.  The problem of how a protein is biosynthesized is one  of the most exciting in biochemistry, and one in which rapid advances have been made in the last f i v e years.  Within this f i e l d , the elucidation of  the role played by s-RNA, and how it tion.  is played, is receiving much atten-  The p a r t i c i p a t i o n of s-RNA in protein biosynthesis was f i r s t  dicated, in 1958, by its amino acid accepting a c t i v i t y ,  and its  in-  ability  to transfer the attached amino acid to a ribosomal f r a c t i o n ( 8 3 ) .  This  amino acid acceptor RNA seemed to f u l f i l l the requirements for an "adaptor" between template RNA and protein postulated by Crick (44). time, it  At the present  is generally believed that the biosynthesis of protein proceeds  through the following steps: AAj + Enzj + ATP  s  \  Enzj(AAj— AMP) + PP  Enzj (AMP~AA) + sRNAj *  \  AAj~sRNAi + AAj~sRNAj  (1)  AMP + Enz + A A ; ~ s R N A f  ^  AA;-AAj + sRNAj + sRNAj  (2) (3)  where AAj is a p a r t i c u l a r natural amino a c i d , Enzj is an enzyme s p e c i f i c for the activation of AAj, s-RNA is a molecule of soluble RNA s p e c i f i c for AAj, and AAj-AAj represents the growing peptide chain. catalysed by guanosine triphosphate and ribosomes. Reactions  It  Reaction (3)  is  is now agreed that  (1) and (2) are catalysed by the same enzyme, and s p e c i f i c en-  zymes have been found which activate p a r t i c u l a r amino acids tor form aminoacyl-s-RNA's  (16).  Data have also accumulated which indicate that there  are d i f f e r e n t s-RNA's for d i f f e r e n t amino acids In the last three years,  (132).  interest has been aroused in the  s p e c i f i c reactions between s-RNA's and transfer enzymes.  inter-  Most of the  reports concern comparisons of the heterologous reactions between yeast, mammalian and E. c o l i systems.  It appears that there is often a cross  21. reaction between mammalian enzymes, and yeast s-RNA's, or vice versa, but that yeast and E. c o l i systems generally react poorly, i f at a l l .  The  reaction between the bacterial and mammalian systems is v a r i a b l e .  Thus,  yeast and hog enzymes yielded either yeast or hog tyrosyl-s-RNA, whereas the E. c o l i enzymes reacted only in homologous systems (31).  Mammalian  enzymes catalysed the synthesis of yeast valyl-s-RNA, but not E.  col?  leucyl-s-RNA, according to Z i l l i g et. ah, (182), and this was confirmed by Doctor and Mudd (51).  Benzer and Weisblum (14) reported that there was  l i t t l e or no cross reaction between yeast and JE. c o l i  in the formation of  t y r o s y l - or argininyl-s-RNA, but that there was a cross reaction in the formation of  lysyl-s-RNA.  Interbacterial  crosses for the leucine system, as well as cross  reactions between yeast, rat l i v e r , and £. c o l i . were studied by Rendi and Ochoa (129), who found that enzymes from Lactobacillus arabinosus, Propionibacterium shermanii, S_. faecal i s , and A. vineland? i reacted with the s-RNA of £. c o l i with e f f i c i e n c i e s ranging from 10 to 50% of the homologous system, but did not incorporate the amino acid into the s-RNA's of yeast or rat l i v e r .  There was no cross reaction between the E_. col ?  enzyme system and either the yeast or rat l i v e r s-RNA's or vice versa. According to Doctor and Mudd (51), however, yeast enzyme reacts as well with JE. c o l i and rat l i v e r  leucine s-RNA as it does with its own, suggest-  ing to these authors that the E. c o l i s-RNA used by Rend? and Ochoa might have lost some of its a c t i v i t y during i s o l a t i o n .  This is supported by the  findings of Keller and Anthony (94), who demonstrated that rat l i v e r enzyme incorporated leucine into E. c o l i s-RNA quite r e a d i l y . therefore,  One must,  interpret r e s u l t s , especially those obtained with one amino  acid only, with caution, since either the enzyme or the s-RNA may have  22. been damaged during i s o l a t i o n . Doctor and Mudd (51) have attempted to overcome this problem by a comprehensive study on the i n t e r s p e c i f i c reactions between the s-RNA's and enzymes from yeast, E. col? and rat  l i v e r , for 14 amino acids.  most of the cases studied, there was some reaction, indicating that  In there  seems to be no absolute s p e c i f i c i t y between the enzymes and s-RNA's of a system.  In a d d i t i o n , in some of the cross reactions, the heterologous  system was more active than the homologous one, especially with the rat l i v e r enzymes.  The authors have several suggestions to explain this anom-  alous f i n d i n g :  damage to s-RNA's or enzymes of the homologous system  during i s o l a t i o n ; incorporation of an amino acid into a d i f f e r e n t acceptor RNA ( i . e . , the s p e c i f i c i t y between amino acid and s-RNA is not complete); d i f f e r e n t amounts of s-RNA's  in different species; o r , f i n a l l y , recogni-  tion of more than one component of a p a r t i c u l a r amino acid s p e c i f i c s-RNA by the heterologous system, whereas only one is recognized by the homologous system. That there is more than one component in several s-RNA's c o r r e s ponding to a s i n g l e amino acid is well established. Merrill  Apgar, Hoi ley and  (8) have used counter current d i s t r i b u t i o n to achieve some separa-  tion of some acceptor s-RNA's, while Sueoka and Yamane (147) the aminoacyl-s-RNA's on methylated albumin columns.  fractionated  By these methods,  leucine, isoleucine, s e r i n e , threonine, glutamic acid and valine s-RNA's from E_. col i have been shown to consist of more than one component.  In  addition, Berg et a l .  E.  (17) have demonstrated by enzymatic means that  c o l i methionine s-RNA has at least two components, since yeast enzyme r e acted to only one-third the extent with E. c o l i s-RNA as did the homologous enzymes.  Sueoka and Yamane (148) further reported that, again in E..  23. c o l i , this heterogeneity of s-RNA's for certain amino acids was reproducible, and that the r e l a t i v e amount of each component was rather constant under different growth conditions.  Moreover, in d i f f e r e n t microorganisms  (E. col i, Micrococcus lysodeikt icus. B_. subt i 1 i s . and y e a s t ) , the elution p r o f i l e s of aminoacyl-s-RNA's were d i f f e r e n t , although the elution p r o f i l e of each aminoacyl-s-RNA in an organism tended to resemble that of the same aminoacyl-s-RNA in the others. An interesting experiment also reported by Sueoka (148) has added a complication to our knowledge of the heterologous reaction.  The  leucine s-RNA's resulting from the reaction of the yeast homologous system, and from that of the yeast s-RNA and the E. col? enzyme, were subjected to column chromatography.  The elution p r o f i l e s of the two leucyl-s-RNA's  proved to be e n t i r e l y d i f f e r e n t , although there was a small amount of overlap. since,  However, the s i g n i f i c a n c e of this anomalous reaction is not c l e a r , in a later paper, Yamane and Sueoka (176)  investigated heterologous  systems for other amino acids, and found that leucine was the only compound to react  in this way.  Moreover, the leucyl-s-RNA obtained constituted only  one percent of the normal yeast It  leucyl-s-RNA.  is not yet known whether there is a corresponding enzyme for  each component s-RNA, but this periments of Berg et a l .  is undoubtedly being investigated.  The ex-  (17) reported above, would tend to support mult-  iple enzyme systems, since one would consider that E. c o l i contains two enzymes, one for each methionine s p e c i f i c s-RNA, while the yeast contains only one enzyme, and therefore can react with only one of the component s-RNA's.  Further supporting evidence is found in the experiments of  Doctor and Mudd (51), and of Benzer and Weisblum (14), where, although a cross reaction may work one way, the reverse reaction is often  inactive.  2Z  *.  The existence of several s-RNA's for one amino acid becomes more interesting when t h i s is considered together with the coding problem. Speyer_et_ a_l_. (141), and Nirenberg and coworkers (114,117), have independent evidence for degeneracy in the amino acid code for asparagine, leucine, threonine, and s e r i n e .  Sueoka and Yamane (148) have reported multiple  components for a l l these amino acids except asparagine, which has not yet been investigated by t h e i r methods.  Using the counter current d i s t r i b u -  t i o n technique, Weisblum, Benzer and Hoi ley (166) have found that of two leucine acceptor components in E. c o l i . one responds to poly UC, and the other to poly UG, in the incorporation of leucine into acid insoluble p o l y peptides.  It may be that there are alternate codes for amino acids, and  the one used depends on the activating enzyme which is favoured in a given s ituation. L o f t f i e l d and Eigner (105),  in a recently published paper, take  the opposite view of the s p e c i f i c i t y problem.  Their idea of the s i t u a -  tion is not a multicomponent system with species differences governing the components present, but a system depending on kinetics for s p e c i f i c i t y . In k i n e t i c experiments with s-RNA's and enzymes from yeast and E.  coli.  using valine incorporation as the t e s t , L o f t f i e l d and Eigner reported that, although the rate of the heterologous reaction between the E. c o l i enzyme, and the yeast s-RNA was much slower than that of the homologous system, the Michael is constants were very s i m i l a r .  The lower incorporation at a  given time, then, was due to the slower rate of reaction of the heterologous enzyme-substrate complex to y i e l d valyl-s-RNA. mit that this  These authors ad-  is a consequence of structural differences, but take the  stand that these are not necessarily, or even l i k e l y to be in the enzyme recognition area.  25. Another approach t o s t u d i e s s-RNA's  is through hybrid  deoxyribonucleic acid and  (72)  Rich  (DNA).  Giacomoni that  f o r m a t i o n when Ej_ c o l ?  The r e s u l t a n t  the  s - R N A was s e n s i t i v e t o t h i s  system,  0.024% o f t h e  t o the s-RNA. extents (72)  the  DNA's o f u n r e l a t e d £.  (68),  microorganisms.  E. coli,  but  RNA w i t h t h e  in the  ings t h a t  with  E. col?  the E.  complementary  to different,  i t can s t i l l  be  with  lesser  than  Rich  DNA's o f  the  15% a s much w i t h B_. c e r e u s . B_.  hybridization of  DNA o f E . c o l i .  s-RNA can t r a n s m i t  coli  McCarthy and B o l t o n  (107)  heterolog-  The s-RNA-DNA h y b r i d -  m i c r o o r g a n i s m s by G i a c o m o n i  and  D N A ' s o f P . a e r u g i n o s a and B_.  B . megaterium s-RNA,  i n t o h e m o g l o b i n (159), a n d r e a c t microorganisms,  r e s u l t was o b t a i n e d  With  B_. m e g a t e r i u m ,  homologous B . m e g a t e r i u m s y s t e m .  the  whereas  T h u s , Goodman and  less  and M . l y s o d e i k t i c u s .  who f o u n d t h a t when t h e  megaterium were heated only  (68).  s-RNA h y b r i d i z e d  experiments were extended to other  Spiegelman  (RNase) r e s i s t a n t , A similar  megater?urn  an a n a l o g o u s s i t u a t i o n f o r t h e  DNA a n d m e s s e n g e r  ization  enzyme.  o r g a n i s m s s u c h as  fluorescens,  have r e p o r t e d  a n d Goodman  t h e r e was o v e r 50% as much r e a c t i o n w i t h t h e  E n t e r o b a c t e r i a c e a e as w i t h  abortus,  ous  DNA's o f o t h e r  denatured  reproducible  DNA was f o u n d t o c o n s i s t o f s e q u e n c e s  However, t h e E . c o l ?  found t h a t  other the  with  but  in various  s - R N A a n d DNA w e r e c o m b i n e d a n d  h y b r i d was r i b o n u c l e a s e  w i t h t h e s - R N A and DNA o f B a c i l l u s  heat  a n d S p i e g e l m a n (68)  t h e r e was a s m a l l ,  heated. free  differences  formation with heterologous,  have r e p o r t e d  amount o f h y b r i d  of species  the  hybridization  Therefore,  occurred  despite  g e n e t i c message o f a  the  find-  rabbit  t h e enzyme s y s t e m s o f a number o f  identified  with the  genome o f  its  origin.  26. MATERIALS AND METHODS I.  Oxidative Assimilation Into Whole C e l l s of Aerobic A.  Bacteriological  Bacteria  methods  JP. aeruginosa 120 Na and P. fluorescens A 3.12 were grown in a glucose-ammonium phosphate-salts medium as described by Warren et a l . (163).  The c e l l s were harvested by centrifugation  in the cold after  growth for 20 hr at 30 C, washed twice in cold 0.05 M t r i s methyl)  aminomethane ( t r i s ) buffer  the growth concentration. throughout the t h e s i s .  (hydroxy-  (pH 7.2), and resuspended to ten times  Unless otherwise noted, this buffer was used  This procedure was found to y i e l d a c e l l suspen-  sion containing approximately 5 mg (dry weight) of c e l l s per ml.  When  diluted with 19 volumes of buffer, the suspension gave a reading of 20% transmission at 660 mu in a Beckman model B spectrophotometer.  Accord-  ingly, the other microorganisms used were resuspended so that a 1:20 d i l u t i o n gave the same reading.  This was generally found to correspond  to 5 mg (dry weight) per ml; 1 ml of the suspension of the pseudomonads was used per Warburg v e s s e l .  Dry weights were established by drying 5 ml  of the c e l l suspensions to constant weight at 100 C. Achromobacter B8l was grown and harvested as above.  The e e l l s  were resuspended to 40 times the growth concentration, and 1 ml was used per v e s s e l . Achromobacter viscosus ATCC 12448 f a i l e d to grow in the glucosemineral s a l t s medium used for the other species, but this situation was remedied by the addition of 0.2% yeast extract harvested after  (Difco).  The c e l l s were  17 hr of growth, washed as previously described, and r e -  suspended to 25 times the growth concentration; 2 ml of this suspension  27. were used per v e s s e l . Azotobacter a g i l i s and Azotobacter vinelandi i were obtained through the courtesy of Or. J . Basaraba, Dept. of Soil Science, The Unive r s i t y of B r i t i s h Columbia, and originated from the University of Wisconsin stock culture c o l l e c t i o n .  They were grown in the medium of Warren et  (163), except that 0.5% glucose was used as a carbon source.  ah  The c e l l s  were harvested and washed in the same manner as has already been d e s c r i b ed.  Incubation time for A. agi1 is was 20 hr at 30 C, and for A. vineland i i  hO hr.  A. agi1 is c e l l s were resuspended at 67 times growth concentration,  whereas A. vineland? i required resuspens ion at 100 times growth. Acetobacter aceti ATCC 8303 was cultured in a medium consisting of 2% glucose, 0.2% yeast extract f i n a l pH 6.0.  (Difco), with s a l t s added as above,  The medium was dispensed in 100 ml portions in 500 ml  Florence f l a s k s , and the cultures were incubated for 17 hr on a rotary shaker.  The c e l l s were harvested and washed as before, except that the  buffer used was 0.05 M t r i s , pH 6.5.  The c e l l s were resuspended to 60  times t h e i r growth concentration, and 2 ml were used per Warburg v e s s e l . Acetobacter xylinum ATCC 10245 was grown in the same manner as A. acet i, but the harvesting procedure required modification because of the formation of c e l l u l o s e . tion through several 6.5 t r i s .  The culture was freed of c e l l u l o s e by f i l t r a -  layers of cheesecloth, followed by washing with pH  The c e l l s were then harvested and washed as for A. acet ?, and  resuspended to 100 times growth concentration in fresh buffer.  One ml of  the suspension was used per Warburg v e s s e l . B.  Assimilation studies Manometric experiments were carried out in a Warburg apparatus  at 30 C, using conventional techniques for measuring oxygen consumption.  28. Each v e s s e l of  contained  5 Mmoles o f s u b s t r a t e  ( a p p r o x i m a t e l y 3 t o 3.5 Mc  glucose-U-Cl4 i n r a d i o a c t i v e experiments),  cell  suspension,  3 ml.  a n d 0.05 M t r i s  buffer  the appropriate  (pH 7.2), t o a f i n a l  In t h e c a s e o f t h e t w o A c e t o b a c t e r s p e c i e s ,  was u s e d  i n p l a c e o f t h e pH 7.2 b u f f e r .  was c h o s e n s o t h a t intervals,  t e n t s were p i p e t t e d centrifuged  into  yielded  five  alcohol  s o l u b l e , and r e s i d u a l  C.  cold  Starved c e l l 1.  buffer  in cold  (55), e x c e p t  that  washed t w i c e w i t h s t e r i l e  placed  t h e e x t r a c t i o n w i t h hot This  procedure  a n d h o t 5% t r i c h l o r o a c e t i c a c i d s o l u b l e , ;  lipid,  fractions.  procedure  Twenty hour o l d c e l l s were h a r v e s t e d  in fresh  t u b e s and  experiments  Starvation  concentration  At appropriate  centrifuge  a c i d was c a r r i e d o u t a t 90 C f o r 10 m i n .  fractions:  suspension  From h e r e o n , t h e c e l l s w e r e f r a c t i o n a t e d a c -  c o r d i n g t o Duncan and C a m p b e l l trichloroacetic  i n 120 m i n .  pH 6.5  f o r c h e m i c a l a n a l y s i s , 2 ml o f t h e c u p c o n -  1 ml o f t r i s  immediately.  volume o f  0.05 M t r i s ,  The volume o f c e l l  o x y g e n u p t a k e was c o m p l e t e  t o o b t a i n samples  volume o f  tris,  buffer.  under s t e r i l e  and resuspended  t o t e n times  T w e n t y - f i v e ml o f t h i s  conditions, their  suspension  growth were  i n a s t e r i l e 250 ml E r l e n m e y e r f l a s k c o n t a i n i n g 2 ml o f 20% K0H  in t h e c e n t r e w e l l ,  and shaken  bath  h a v i n g a speed  o f 110-120 s t r o k e s  iod,  the cell  suspension  15 m i n i n t h e c o l d , This c e l l  resuspended  per min.  was r e m o v e d by p i p e t t e ,  washed o n c e w i t h  s u s p e n s i o n was t h e n  also starved  f o r 3 h r a t 30 C i n a L a b i i n e s h a k e r  tris  buffer,  A t t h e end o f t h i s centrifuged  by i n c u b a t i o n w i t h o u t s h a k i n g , b e i n g h a r v e s t e d , as d e s c r i b e d  a t t h e end o f t h e 3 h r p e r i o d .  per-  a t 6000xg f o r  and resuspended  used f o r a s s i m i l a t i o n s t u d i e s .  water  t o 25 m l .  Cells  were  washed, and  29. 2.  Assimilation  experiments  These were performed the waterbath two f l a s k s , U-c'4  i n t h e same t y p e o f f l a s k a n d i n c u b a t e d i n  as o u t l i n e d a b o v e .  A total  c o n s i s t i n g o f 8.3 m l o f c e l l  after  10 m i n e q u i l i b r a t i o n .  with nonradioactive glucose or other Warburg t e c h n i q u e , strate  in a total  was 20 m i n .  chilled  centrifuge  k C.  D.  tube,  1•  of residual  with  radioactive  radioactivity,  0.8x10cm).  by 75 ml o f 1 N NH^OH.  determination  were recovered  glucose, in a  in Section  IB.  fractions f r a c t i o n s was  ampoule.  The hydrb-  i n a v a c u u m d e s i c c a t o r o v e r NaOH a n d C a S O ^ ; an a l i q u o t t a k e n  and t h e r e m a i n d e r  evaporator;  time  i m m e d i a t e l y f o r 15 m i n a t 6000xg  (2 m l ) o f e a c h o f t h e 120-min r e s i d u a l  1 m l o f w a t e r was a d d e d ,  100 m e s h ;  were used, p r e i n c u b a t i o n  w e r e r e m o v e d , a d d e d t o 1 ml o f c o l d t r i s  and c e n t r i f u g e d  l y s a t e was t a k e n t o d r y n e s s  for  When c o f a c t o r s  1 N HC1 f o r h h r a t 108 C i n a s e a l e d  hydrolysed with  experiments,  methods  Analysis  A portion  to  a n d 5 Mmoles o f s u b -  From h e r e o n , t h e p r o c e d u r e was t h a t d e s c r i b e d  Analytical  buffer  The s u b s t r a t e  Parallel  suspension,  At i n t e r v a l s during t h e experiments  2 ml o f t h e f l a s k c o n t e n t s  flash  and t r i s  s u b s t r a t e s , w e r e r u n by t h e u s u a l  u s i n g 1 ml o f c e l l of 3 nil.  i n each o f  0.83 ml o f g l u c o s e -  Two ml o f 20% KOH w e r e a d d e d t o t h e c e n t r e w e l l .  was a d d e d by p i p e t t e  then,  suspension,  (50 u m o l e s p e r m l ) , a p p r o x i m a t e l y 29 u c u r i e s ,  volume.  at  o f 25 m l w e r e p l a c e d  f o r the determination o f  a p p l i e d t o a Dowex-50  (H) c o l u m n (50 t o  T h e c o l u m n was w a s h e d w i t h 50 m l o f w a t e r , Both eluates  were c o n c e n t r a t e d  1 m l o f w a t e r was a d d e d t o e a c h , of radioactivity.  i n t h e two f r a c t i o n s .  followed  t o dryness with a  a n d a s a m p l e was t a k e n  C l o s e t o 100% o f t h e a p p l i e d Both f r a c t i o n s  counts  o f each r e s i d u e  were  30. subjected  to analysis  by p a p e r  chromatography  and e l e c t r o p h o r e s i s ,  r a d i o a c t i v e a r e a s on t h e p a p e r w e r e d e t e r m i n e d on p a p e r  chromatography.  Control  fi - h y d r o x y b u t y r i c a c i d showed t h a t t h e h y d r o l y s i s , and t h a t the  experiments  as d e s c r i b e d  in the  section  c a r r i e d o u t w i t h g l u c o s e and  these substances  b o t h compounds  and  appeared  were not  destroyed  in the water  eluate  by  from  Dowex-50 c o l u m n . 2.  Analysis of cold t r i c h l o r o a c e t i c acid  T h r e e ml p o r t i o n s  of the cold  trichloroacetic acid  The e x t r a c t s ,  of ether to  which were s t i l l  g r a p h y and e l e c t r o p h o r e s i s .  fractions  15 m i n w e r e e x t r a c t e d w i t h  c e l l s w h i c h had m e t a b o l i z e d g l u c o s e f o r s u c c e s s i v e 3 ml p o r t i o n s  fractions  remove t h e t r i c h l o r o a c e t i c  acidic,  were subjected  t o paper  of  four  acid. chromato-  R a d i o a c t i v e a r e a s on t h e p a p e r were  located  by s c a n n i n g . In an e f f o r t acetic  to  i s o l a t e the polymer from the c o l d  a c i d s o l u b l e f r a c t i o n of Achromobacter B81, the contents  l a r g e Warburg v e s s e l s ,  containing a total  15 m i n o n t h e W a r b u r g a p p a r a t u s a t the of  a s s i m i l a t i o n experiments, the t r i c h l o r o a c e t i c acid  from t h i s  concentrated  extraction  repeated.  (pH 5 . 4 ) ,  presence  added t o t h e  tenfold  was c o n c e n t r a t e d by t h e  with  into cold  ether  solution  a t 4 0 C , and t h e  point  extractor ether  (30 t o 35 m l ) w h i c h was  method  (153).  Ethyl  for  (330 m l )  fraction  t o 8 m l , and a s a m p l e t e s t e d  anthrone  as  the  in a l i q u i d - l i q u i d  nine  after  buffer  c a r r i e d through  The s u p e r n a t a n t  by e v a p o r a t i o n  The r e s i d u a l  of carbohydrate  30 C , p i p e t t e d  extraction.  from  162 m l , w e r e removed  of  and t h e p r o c e d u r e  e x t r a c t i o n was e x t r a c t e d  overnight,  acidic  trichloro-  for  still the  a l c o h o l was  r e m a i n d e r o f t h e s o l u t i o n , t o a c o n c e n t r a t i o n o f 70%  (v/v).  T h e f l o c c u l e n t p r e c i p i t a t e w h i c h f o r m e d on s t a n d i n g was c o l l e c t e d by centrifugation,  washed,  and r e d i s s o l v e d  in water.  This opalescent  solu-  31. tion was used for the following t e s t s : reaction with iodine, periodate (by paper chromatography) s t a b i l i t y to a l k a l i after hydrolysis at 100 C, composition as revealed by acid hydrolysis, and s u i t a b i l i t y as a substrate for phosphorylase. 3.  Chemical methods  Glucose in the supernatant f l u i d was determined by the "glucos t a t " method of Worthington Biochemical Corp., Freehold, N.J.;  gluconic  acid by the method of Hestrin (82); and keto acids by the technique of Friedemann (64) as modified by Duncan and Campbell (55).  Derivatives were  prepared from keto acids by use of 2,4-dinitrophenylhydrazine (56).  For  ° ( - k e t o g l u t a r a t e , pyruvate, oxalacetate and glyoxylate, the reaction mixtures were allowed to stand for 4 hr at room temperature; for 2-ketogluconate, the incubation time was 16 hr. Conway microdiffus ion technique 4.  Ammonia was determined by the  (40).  Paper chromatography and electrophoresis  Paper chromatography of the fractions obtained from the supernatant f l u i d s was carried out on Whatman no. 1 paper, by use of secondary butanol-formic acid-water water (EMW; 45:45:10, v/v;  (BFW;  70:10:20, v/v;  120).  130) or ethanol-methanol-  EMW separates glucose and gluconic a c i d ,  whereas BFW distinguishes between Krebs cycle acids.  Gluconic and 2-keto-  gluconic acids were d i f f e r e n t i a t e d by chromatography in ethyl pyridine-sat. aq. boric acid (EPB;  60:25:20, v/v;  73).  acetate-  For chromatography  of the 2,4-dinitrophenylhydrazone derivatives of keto a c i d s , the solvent system used was n-butanol-ethanol-ammonia (70:10:20, v/v; 56).  Paper  electrophoretograms were run for 2 hr in 0.05 M NH^HCO^ (pH 7.7) at 13 v per cm.  For location of compounds, the following reagents were used (138):  amino acids: 0.2% ninhydrin in acetone; sugars and sugar a c i d s : s i l v e r  3?. nitrate-sodium hydroxide-thiosulfate d i p ; organic a c i d s : a n i l i n e xylose d i p ; reducing substances: a n i l i n e phosphoric acid d i p ; and carbohydrates: periodate-benzidine spray. Radioactive areas on paper chromatograms or electrophoretograms were determined by running s t r i p s through a Nuclear-Chicago Model C 100 B Actigraph II,  with a gas-flow counter, a Model 1620 B Analytical  Ratemeter, and a Chart Recorder.  Count  In later experiments, the apparatus was  modified by replacing the gas-flow counter with a Nuclear-Chicago Model 1032 B 4-pi Counter assembly. E.  Isotopic methods These were as described by Duncan and Campbell (55).  Uniformly  labelled g l u c o s e - c ' ^ was diluted so that it had a s p e c i f i c a c t i v i t y of 0.6 to 0.7 He per "mole, and 5 "moles were added per reaction v e s s e l . II.  Inorganic Nitrogen Assimilation by Pseudomonas aeruginosa and Pseudomonas fluorescens Nitrogen assimilation studies were performed in the conventional  Warburg apparatus, using 1 ml of c e l l suspension (prepared as described in Section  I A from 20 hr c e l l s ) per Warburg f l a s k .  used as the source of ammonia.  Ammonium sulphate was  The keto acids, i n h i b i t o r s , and ammonium  sulphate solutions were neutralized to pH 7.2 before use.  Ammonia was de-  termined by the Conway microdiffus ion technique (40), on supernatant  fluids  from the Warburg f l a s k s . III.  Oxidative Assimilation into the Cytological Fractions of Normal. Starved, or Chloramphenicol Treated Cells of £. aeruginosa ATCC 9027  33. A.  Assimilation studies C e l l s were grown for 20 hr at 30 C, harvested, and resuspended  as before (Section I A).  The starved c e l l s were ^prepared as previously  described (Section I C 1), by shaking a 10 times growth suspension under s t e r i l e conditions for 3 hr at 30 C, washing, and resuspending the c e l l s to the same volume in fresh buffer. procedure outlined in Section  For the assimilation studies, the  I C 2 was followed.  Chloramphenicol (200  Hg per ml) was in contact with the c e l l s for 30 min p r i o r to the addition of the substrate.  Oxygen uptake was followed with a Warburg respirometer,  using an appropriate aliquot of c e l l s . At predetermined intervals throughout the experiment (5, 15, 30 and 120 min), 5 ml were removed from each Erlenmeyer f l a s k , added to 2.5 ml of cold t r i s buffer in a c h i l l e d centrifuge tube, and then centrifuged immediately at 6000xg in the c o l d .  The c e l l s from each pair of centrifuge  tubes were pooled, resuspended to 6 ml in t r i s containing 3.7x10  M Mg , ++  an aliquot removed for counting, and the remainder held in an ice bath until the end of the experimental period. B.  Preparation of c e l l  fractions  The c e l l s removed at each time interval were broken separately in a c h i l l e d French pressure c e l l at 15,000 to 17,000 lbs pressure, the extracts collected in cold centrifuge tubes, and 0.1 ml of deoxyribonuclease (ONase) (1 mg per ml) added to reduce their v i s c o s i t y .  The extracts  were s t i r r e d intermittently for f i v e min at room temperature, and then centrifuged twice at 6000xg for 15 min to remove whole c e l l s .  An aliquot  of each extract was removed for counting, and the remainder fractionated by the following scheme:  34.  Cel1 free extract 25,000xg 30 min twice Servall centrifuge  1  1  prec ip i t a t e wash with .05 M t r i s b u f f e r , pH 7.2 resuspend in buffer I precip i t a t e ("Membranes' IX) ("Membranes' 2X)  1 ,  „  supernatants (Washes)  supernatant 40,000xg 30 min Spinco Model L ultracentrifuge No. 40 head I precipitate (40,000xg fraction)  1  supernatant 105,OOOxg 2 hr Spinco Model L ultracentrifuge  1  rr"—  supernatant  prec ip itate (R i bosomes) C.  Analysis of cytological  (Cytoplasm)  fractions  Large scale preliminary experiments were done, in which the fractions were analysed for RNA (orcinol method) (131), DMA (diphenylamine method) (131), glucose oxidizing a c t i v i t y tein  (manometrically),  and for p r o -  (Lowry method) (106), as a means of establishing the purity of v a r i -  ous fractions D.  (30).  Chemical fractionation of the cytological  fractions  The "membranes," ribosomes, and cytoplasm were chemically f r a c tionated as outlined in Section I B. E.  Analysis of chemical fractions 1.  "Membrane" residual  fraction  35. The 30 and 120 min residual fractions from the "membranes" were hydrolysed and subjected to column chromatography as previously described (Section I D 1), except that the dimensions of the Dowex-50 columns were 1.2 x 30 cm.  However, on paper chromatography of the water eluates of  these columns from the 120 min fractions of control and chloramphenicol treated c e l l s , that  it was found that there was so l i t t l e  r a d i o a c t i v i t y present  it was impossible to determine whether these materials were a c i d i c or  neutral.  The water eluates from the 30 min fractions of control and a n t i -  b i o t i c treated c e l l s , and those from both the 30 min and 120 min starved c e l l s , were therefore rechromatographed on Dowex-1 (acetate) columns  (1.2  x 30 cm), which were washed with 80 ml of water (eluted neutral compounds), followed by 80 ml of IN HC1 (eluted a c i d i c compounds).  Each of these e l -  uates was taken to dryness in a f l a s h evaporator, then 1 ml of water was added, and an aliquot counted for its  content.  Paper chromatography  and electrophoresis were carried out as previously described (Section I D 4). 2.  Cytoplasmic cold t r i c h l o r o a c e t i c acid soluble fractions  The cold t r i c h l o r o a c e t i c acid soluble extracts from the c y t o plasmic fractions were ether extracted, and subjected to paper chromatography and electrophoresis as given in Section I D 2. F.  Preparation of P_. aeruginosa s-RNA P. aeruginosa was cultured in a 20 1 bottle containing 17 1 of  medium, which was aerated by a stream of a i r from a pump, the a i r being passed through a s t e r i l e cotton f i l t e r . agitated by means of an overhead s t i r r e r .  In addition, the culture was The medium used consisted o f :  enzymatic casein hydrolysate (Difco) 3%, glucose 0.3%, I^PO^  0.2%,  36. K2HP0 .3H 0 0.3%, FeS0 4  2  4  0.0005%, pH 7.2.  After autoclaving, 10 ml of  s t e r i l e MgS0 .7H20 per 1 of medium was added a s e p t i c a l l y . 4  Antifoam" was added at  intervals as needed.  Sterile "G.E.  The medium was inoculated  with 500 ml of 2k hr c e l l s , and the culture was incubated for 20 hr at approximately 30 C.  The purity of both the inoculum and the bulk culture  was checked by Gram staining and p l a t i n g .  Harvesting was done with a  Sharpies centrifuge at top speed at room temperature.  The c e l l s were  c o l l e c t e d from the c y l i n d e r , washed twice with cold t r i s buffer in a r e frigerated Servall centrifuge, the wet paste weighed, and stored frozen. After being thawed, the bacterial c e l l s  (about 150 g) were  mixed with enough alumina to give a doughy consistency, and were then broken by grinding in a c h i l l e d mortar u n t i l  liquefaction occurred.  Gen-  e r a l l y , this process required about 10 to 15 min.  The mixture was ex-  tracted with s u f f i c i e n t t r i s buffer, containing 1 0  _il  of suspension.  M Mg' to give 400 ml  The viscous suspension was poured into a beaker, 3 mg  DNase added, and the mixture was s t i r r e d at room temperature for 10 min. By this time the v i s c o s i t y was greatly reduced.  Alumina and unbroken c e l l s  were removed by centrifugation twice at 3000xg for 15 min in a refrigerated Servall centrifuge.  The supernatant from this c e n t r i f u g a t i o n , amounting  to between 170 and 200 ml, was then centrifuged for 2 hr. batchwise, at 105,000xg in a Spinco Model L preparative u l t r a c e n t r i f u g e .  The upper two-  thirds of each tube was removed, and stored frozen overnight.  After thaw-  ing, t h i s f r a c t i o n was used for the preparation of s-RNA by the phenol method of T i s s l e r e s (151).  The y i e l d was approximately 1 mg of s-RNA (dry  weight) per 1 g (wet weight) of c e l l s . G.  Assay procedure for incorporation of  amino acids into s-RNA  37. A reaction mixture which contained the following components was prepared: MgCl  2  t r i s acetate buffer 0.5M pH 7.4, 1.5 ml; ATP 0.1M, 0.3 ml;  1M, 0.075 ml; KC1 2M, 0.075 ml; ethyl mercaptan 5%, 0.15 ml;  C h l o r e l l a c'^ amino acid hydrolysate (Merck, Sharpe and Dohme Co. L t d . , Montreal), 0.03 ml (3 He).  This mixture was stored frozen.  The incuba-  t i o n mix consisted of - reaction mixture, 0.15 ml; cytosine triphosphate 0.3M, 0.02 ml; s-RNA 10 mg per ml, 0.05 ml; enzyme, as required. tion was for 30 min at 35 C.  Incuba-  The reaction was stopped by the addition of  0.03 ml of cold 95% ethanol, followed by 1 ml of alcohol-salt solution (2 parts 95% ethanol to 1 part 1.5M NaCl) with vigorous s t i r r i n g after each ad d itio n .  After 10 min in an ice bath, the samples were centrifuged,  the supernatants decanted, 1 ml of alcohol-salt solution added, the mixture s t i r r e d vigorously, allowed to stand 10 min in an ice bath, and then centrifuged.  This washing procedure was repeated twice, the t h i r d wash  consisting of c o l d , 95% ethanol.  The f i n a l p r e c i p i t a t e was dissolved in  0.2 ml of IN NH^OH, and two 0.02 ml portions were plated at thinness for determination of the c'^ H.  infinite  incorporation into the s-RNA.  Isotopic methods These were as previously described (Section I E).  IV.  Species S p e c i f i c i t y of s-RNA's and Amino Acyl-s-RNA Synthetases The microorganisms used in this study were P_. aeruginosa ATCC  9027, JP. aeruginosa 120 Na, P. fluorescens A 3.12, E. col? B, Achromobacter B81, and Saccharomyces cerevis iae (bakers' A.  yeast).  Preparation of s-RNA's The bacteria were grown, harvested, and c e l l extracts prepared  38. as outlined in Section IV A.  For E. c o l i and Achromobacter B81, the con-  centration of glucose in the medium was raised to 1%.  Soluble RNA s were 1  prepared from the 105,000xg supernatant fractions of the c e l l extracts by the phenol extraction method of T i s s i e r e s (151). of Drs. G.M. Tener and B.  Yeast s-RNA was a g i f t  R.V. Tomlinson (13).  Preparation of enzymes The bacteria were grown for 20 hr at 30 C in a glucose-ammonium  s a l t s medium (163).  For JE. col ?, 1% glucose was used, instead of the  0.2% normally employed.  The c e l l s were harvested, washed, resuspended in  buffer, and disrupted by passage through a French pressure c e l l , and the c e l l extracts fractionated as before (Section III  B).  The I05,000xg  supernatant f r a c t i o n s , which were stored at 0 C, were used as the synthetase systems. The yeast enzyme was obtained from Dr. R.V.  Tomlinson, and was  prepared in e s s e n t i a l l y the same way as described for the bacterial enzymes, the s t a r t i n g material being Fleischmann yeast cakes. A l l the enzymes were tested for a c t i v i t y on the same day that they were prepared. their activity C.  As long as they were not frozen, the enzymes retained  for several days in the c o l d ,  Preparation of aminoacyl-s-RNA's cJ^ aminoacyl-s-RNA's were prepared using the assay procedure in  Section  III  G.  For the preliminary experiments, it was found that 0.5-  0.075 mg of S-RNA gave enough incorporation of  to determine the r e -  a c t i v i t y of the systems being tested, but in order to obtain s u f f i c i e n t incorporation so that individual amino acids could be detected, the amount of c'^  added was increased to 12 uc per sample, and an excess of both enzyme  39. (0.05 ml), and s-RNA (1.5 mg) were used. D.  Paper chromatography The amirioacyl-s-RNA's were hydrolysed by allowing the NH^OH  solutions from incorporation experiments to stand at room temperature for 1 hr. after  Two volumes of cold ethanol were then added to each sample, and 10 min in an ice bath, the tubes were centrifuged, decanted, stored  at -15 C, and later used for paper chromatography. made of the r a d i o a c t i v i t y  A determination was  in each of these alcoholic s o l u t i o n s , and it  was found that there was complete recovery of the c'^ present p r i o r to the r e p r e c i p i t a t i o n .  Chromatograms were run, in the descending manner,  on Whatman no. 1 f i l t e r paper, for 30 hr, with nrbutanol: g l a c i a l a c i d : water (120:20:50, v/v) sample of c'^  as the solvent.  acetic  About one-third of each  amino acid was applied to the paper, d r i e d , and then co-  chromatogrammed with a set of amino acid standards.  After development of  the chromatograms, they were cut into s t r i p s , and analysed by an A c t i graph Model 1032B 4-pi scanner. (0.2% in acetone),  The s t r i p s were dipped into ninhydrin  heated at 60 C for 2 min, and the radioactive peaks  were i d e n t i f i e d by comparison with the standards.  The percentage  incor-  poration of each amino a c i d , or group.of amino acids, into the s-RNA was determined by the weight of each peak in the chart, compared to the total weight of a l l the amino acid peaks.  40.  RESULTS AND DISCUSSION I.  Oxidative Assimilation into Whole Cells of Aerobic Bacteria A.  Patterns of oxidative assimilation into strains of Pseudomonas and Achromobacter 1.  Manometric observations, ammonia production and uptake. and excretion of radioactive products into the supernatant f l u i d s during glucose-U-C^ oxidation  f_. aeruginosa 120 Na oxidized glucose rapidly for about 45 min, at which time 58% of the amount of oxygen required for complete oxidation had been consumed (Figure 1A).  Beyond this point, a slow secondary rate  obtained u n t i l 67% of the theoretical value was reached.  Gluconate was  oxidized in s i m i l a r fashion, whereas °<-ketoglutarate was oxidized slowly and after a period of induction.  The r e l a t i v e  values for the o x i d a -  tion of glucose, gluconate, and ° ( -ketoglutarate were 131, 110, and 7.2, respectively.  Glucose had disappeared from the supernatant f l u i d by  15 min, and it would appear that oxygen uptake from this point on was due to oxidation either of intermediates which had been secreted into the supernatant f l u i d , or of assimilated material.  The former explanation  is at least p a r t l y c o r r e c t , since gluconate, pyruvate, and °C -ketoglutarate were present in the supernatant f l u i d at 15 min and gradually decreased in concentration after t h i s time, until a l l had almost completely disappeared by 45 min (Table 1). The break in the oxygen uptake curve at 45 min came, therefore, at a point where exogenous substrate and i n t e r mediates were v i r t u a l l y  exhausted.  No 2-ketogluconate was detected by  treatment of the supernatant f l u i d with 2,4-dinitrophenylhydrazine,  41.  Table 1. Radioactive compounds in the supernatant  f l u i d during glucose oxidation  Compounds present at Microorganism 15-45 min pseudomonas  Gluconate Pyruvate ++ •"C-Ketog1ut arat e+  P.  Glucose++  aeruginosa 120 Na  fluorescens A 3.12  60 min'  120 min*  Succinate++ Fumaratet+  Succinate++ Fumarate++  Gluconate*++ Neutral  Achromobacter B81  A. viscosus  compound A+++ Glucose++ ''C-Ketogl utarate++ Succinate+ Fumarate+ Glucose+++ Neutral  compounds B++  Glucose+ Fumarate (trace) D i carboxylie ac i ds (trace) Neutral Neutral compound B+++ compound B+  Radioactive UV-absorbing material was present 60 and 120 min.  in a l l cases at  hi.  Minutes  . Minutes  FIG. 1 A. Oxygen uptake with 5 F oles of substrate and disappearance of glucose and C14 from supernatant f l u i d s during manometric experiments with washed-cell suspensions of Pseudomonas aeruginosa. Oxygen uptake with glucose, P ; with ° f - k e t o g l u t a r a t e , A ; endogenously, 0 . D i s appearance from supernatant f l u i d s of glucose, • , and jC.14, • . Endogenous oxygen uptake values have been subtracted from the values reported for substrate oxidation. m  FIG. 1 B. Time course of NH3 production, keto acid formation, and cJ4 i n corporation into c e l l s of Pseudomonas aeruginosa during oxidation of 5 umoles of glucose-U-Cl4 by washed-cell suspensions. Production of NH3 endogenously, 0 : and in presence of glucose, • . Keto acid product i o n , Q , and C'4 incorporation into c e l l s , A .  43. followed by extraction, chromatography, and scanning.  Endogenously  produced ammonia was incorporated into the c e l l s on the addition of glucose, and none could be detected in the supernatant f l u i d until glucose and °f-ketoglutarate had disappeared (Figure  IB).  P. fluorescens oxidized glucose in a different manner, there being no sharp break in the curve (Figure 2A).  The i n i t i a l  rate was  slower than that of P. aeruginosa, and decreased at 25 min, at which time 28% of the amount of oxygen required for complete oxidation had been consumed.  Oxygen uptake ceased at 68% of the theoretical t o t a l .  glutarate was oxidized at an i n i t i a l  rate identical to that of glucose,  whereas gluconate was attacked more slowly. gluconate, and  °< -Keto-  The  values for glucose,  -ketoglutarate were 124, 65, and 124, respectively.  As one might conclude  from the manometric data, analyses of the super-  natant f l u i d s showed that gluconate was present and keto acids were absent.  From radioactive scanning of chromatograms and electrophoreto-  grams, it was found that gluconate concentration was highest at 15 min and then gradually decreased, whereas a second product increased in concentration u n t i l 45 min and then disappeared f a i r l y r a p i d l y .  This  second product, designated as neutral compound A in Table 1, had an Rf of 0.14-0.16 in BFW (R  f  of glucose = 0.22), gave a p o s i t i v e s i l v e r  reaction, and did not migrate e l e c t r o p h o r e t i c a l l y .  nitrate  Similar results were  obtained with disaccharides, but attempts to isolate and characterize the unknown compound f a i l e d .  Oxygen uptake after 30 to 45 min, at which  time glucose was exhausted, can be correlated, therefore, with the d i s appearance of these two products, which apparently are oxidized at a slower rate than is glucose.  Endogenously produced ammonia was incor-  porated into c e l l s on the addition of glucose; however, some remained in  FIG. 2 A. Oxygen uptake with 5 H ° J ° f substrate and disappearance of glucose and from supernatant f l u i d s during manometric experiments with washed-cell suspensions of Pseudomonas fluorescens. Oxygen uptake with glucose, © ; with of-ketoglutarate, 4k ; endogenously, 0 . Disappearance from supernatant f l u i d s of glucose, • , and C l 4 0 Endogenous oxygen uptake values have been subtracted from the values reported for substrate oxidation. m  e s  >  FIG. 2 B. Time course of NH3 production and cJ4 incorporation into c e l l s of Pseudomonas fluorescens during oxidation of 5 Fmoles of glucose-U-Cl4 by washed-cell suspensions. Production of NH3 endogenously, 0 ; and in presence of glucose, 0 . cJ^ incorporation into c e l l s , A .  45. the supernatant f l u i d at a l l times (Figure 2B). Achromobacter B81 oxidized glucose at a rapid i n i t i a l  rate  with a break in the curve at 30 min, at which time 28% of the amount of oxygen required for complete oxidation had been taken up (Figure 3A). This was followed by a slower secondary oxidation which ceased at 59% of the theoretical t o t a l .  After a 5 to 7 min period of induction, gluconate  was oxidized r a p i d l y , whereas <K -ketoglutarate oxidation was characterized by a very slow rate of induction and subsequent o x i d a t i o n . for glucose, gluconate, and  The QQ^ values  -ketoglutarate were 101, 86, and 6.7 r e -  spect i v e l y . The main product which accumulated during glucose oxidation was found to be ° f - k e t o g 1 u t a r a t e , which disappeared slowly from the supernatant f l u i d during the interval  between 30 and 120 min (Table 1).  Pyruvate could not be detected in the supernatant f l u i d s obtained at 15 and 30 min.  Succinate, fumarate, and other doubly charged acids  identif-  ied by cochromatography and scanning, were present but did not disappear from the supernatant f l u i d s . was given by the u l t r a v i o l e t  Support for the accumulation of the acids (UV) spectra, which showed a high end  absorption c h a r a c t e r i s t i c of dicarboxylic acids.  Endogenously produced  ammonia was incorporated into the c e l l s on the addition of glucose, and none could be detected in the supernatant f l u i d until glucose and •C-ketoglutarate had disappeared (Figure 3B). h.» viscosus. which required yeast extract for growth, proved to have a delayed oxidation of glucose (Figure 4A).  There was no apparent  secondary o x i d a t i o n , and leveling of the curve occurred at 54% of theoretical oxygen uptake.  Gluconate and ° ( - k e t o g l u t a r a t e were oxidized after  an induction p e r i o d , the latter being oxidized very slowly.  The QQ  46.  FIG. 3 A. Oxygen uptake with 5 pmoles of substrate and disappearance of glucose and from supernatant f l u i d s during manometric experiments with washed-cel1 suspensions of Achromobacter B81. Oxygen uptake with glucose, 9 , with ° ( - k e t o g l u t a r a t e , & ; endogenously, 0 . Disappearance from supernatant f l u i d s of glucose, • , and C ^ , E3 . Endogenous oxygen uptake values have been subtracted from the values reported for substrate oxidation. FIG. 3 B. Time course of NH3 production, keto acid formation, and incorporation into c e l l s of Achromobacter B81 during oxidation of 5 Hmoles of glucose-U-C^ by washed-cel I suspensions. Production of NH3 endogenously, 0 ; and in presence of glucose, © . Keto acid production, • ; and c'^ incorporation into eel Is,A. .  47.  Minutes  Minutes  i FIG. 4 A. Oxygen uptake with 5 (Jmoles of substrate and disappearance of glucose and c'4 from supernatant f l u i d s during manometric experiments wit washed-cell suspensions of Achromobacter viscosus. Oxygen uptake with glucose, 0 ; with q(-ketoglutarate,i& ; endogenously, 0 . Disappearance from supernatant f l u i d s of glucose, • , and C^, M . Endogenous oxygen uptake values have been subtracted from the values reported for substrate oxidation. FIG. 4 B. Time course of NH3 production and C'4 incorporation into c e l l s of Achromobacter viscosus during oxidation of 5 Mmoles of glucose-U-Cl4 by washed-cell suspensions. Production of NH3 endogenously, 0 ; and in the presence of glucose, 0 . c ' ^ incorporation into c e l l s , A .  h8. values for the oxidation of glucose, gluconate, and °C-ketoglutarate were 17, 13, and 2.1, respectively.  Glucose persisted in the supernatant  f l u i d for 60 min, and there was no keto acid or gluconate present at any time.  Comparison of the rates of disappearance of r a d i o a c t i v i t y and  glucose from the supernatant f l u i d revealed that the level of glucose f e l l more r a p i d l y , indicating that some intermediate compound must be accumulating in the supernatant f l u i d .  Chromatography and electrophoresis  of the supernatant fractions revealed the presence of neutral compound B, which increased in amount u n t i l 60 min, and then decreased rapidly (Table 1).  This compound had properties similar to those of compound A  of f_. fluorescens. and attempts at its  isolation also f a i l e d .  After 60  min, traces of fumarate and other acids, in addition to compound B, were detected. The rate of glucose oxidation by this organism was so slow and the endogenous production of ammonia so rapid, that the addition of glucose did not have the pronounced effect on the ammonia content of the supernatant f l u i d that was evident with the other organisms.  However,  A. viscosus appeared to take up the largest amount of ammonia of any of the organisms under study (Figure  kB).  In every instance, some r a d i o a c t i v i t y remained in the supernatant f l u i d at the termination of the experiment.  Most of this could be accounted  for by the increase, with time, of UV-absorbing material.  Viable-cell  counts were not carried out, and so the p o s s i b i l i t y of some c e l l cannot be eliminated.  lysis  In the case of A. viscosus. a comparatively  large  amount of UV-absorbing material was present at the end of the experiment. Incubation of the supernatant f l u i d with DNase or RNase, with concurrent determination of changes in 0D at 260 mu, showed that the UV-absorbing  material was a substrate for RNase only. least  in this  This would suggest that,  at  instance, the increase in UV-absorbing material was due to  the secretion of RNA and not to c e l l  lysis.  A comparison of the results of the oxidation of glucose-U-C^ by resting c e l l s of f_. aeruginosa. f_. fluorescens, Achromobacter B81, and A. viscosus with those obtained by Duncan and Campbell (55), using P. aeruginosa ATCC 9027, shows that there are basic s i m i l a r i t i e s between the bacteria.  With a l l the s t r a i n s , the two stage glucose oxidation c o r r e s -  ponded to the accumulation of intermediate compounds, whose rates of oxidation were l i m i t i n g for the conversion of glucose to C0£ and water. With P. aeruginosa these were pyruvate, gluconate, and ° f - k e t o g l u t a r a t e , with Achromobacter B81 it was ° C - k e t o g l u t a r a t e , whereas with P. it was gluconate.  fluorescens  Both P. fluorescens and A. viscosus also accumulated,  and later oxidized, unknown neutral compounds.  Assuming that a l l  bacteria studied continued to oxidize their endogenous reserves presence of exogenous substrate,  four  in the  (as does P. aeruginosa ATCC 9027 with  glucose (121)), with the resultant production of ammonia, then it would appear that each organism reincorporated some ammonia by way of intermediates of glucose oxidation.  Since f_. aeruginosa and Achromobacter B81  formed °C-ketoglutarate, as shown by its excretion into the supernatant f l u i d , ammonia incorporation could have occurred through the action of glutamic dehydrogenase. glutarate;  P_. fluorescens did not accumulate ° f - k e t o -  however, the rate of oxidation of this compound was similar  to that of glucose, and one could not, therefore, expect it to be in excess.  A. viscosus also f a i l e d to accumulate ° < - k e t o g l u t a r a t e , and  because the rate of oxidation of the keto acid was very slow,  if it had  been produced in excess within the c e l l , and then excreted, it should have  50. accumulated in the supernatant f l u i d during the experiment.  However, the  high u t i l i z a t i o n of endogenously produced ammonia indicated that the rate of removal of °C-ketoglutarate by amination would be s u f f i c i e n t l y rapid to prevent the e x t r a c e l l u l a r accumulation of this keto a c i d .  Despite  the finding that keto acids were not excreted into the medium in a l l cases, each of the bacteria possessed a mechanism for the uptake of ammonia. The excretion of keto acids into the supernatant f l u i d s occurred in the instances where the supply of ammonia from endogenous respiration was low, thus permitting a more e f f i c i e n t u t i l i z a t i o n of ammonia and glucose for synthetic purposes. Although there were s i m i l a r i t i e s among the organisms under study, there were also some differences that are perhaps pertinent to this d i s cussion.  P_. f 1 uorescens and A. viscosus synthesized neutral, low molecular  weight polymeric compounds which were excreted into the supernatant f l u i d s , and, in each case, the compounds were u t i l i z e d when the parent substrate had disappeared, but unlike f_. aeruginosa and Achromobacter B81 they did not excrete keto a c i d s .  Both strains of Achromobacter formed dicarboxylic  acids from glucose, although these compounds did not disappear with time as did the other products detected in the supernatant f l u i d .  The produc-  tion of these acids was also found in several Achromobacter species by Sguros and Hartsell  (133), when the r a t i o of carbon to nitrogen was high,  as it was in the Warburg cup during glucose oxidation. 2.  Incorporation of  j t o c e l l s during oxidative assimilation n  The amount and patterns of incorporation of r a d i o a c t i v i t y c e l l s of the bacteria are shown in Tables 2a, 2b, 3a and 3b.  into  In each case,  the amount of assimilated material was much less than would be expected from the oxygen uptake, a result s i m i l a r to that found in P. aeruginosa  51.  Table 2 a . Incorporation of from 5 "moles of glucose-U-C^ into washed-cel1 suspensions of P. aeruginosa  Time (min)  Coldsoluble*  Lipid  Alcoholsoluble protein  Hot soluble*"  Per cent of total C  l Z f  Residue  Total in fractions  Unfractionated cells  added to vessel  15  2.0  2.0  0.48  0.78  2.6  7.9  7.2  30  2.8  2.7  0.78  1.40  4.5  12.2  11.4  120  2.7  2.9  0.70  1.70  5.6  13.6  12.6  Per cent of total C ^  incorporated into eel 1 fract ions  15  25  25  6.1  10  34  100  30  23  22  6.4  12  37  100  120  20  20  5.6  13.4  41  100  Cold and hot t r i c h l o r o a c e t i c acid soluble f r a c t i o n s .  52.  Table 2b. Incorporation of c'^ from 5 Fmoles of glucose-U-c'^ into washed-cell suspensions of P. fluorescens  Time (min)  Coldsoluble*  Lipid  Alcoholsoluble protein  Hot soluble*  Residue  Total in fractions  Unfractionated cells  Per cent of total C'^ added to vessel  15  5.4  1.9  1.1  1.2  4.8  14.4  14.4  30  5.9  2.5  1.2  1.5  6.4  17.3  17.7  120  5.3  3.0  0.79  1.7  8.5  19.3  19.2  Per cent of total  C * incorporated into eel 1 fract ions ]l  15  38  13  7.4  8.4  33  100  30  34  14  6.9  8.5  37  100  100  27  16  4.2  8.8  44  100  * Cold and hot t r i c h l o r o a c e t i c acid soluble f r a c t i o n s .  53.  Table 3a. Incorporation of C ^ from from 5 5 H "moles of glucose-U-C ^ 1  m(  into washed-cell suspensions of Achromobacter B81  Time (min)  Coldsoluble"  Lipid  Alcoholsoluble protein  Hot soluble'  Per cent of total C  , Z f  Residue  Total in fractions  Unfractionated cells  added to vessel  15  4.3  0.51  0.31  2.6  3.6  11.3  11.0  30  5.4  0.55  0.39  3.6  5.9  15.8  15.5  120  5.3  0.65  0.56  3.3  9.6  19.4  19.4  Per cent of total C ^  incorporated into eel 1 f r a c t ions  15  38  4.5  2.7  23  32  100  30  34  3.5  2.5  23  37  100  120  27  3.4  2.8  17  50  100  " Cold and hot t r i c h l o r o a c e t i c acid soluble fractions  54.  Table 3b. Incorporat ion of C ^ from 5 pmoles of glucose-U-C' into washed-cel1 suspensions of A. viscosus  Time (min)  Coldsoluble" rf  Lipid  Alcoholsoluble protein  Hot soluble*  Per cent of total c'^  Residue  H  Total in fractions  Unfractionated cells  added to vessel  15  6.8  0.29  0.25  0.80  2.0  10.1  30  9.5  0.49  0.50  2.0  3.5  16  16  120  7.0  1.1  0.60  5.0  12.5  26.2  26  Per cent of total C 15  67  30  59  120  27  . 2.9  1 4  incorporated into c e l l  fractions  2.5  7.9  20  100  3.1  3.2  12.5  22  100  4.2  2.3  19  48  100  * Cold and hot t r i c h l o r o a c e t i c acid soluble f r a c t i o n s .  9.5  55.  ATCC 9027 ( 5 5 ) .  The products of glucose oxidation which were excreted  into the supernatant f l u i d s appeared to be important in determining the extent of a s s i m i l a t i o n , not only °<*-ketoglutarate for its  involvement  in  ammonia uptake, but also compounds such as pyruvate, gluconate, and the unidentified neutral substances formed by f_. fluorescens and A. viscosus. There was a c o r r e l a t i o n between the time required for the disappearance of these oxidizable substrates from the supernatant f 1 uJld, and the amount of material assimilated.  In the case of A. viscosus, an unusually high  degree of oxidative assimilation was achieved by a slow rate of glucose oxidation, and the presence of large amounts of endogenously produced ammonia.  The other three organisms oxidized glucose r a p i d l y , but the  accumulation of the "pacemaker compounds" ensured that substrate was available over a prolonged period, thus resulting in the reincorporation of endogenously produced ammonia and a f a i r l y high level of oxidative ass i m i l a t i o n . 3.  Assimilation of C ^ into the c e l l f r a c t i o n soluble in cold t r i c h l o r o a c e t i c acid  The compounds removed from the c e l l s by treatment with t r i c h l o r o acetic acid in the c o l d , and therefore considered to be " p o o l " constituents, made up a rather large proportion of the total the c e l l s .  r a d i o a c t i v i t y present  As might be expected, if oxidative assimilation  in  involved  protein synthesis, labelled glutamate was a usual pool constituent. However, f_. f 1 uorescens did not contain free glutamate, perhaps because it possessed a strong a b i l i t y to oxidize ° f - k e t o g l u t a r a t e , and therefore did not accumulate t h i s keto acid during glucose oxidation.  P.  fluorescens  d i d , however, contain a permease which brought about the passage of a high concentration of glucose into the t r i c h l o r o a c e t i c acid soluble p o o l .  56. Other pool components were neutral amino acids and a npolymer in P. aeruginosa and f_. fluorescens; a polymer in Achromobacter B81; and glucose, amino acids, a neutral compound, and a polymer in A. viscosus.  The  Identity of the polymeric substance present in the ccold t r i c h l o r o a c e t i c acid soluble fractions of each bacterium could not be established, owing to lack of material.  However, from a large-scale, non-radioactive  experiment with Achromobacter B81, evidence was found that, the polymer was carbohydrate in nature, but not glycogen.  in this case, Treatment of  whole c e l l s with the anthrone reagent before and after assimilation of glucose, showed that 5% of the dry weight of the c e l l s was made up of carbohydrate material which did not increase during the experiment. . A similar analysis on the cold t r i c h l o r o a c e t i c acid extract revealed that 20% of the carbohydrate was present  in this f r a c t i o n .  The remaining  carbohydrate was distributed between the hot t r i c h l o r o a c e t i c acid extract and residue.  The alcohol p r e c i p i t a b l e material from the cold t r i c h l o r o -  acetic acid extract reacted with periodate, did not react with iodine, was stable to a l k a l i n e hydrolysis, and was not hydrolysed by phosphorylase. The unit compound of the polymer behaved l i k e glucose when analysed by paper chromatography. react s i m i l a r l y .  However, other hexoses, notably galactose, would  In addition to the polymeric carbohydrate, there was  present, before acid hydrolysis, as well as a f t e r , a periodate-oxidizable compound, having an Rf in BFW s i m i l a r to that of g l y c e r o l . k.  Assimilation of C ** into the c e l l fractions 1  insoluble in  cold t r i c h l o r o a c e t i c acid The assumption that a l l four organisms reincorporated some ammonia during glucose oxidation would seem to be v a l i d , for analysis of the c e l l s by the techniques of Roberts et a h  (130), as modified by  57. Duncan and Campbell (55), revealed that  appeared in a l l  fractions  containing nitrogenous components, as well as the l i p i d containing ones, (Tables 2 and 3).  Moreover, the residual f r a c t i o n containing the  protein was the one which continued to increase in r a d i o a c t i v i t y time, and which, in a l l cases, accounted for almost half the of the c e l l at 120 min.  with  radioactivity  When these residual protein-containing fractions  from c e l l s which had respired for 2 hr were hydrolysed and applied to Dowex-50 (H) columns, some of the r a d i o a c t i v i t y was removed by elution with water (Table 4 ) .  The small amount of radioactive material  Table  in the  k.  Acid hydrolysis and separation by ion exchange of res idual*f ract ion  "Glucose content**  11  Microorgan ism  Eluted by water (neutral compounds)  Eluted by NH4OH  i(ninhydrin • compounds)  %  %  Pseudomonas a e r u g i nosa  0.6  10  90  P.  fluorescens  0.3  7  93  Achromobacter B81  1.0  30  70  A. viscosus  0.5  7.5  insoluble in hot 5% t r i c h l o r o a c e t i c  92.5  acid.  Glucose content was measured by the anthrone t e s t . Results are expressed as per cent of dry weight of eel Is.  58.  water eluates of three of the organisms could have been due to c e l l wall constituents; however,  in the case of Achromobacter B81, 30% of the  residual material was eluted.  This high value agrees with the fact that  this organism synthesized a carbohydrate polymer which was soluble in cold t r i c h l o r o a c e t i c a c i d , as is free glycogen.  The compounds in the  water eluates should be either neutral or a c i d i c in nature; however, paper chromatography and electrophoresis of these eluates never revealed any a c i d i c components.  The sole radioactive peak in each of the water eluates  reacted s i m i l a r l y to glucose upon chromatography or paper electrophoresis of a mixture of the eluate with standard glucose.  However, the systems  used would not distinguish between glucose and galactose. ft -Hydroxybutyrate, which, i f present, should be in this eluate, was never detected. Elution of the columns with ammonia removed 70-93% of the total of the f r a c t i o n s .  Paper chromatography and scanning of the ammonia  eluates showed that a l l radioactive areas were ninhydrin p o s i t i v e .  The  individual amino acids were not i d e n t i f i e d . To a lesser extent than the protein f r a c t i o n , the f r a c t i o n soluble in hot t r i c h l o r o a c e t i c a c i d , which contained primarily nucleic acids, also increased with time. high percentage of the  A. viscosus assimilated an unusually  of glucose, a considerable amount of which was  due to its containing a s u r p r i s i n g l y large amount of radioactive material soluble in cold or hot t r i c h l o r o a c e t i c a c i d . radioactivity  The r e l a t i v e amount of  in the hot t r i c h l o r o a c e t i c acid soluble fractions doubled  between 15 and 120 min, and accounted for 19% of the total c e l l s at the completion of the experiment.  of the  Achromobacter B81 also as-  similated a large percentage of the added r a d i o a c t i v i t y  into the nucleic  59. acid f r a c t i o n , but in t h i s portant  instance the f r a c t i o n appeared to be most im-  in the early stages of oxidative a s s i m i l a t i o n . In confirmation of the observations of Duncan and Campbell  (55),  the l i p i d of P. aeruginosa appeared to be of s i g n i f i c a n c e in the early stages of oxidative a s s i m i l a t i o n .  This suggestion is also true for Achromo-  bacter B81, but not for the other two organisms. The pattern of assimilation of glucose found in these e x p e r i ments was s i m i l a r to that reported recently by Duncan and Campbell (55) C l i f t o n (36),  i.e.,  the r a d i o a c t i v i t y appeared f i r s t  and  in the cold t r i c h l o r o -  acetic acid soluble components, from where it was distributed into the other f r a c t i o n s .  A l l four bacteria incorporated a large proportion of the  assimilated carbon into nitrogenous c e l l components, of which protein contained most of the C'4  in each case.  The concept (55)  that oxidative a s -  s i m i l a t i o n occurred by way of reincorporation of endogenously produced ammonia was found to be tenable.  The two Achromobacter species a s s i m i l -  ated a high proportion of the r a d i o a c t i v i t y at the expense of the l i p i d .  into the nucleic acid f r a c t i o n s ,  This is p a r t i c u l a r l y  interesting in the case  of A. viscosus. since this organism w i l l not grow in an inorganic s a l t s medium with glucose as the sole source of carbon and without organic nitrogen.  Growth experiments with A. viscosus showed that both amino acids  and vitamins were required to replace the yeast extract used in the growth medium for assimilation experiments.  Despite the fact that this micro-  organism cannot grow in the inorganic medium, resting c e l l s with no added vitamins or amino acids exhibited the highest amount of oxidative a s s i m i l a tion of any organism studied. The only bacterium investigated which appeared to have a primary storage product was Achromobacter B81.  This conclusion is supported by  6p. the r a t i o (10.6) of endogenous oxygen uptake to ammonia production, which is a figure twice that normally obtained with obligate aerobes, and s i m i l a r to that found by Gronlund and Campbell (75) with E. c o l i .  In addition,  the apparent incorporation of ammonia by Achromobacter B81 was very low in relation to the amount of material assimilated.  Analysis of the cold  t r i c h l o r o a c e t i c acid-soluble pool in the early stages of glucose oxidation, and the f r a c t i o n insoluble in hot t r i c h l o r o a c e t i c acid at the end of the experimental period, revealed that the storage product was a polymeric carbohydrate that was not identical to glycogen. B.  Patterns of oxidative assimilation into strains of Acetobacter and Azotobacter 1.  Manometric observations, ammonia production, and excretion of radioactive products into the supernatant f l u i d s during qlucose-cl4 oxidation  Acetobacter aceti oxidized glucose slowly, and for the most part only to the gluconic acid stage.  A total of 68 Ml of oxygen were taken  up, while a value of 56 Ml would indicate complete conversion of gluconate (Figure 5A).  Gluconate was not oxidized by whole c e l l s of this organism,  and 91% of the added c'4 remained in the supernatant f l u i d at the end of the experiment.  Moreover, more than 4 Mmoles of gluconic acid were present  in the supernatant f l u i d at 120 min (Table 5).  The production of almost  stoichiometric amounts of gluconate from glucose by A. acet ? was also found by De Ley and Schell  (48).  The oxygen uptake value, the amount of  radioactive material which disappeared from the supernatant f l u i d , and the s p e c i f i c quantitative analysis for gluconate, indicated that approximately 10% of the glucose was oxidized beyond the gluconic acid stage.  This  61.  Table 5. Radioactive compounds in the supernatant f l u i d during glucose oxidation  Compounds present at Microorgan ism  15-45 min  60  min  120  min''  Acetobacter aceti  Glucose*** Gluconate***  Glucose* Gluconate****  Gluconate****  Acetobacter xy1inum  Glucose++ Gluconate***  Glucose* Gluconate* Cellulose*  Gluconate* Cellulose**  Azotobacter agi1 is  Glucose*  Azotobacter vinelandii  Glucose*  " Radioactive UV-absorbing material was present at 60 and 120 min.  in a l l  cases  62.  B  A  500J  I  0  0  _I00  F —  0}  80  •g 400. Q. g  T3  « .2.0  T3 O  300.  60 2 o  200.  40  100.  20  30  60  90  30  Minutes  6.0'  1.5  1o 3 "O O  O.  X  0.5  90  Minutes  FIG. 5 A. Oxygen uptake with 5 Hmoles of substrate and disappearance of glucose and Cl4 from supernatant f l u i d s during experiments with washedc e l l suspensions of Acetobacter a c e t i . Oxygen uptake with glucose, 0 ; endogenously, 0 . Disappearance of glucose,Q,and CJ^, from supernatant f l u i d s . Endogenous oxygen uptake values were subtracted from the values reported for glucose oxidation. FIG. 5 B. Time course of NH3 production and incorporation into c e l l s of Acetobacter aceti during oxidation of 5 H ' of glucose-U-C^ by washedc e l l suspensions. NH3 production endogenously, 0 ; NH3 production in presence of glucose, 0 ; Cl4 incorporation into c e l l s , & . m o  e s  63. could have been by pathways not involving gluconate, for whole c e l l s and c e l l extracts, prepared with a 10 Kc Raytheon sonic o s c i l l a t o r , f a i l e d to oxidize this compound,  -Ketoglutarate was oxidized at almost the same  rate as glucose, the Qn^'s for glucose, gluconate and of*-ketoglutarate being 19, 0, and 15, respectively.  There were traces of glucose present  in the reaction mixture at 90 min, and it would appear that the two stage oxidation with A. acet i was due to a decrease in the rate of glucose u t i l ization.  There was no obvious reason for the change in rate of glucose  disappearance, since gluconolactone, which would be in enzymatic e q u i l i b rium with the glucose, and thus might slow the rate of its oxidation, was not detected.  There were no keto acids excreted into the medium.  the very low endogenous oxygen uptake, ammonia production was high (Figure 5B).  Despite  relatively  However, the presence of glucose did not result  in  ammonia reincorporation; in f a c t , ammonia production appeared to be s l i g h t ly stimulated by glucose.  Not s u r p r i s i n g l y , only 2% of the  radioactivity  was incorporated into the c e l l s . The observation that ammonia was not reincorporated by A. acet ? was to be expected, since glucose was oxidized only as far as gluconate, and therefore no of-ketoglutarate would have been available for reductive amination to glutamate.  Moreover, experiments on ammonia uptake during the  oxidation of «C-ketoglutarate and pyruvate by resting c e l l s of A. acet i showed that, although both keto acids were oxidized, the amount of ammonia was the same in the presence and absence of these substrates.  Therefore,  one can conclude that ammonia cannot be reincorporated into the c e l l  even  when the substrate is being oxidized by way of the t r i c a r b o x y l i c acid cycle.  This finding is in agreement with the fact that A. acet ? required  an organic nitrogen source for growth, apparently because it was unable to  Sh. u t i l i z e inorganic nitrogen for the synthesis of organic compounds. In contrast to A,, acet ?, Acetobacter xyl inum oxidized glucose at a rapid, constant rate, u n t i l the break in the curve at 90 min, when 80% of the theoretical of glucose was achieved  amount of oxygen required for complete oxidation (Figure 6A).  Gluconate and ° C - k e t o g l u t a r a t e were  oxidized by induced enzymes, the Qn^'s for glucose, gluconate and ° C - k e t o glutarate oxidation being 78, Ih, detected  and 39, respectively.  The products  in the supernatant f l u i d during glucose oxidation were gluconic  a c i d , which increased u n t i l 30 min, and then decreased slowly, and c e l l ulose, which was produced in quantity after 60 min (Table 5 ) .  It  appeared  that glucose and gluconate were being oxidized simultaneously, for some glucose was present until 60 min. medium.  No keto acids were detected in the  The endogenous oxygen uptake followed a most unusual pattern,  there was a rapid i n i t i a l  rate, but a l l oxidation soon stopped.  result was obtained repeatedly.  The same  Despite the fact that A. xylinum required  a complex nitrogen source for growth, endogenously produced ammonia appeared to be reincorporated during the oxidation of glucose, without, the excretion of keto acids  into the medium (Figure 6B).  however,  This is a s i m i l a r  s i t u a t i o n to that found with Achromobacter viscosus. The oxidation of glucose by Azotobacter vinelandii proceeded at a very rapid rate without the accumulation of any intermediate product  in  the supernatant f l u i d (Table 5, Figure 7A). However, there was a secondary rate of oxidation which began after 67% of the oxygen for complete oxidation of glucose had been consumed, and continued l i n e a r l y until  the  experiment was terminated at 120 min, at which time 89% of the theoretical oxygen uptake had been reached.  Part of this oxygen uptake could have  been due to the oxidation of glucose which was found in the metabolic pool,  65.  Minutes  Minutes  FIG. 6 A. Oxygen uptake with 5 Rnoles of substrate and disappearance of glucose and Cl4 from supernatant f l u i d s during experiments with washedc e l l suspensions of Acetobacter xylinum. Oxygen uptake with glucose, # ; endogenously, 0 . Disappearance of glucose, Q , and C ^ , • from supernatant f l u i d s . Endogenous oxygen uptake values were subtracted from the values reported f o r glucose o x i d a t i o n . FIG. 6 B. Time course of NH3 production and C ' 4 incorporation into c e l l s of Acetobacter xyl inum during oxidation of 5 Mmoles of glucose-l)-Cl4 by washed-cell suspensions. NH3 production endogenously, 0 ; ; NH3 production in presence of glucose, • ; incorporation Into c e l l s , A .  66.  Minutes  Minutes  FIG. 7 A. Oxygen uptake with 5 Hmoles of substrate and disappearance of glucose and from supernatant f l u i d s during experiments with washedcel 1 suspensions of Azotobacter v i n e l a n d i i . Oxygen uptake with glucose, 0 ; endogenously, 0 . Disappearance of glucose, Q , and C ^ , g§§ from supernatant f l u i d s . Endogenous oxygen uptake values have been subtracted from the values reported for glucose oxidation. FIG. 7 B. Time course of NH3 production and C^^ incorporation into c e l l s of Azotobacter v i n e l a n d i i during oxidation of 5 Hmoles of glucose-U-C^ by washed-cel1 suspensions. NH3 production endogenously, 0 ; NH3 production in presence of glucose, 6 ; Cl4 incorporation into c e l l s , & .  67. but for the r e s t , one can only conclude that the presence of glucose allowed the organism to draw on some previously acquired reserve material, for this secondary rate of oxidation was considerably in excess of the normal rate of endogenous r e s p i r a t i o n . of r a d i o a c t i v i t y  Yet, there was no detectable  from either the c e l l s or from the supernatant f l u i d  loss after  45 min, although enough oxygen was consumed to oxidize completely 1 umole of glucose during this p e r i o d .  While this secondary oxidation was going  on, A. vineland? ? did not release any ammonia, thus indicating that the reserve material being oxidized was non nitrogenous (Figure 7B). Gluconate was oxidized by induced enzymes, whereas arate oxidation proceeded at a slow, steady rate.  The Qo^'s  °<-ketoglutfor glucose,  gluconate, and «C-ketoglutarate oxidation were 180, 20, and 23, respectively.  There appeared to be ammonia taken up, because there was a decrease  in the amount of ammonia in the early stages when glucose was present. Since the presence of an available energy source such as glucose w i l l  in-  crease the f i x a t i o n of nitrogen by this organism, the ammonia incorporated during glucose oxidation may be much greater than is apparent from Figure 7B. Azotobacter agi1 is also oxidized glucose r a p i d l y , but without a secondary rate of oxidation (Figure 8A).  Again there was very l i t t l e a s -  s i m i l a t i o n of the glucose supplied, for at 120 min 83% of the theoretical amount of oxygen for complete oxidation had been consumed.  Both gluconate  and « ^ - k e t o g l u t a r a t e were oxidized slowly and by induced enzymes.  The  Qn^'s for glucose, gluconate, and ©C-ketoglutarate were 220, 43, and 8.6, respectively.  There was a decrease of ammonia in the medium during the  early stages of glucose oxidation, suggesting that ammonia was being i n corporated into c e l l u l a r material  (Figure 8B).  However, A. ag?1 is ass?m-  68,  FIG. 8 A. Oxygen uptake with 5 Hmoles of substrate and disappearance of glucose and Cl4 from supernatant f l u i d s during experiments with washedc e l l suspensions of Azotobacter a g i l i s . Oxygen uptake with glucose, © ; endogenously, 0,. Disappearance of glucose, • , and C14, HI from supernatant f l u i d s . Endogenous oxygen uptake values have been subtracted from the values reported for glucose oxidation. FIG. 8 B. Time course of NH3 production and C.14 incorporation into c e l l s of Azotobacter a g i l i s during oxidation of 5 Hmoles of glucose-U-C^ by washedc e l l suspensions. NH3 production endogenously, 0 ; NH3 production in presence of glucose, 0 ; C^4 incorporation Into c e l l s ,  69. ilated only 5% of the of ammonia available to 2.  into c e l l u l a r material,  in s p i t e of the excess  it.  Incorporation of  into c e l l s during oxidative assimila-  t ion The amount and patterns of the incorporation of are shown in Tables 6 a , 6b, 7a and 7b.  from glucose  The amount of assimilated material,  unlike that in most cases in the previous s e c t i o n , corresponded f a i r l y well to that which would be predicted by the oxygen uptakes.  A. vineland? i.  for example, incorporated 11% of the added r a d i o a c t i v i t y , while oxidizing glucose to 89% of the theoretical amount.  None of these b a c t e r i a , however,  excreted compounds into the medium which could be considered to act as "pacemakers' for oxidative a s s i m i l a t i o n , since the gluconate produced by 1  A. acet i. and the c e l l u l o s e by A. xylinum were not metabolized f u r t h e r . Although ammonia was found to be the l i m i t i n g factor in the experiments of Duncan and Campbell (55) with P. aeruginosa, this could not have been the case with Azotobacter species, which are able to f i x nitrogen, and yet they exhibited a very low rate of glucose a s s i m i l a t i o n .  The reason for  t h i s apparent anomaly may be that the energy requirements for nitrogen f i x a t i o n are great enough to limit oxidative assimilation severely.  With  these b a c t e r i a , therefore, the degree of assimilation may be a function of t h e i r e f f i c i e n c y of f i x i n g nitrogen, and it  is true that A. vineland i i  which is known to f i x nitrogen very e f f i c i e n t l y , and produced large amounts of ammonia during glucose oxidation, assimilated much more r a d i o a c t i v i t y than did A. a g i l i s . 3.  Incorporation of C ^ into the c e l l fractions soluble in cold t r i c h l o r o a c e t i c acid  The compounds extracted from the c e l l s by treatment with cold  70  Table 6a. Incorporation of c'^ from 5 H Hmoles of glucose-U-c'^ *™ into washed-cel1 suspensions of A. acet i  Time (min)  Coldsoluble*  Lipid  Alcoholsoluble protein  Hot soluble*  Residue  Total in fractions  Unfractionated cells  Per cent of total C * added to vessel ]l  15  1.9  0  0  0  0  1.90  1.9  30  1.9  0.02  0  0.02  0  1.94  2.0  120  2.2  0.02  0.01  0.02  0.02  2.29  2.5  Per cent of total C  I H  incorporated into c e l l  fractions  15  100  0  0  0  0  100  30  98  1  1  0  0  100  120  96  1  1  0.5  0.5  100  * Cold and hot t r i c h l o r o a c e t i c acid soluble f r a c t i o n s .  71  Table 6b. Incorporation of c'^ from 5 Mmoles of glucose-U-C^ into washed-cel1 suspensions of A. xylinum  Time (min)  Coldsoluble*  Lipid  Alcoholsoluble protein  Hot soluble*  Per cent of total C  I H  Residue  1.5  0.58  0.20  0.90  5.1  30  2  0.87  0.30  1.29  8.3  2.3  1.7  0.64  1.88  Per cent of total  C  ]k  Unfracr tionated cells  added to vessel  15  120  Total in fractions  22  8.28  8.10  12.7  12.5  28.5  29  incorporated into c e l l fract ions  15  18.1  7.1  2.4  11.9  60.5  100  30  15.5  6.8  2.4  10.1  65.2  100  120  8.1  6.0  2.2  6.5  77.2  100  * Cold and hot t r i c h l o r o a c e t i c acid soluble f r a c t i o n s .  72.  .Table 7a. Incorporation of c'^ from 5 Mmoles of glucose-U-C into washed-cel1 suspensions of A. vinelandi ?  Time (min)  Coldsoluble*  Lip id  Alcoholsoluble protein  Hot soluble'*  Res idue  Total in f r a c t ions  Unfract ionated cel Is  Per cent of total C ^ added to vessel 15  3.6  0.50  0.24  0.21  0.8  5.35  5.2  30  5.0  1.48  0.56  0.59  2.1  9.83  10.2  120  3.1  2.06  0.60  1.69  4.2  11.65  11.7  Per cent of total C  1 4  incorporated into cel1 f r a c t ions  15  67  9.5  4.5  3.9  15  100  30  52  15.3  5.7  6.0  21  100  26.5  17.7  5.3  14.5  36  100  120  Cold and hot t r i c h l o r o a c e t i c acid soluble f r a c t i o n s .  73.  Table 7b. Incorporation of  from 5 5 W H<oles of glucose-U-c'^ from m m  into washed-cell suspensions of A. ag?1 is  Time (min)  Coldsoluble*  Lipid  Alcoholsoluble protein  Hot soluble*  Residue  Total in fractions  Unfractionated cells  Per cent of total c'^ added to vessel 15  3.0  0.20  0.10  0.15  0.20  3.65  3.5  30  3.1  0.46  0.34  0.50  0.80  5.20  5.2  120  2.2  0.55  0.55  0.65  1.25  5.20  5.3  Per cent of total C ^ incorporated into c e l l 1  fractions  15  82  5.6  2.7  4.1  5.5  100  30  59.5  8.9  6.6  9.6  15.4  100  10.7  10.7  12.6  24  100  120  42  •A.  " Cold and hot t r i c h l o r o a c e t i c acid soluble f r a c t i o n s .  74.  5% t r i c h l o r o a c e t i c acid accounted for e s s e n t i a l l y a l l of the label of the c e l l s of A. aceti  (Table 6a), and a major amount of the r a d i o a c t i v i t y  in A. agi1 is and A. vinelandi ? (Tables 7a and 7b).  Radioactive neutral  amino acids were present in a l l four organisms, and glutamate was demonstrated in both Azotobacter species.  With the exception of A. a g i l i s .  a l l contained some free glucose at the end of the 2 hr experiments.  In  addition, pools from the Azotobacter species and A. xylinum contained polymeric compounds, and those from the Acetobacter species contained gluconic a c i d . 4.  Incorporation of  into the c e l l fractions  insoluble in  cold t r i c h l o r o a c e t i c acid In each of the bacteria except A. a c e t i . r a d i o a c t i v i t y was d i s tributed among a l l the c e l l f r a c t i o n s .  The two Azotobacter strains ex-  hibited patterns of assimilation very similar to those observed with Pseudomonas species (54,55,152), B. cereus (35), and A. ag?1 is  (140).  Hydrolysis and column chromatography of the residual fractions of c e l l s which had been respiring for 120 min, revealed a c o r r e l a t i o n between the amount of "glucose" as shown by the anthrone reagent, and the percentage of counts eluted with water (Table 8 ) .  The compounds eluted in this man-  ner would be either neutral or a c i d i c in nature, but paper chromatography in two solvent systems, as well as electrophoresis, showed no a c i d i c compounds; the radioactive peaks cochromatogrammed with glucose only. The lack of a c i d i c components in the residual fractions would indicate that poly- /3-hydroxybutyrate was not synthesized during the assimilation experiments, although Sobek and C l i f t o n (140) found low levels of this polymer in A. a g i l i s .  Radioactive amino acids were the constituents of  the ammonia eluates from the columns (Table 8 ) .  Both of the Azotobacter.  75.  Table 8. Acid hydrolysis and separation by ion exchange of r e s i d u a l * f r a c t i o n  Microorgan ism  Per cent Cl4 incorporated  "Glucose" (per cent dry wt)**  Azotobacter aqi1 is  23  0.4  A. vinelandii  3h  1.0  Acetobacter aceti A. xylinum  *  therefore,  0.1 80  Per cent total counts eluted H0  NH^OH  2.5  97.5  2  20  0.3 3.8  80  -  -  20  80  Insoluble in hot 5% t r i c h l o r o a c e t i c As measured by the anthrone t e s t .  acid.  incorporated the major portion of the assimilated material  nitrogenous compounds, including p r o t e i n s .  into  The suggestion of Duncan and  Campbell (55) that oxidative assimilation of glucose in P_. aeruginosa was largely the reincorporation of endogenously produced ammonia can be extended to these Azotobacter species, as well as to the two pseudomonads and the Achromobacter strains previously discussed (152).  Since the incorporation  of ammonia by Azotobacter species into organic material  is known to occur  through glutamate (6), and in these experiments glutamic a c i d - c ' 4 was found in the metabolic p o o l , ammonia was probably assimilated v i a the ° ( - k e t o g l u t a r a t e formed from glucose. The small amount of c e l l u l a r material  insoluble in cold t r i -  chloroacetic acid confirmed the conclusion that A. acet? did not incorporate measurable amounts of ammonia into the c e l l s  (Table 7a).  One must  76,  conclude, therefore, that A. acet? did not exhibit oxidative assimilation when glucose was the substrate, since the c'^ entirely  incorporated was almost  in the pool components, in the form of free glucose and gluconate. The a b i l i t y of A. xylinum resting c e l l s to synthesize c e l l u l o s e  which accumulates e x t r a c e l l u l a r l y , complicated the study of oxidative a s s i m i l a t i o n in this microorganism.  Column chromatography of the hydro-  lysed residual f r a c t i o n revealed that 80% of the r a d i o a c t i v i t y was associated with neutral compounds, which on paper chromatography and e l e c t r o phoresis proved to be glucose and c e l l o b i o s e .  Moreover, a large propor-  tion of the unhydrolysed material was insoluble in water, a c i d , or base, being soluble only in Schweitzer's  reagent.  These observations lead to  the conclusion that c e l l u l o s e was the major component of the residual fraction.  This does not mean that the c e l l u l o s e was formed  intracellular-  l y , because e x t r a c e l l u l a r f i b r i l s would also be in this f r a c t i o n .  The  release of radioactive amino acids by the hydrolysis of the residual f r a c t i o n indicates that protein was synthesized by A. xylinum. also r a d i o a c t i v i t y  There was  incorporated into other nitrogen containing f r a c t i o n s ,  such as the alcohol soluble p r o t e i n , and nucleic acids.  It can be c a l -  culated that about 11% of the added C ^ was assimilated into c e l l material, 1  and of t h i s about half  is nitrogenous, the result of the reincorporation  of endogenously produced ammonia.  C.  Oxidative assimilation by starved c e l l s of P. aeruginosa ATCC  1.  Manometric observations  Starvation of resting c e l l s of P. aeruginosa for 3 hr was found to decrease t h e i r rate of glucose d i s s i m i l a t i o n , perhaps because it slowed  77. down the rate of pyruvate oxidation (Figure 9). glutarate was unaffected.  Oxidation of ©(-keto-  This decrease in the rate of pyruvate oxidation  occurred with c e l l s which had been starved with or without shaking, but shaking gave a more pronounced e f f e c t .  With the shaken c e l l s , the extent  of decrease in pyruvate oxidation was found to vary from experiment to experiment, and probably reflected the state of the c e l l s on harvesting, although conditions were standardized as much as p o s s i b l e .  There was no  stimulation of the rate of oxidation of pyruvate when thiamine pyrophosphate (0.5 Hmole per Warburg f l a s k ) was added.  Since glucose oxidation  was more affected when c e l l s were starved with shaking than without, shaken, starved c e l l s were used in the experiments with glucose-U-C^.  |  n  the ex-  periment quoted below, the oxygen uptake at 120 min with starved c e l l s was 80% of the theoretical  value for the complete oxidation of glucose, as  compared to 67% for freshly harvested  (control) c e l l s .  This higher oxygen  uptake would indicate a lower assimilation of glucose by starved c e l l s . 2..  Ammonia excretion and uptake  When the amount of ammonia excreted into the medium was determined, it was found that shaking the c e l l s had greatly Increased endogenous ammonia production (Table 9, Figure 10).  Moreover, although both shaken  and non shaken c e l l s reincorporated ammonia during glucose oxidation, the amount and the rate of uptake d i f f e r e d greatly.  The curve of ammonia up-  take for the non shaken c e l l s p a r a l l e l e d that for control c e l l s , except that less ammonia was assimilated, not a l l of that available being u t i l i z e d (Table 9, Figure 10).  The c e l l s which had been shaken for 3 hr reincorpor-  ated more ammonia in the f i r s t 5 min of glucose oxidation than did the others, but the calculated uptake then declined, apparently because of a decrease in the rate of endogenously evolved ammonia.  This decrease resulted  Table 9 Ammonia production and uptake by previously starved c e l l s of Pseudomonas aeruginosa during glucose oxidation  Time  Non shaken c e l l s NH3 present per vessel Endogenous  Min  Mmoles  NH3 uptake (calculated)  Glucose umol es  Shaken eel ls NH3 present per vessel  NH3 uptake (calculated  Endogenous  Glucose  umoles  umoles  Mmoles  0.200 0.120 0.140 0.155 0.470  0 0.355 0.350 0.345 0.210  Starvat ion per iod 0 180  0.010 0.080  0.010 0.550  Ass imi1 at ion period 0 5 15 30 120 Total endog NH3 prod'n  0.200 0.225 0.310 0.450 1.100 1.180  0.200 0.110 0.120 0.180 0.320  0 0.115 0.190 0.270 0.780  0.200 0.475 0.490 0.500 0.680 1.230  00  20  40  60 80 minutes  100  120  FIG. 9. Oxygen uptake during oxidation of 5 Hmoles of . pyruvate or glucose by control and starved washed c e l l suspensions of Pseudomonas aeruginosa. . r  80.  20  4.0  60 80 minutes  100  120  minutes  FIG. 10. Production and uptake of NH3 by washed, starved c e l l s of Pseudomonas aeruginosa.  81. in the total amount of endogenously produced ammonia being very nearly equal for the two types of c e l l s . 3.  Excretion of radioactive products into the supernatant fluids  Since pyruvate oxidation was impaired by starving the c e l l s ,  it  is not surprising that large amounts of the keto acid accumulated in the supernatant f l u i d s during glucose oxidation (Figure 11).  In the e x p e r i -  ment where shaken, starved c e l l s oxidized glucose-cJ^, k umoles of pyruvate were present at 30 min, as compared to 2.6 umoles when freshly harvested c e l l s were used.  In a d d i t i o n , chromatography and electrophoresis of the  supernatant f l u i d s , followed by scanning, revealed the presence of "cf-ketoglutarate, gluconate, and 2-ketogluconate.  The sugar acids were most highly  labelled at 5 min, whereas pyruvate and °C-ketoglutarate contained the highest amount of  at 30 min.  This is a pattern s i m i l a r to that found with  control c e l l s , although in this highest at 15 min.  instance, the pyruvate concentration was  There was very l i t t l e °C-ketoglutarate present in the  Warburg supernatants; the r a t i o of optical density readings at:435mu and 390mjj was 2.15 in the keto acid assay throughout the experiment which is c h a r a c t e r i s t i c of pyruvate.  The r a t i o for ° f - k e t o g l u t a r a t e was 1.05.  How-  ever, the s p e c i f i c a c t i v i t y of the °C-ketoglutarate was high, since it could be detected on paper chromatograms by its r a d i o a c t i v i t y .  When non  shaken c e l l s were used, pyruvate also accumulated in greater quantity than with freshly harvested c e l l s , reaching a value of k Mmoles at 15 min. 4.  Distribution of C  Doudoroff and Stanier  l Z f  in the c e l l s  (53) reported that when starved c e l l s of  P.. saccharoph?la were allowed to o x i d i z e glucose, the oxidative a s s i m i l a tion was more than doubled (to 50% of the added C ^), over that of freshly 1  82.  20  40  60 80 minutes  100  120  FIG. 11. Disappearance of C and glucose from, and excretion of keto acid into supernatant f l u i d s during oxidation of glucoseU-C14 by washed, starved c e l l s of Pseudomonas aeruginosa. l i f  83. harvested c e l l s , and that the assimilated material was largely poly-*$hydroxybutyrate.  A similar experiment was performed in our laboratory  with P. aeruginosa, but with different r e s u l t s .  Starved c e l l s of this  pseudomonad assimilated only 9.3% of the added glucose, as compared to 16.3% with freshly harvested c e l l s , and 12.5% in the presence of 200 ug ° f chloramphenicol per ml (Figure  12).  In the early stages of glucose oxidation, the chemical d i s t r i b u tion of the assimilated  in starved c e l l s was more s i m i l a r to that in  chloramphenicol treated, freshly harvested c e l l s control c e l l s  (Table 10),  (55),  than it was to the  in that the cold t r i c h l o r o a c e t i c acid soluble  pool components were highly l a b e l l e d , whereas label However, although the r a d i o a c t i v i t y  in the c e l l s  in the protein was low.  in the presence of the  a n t i b i o t i c remained mainly in the metabolic pool throughout the experiment, in starved c e l l s 5.  it was transferred slowly to protein and nucleic acids.  Analysis of the cold t r i c h l o r o a c e t i c acid soluble fractions  When the cold t r i c h l o r o a c e t i c acid soluble pools from starved c e l l s were investigated by paper chromatography and electrophoresis, and the results compared to those obtained in experiments with freshly harvested and chloramphenicol treated c e l l s , cells  it was found that a l l three types of  incorporated the c'^ mainly into the free amino acid pools.  amino acids were predominantly those which are neutral at pH 7.6 s e r i n e , leucine, glycine, e t c . ) , as well as glutamic a c i d .  The (alanine,  The glutamate  from the control and a n t i b i o t i c treated c e l l s accounted for most of the r a d i o a c t i v i t y of the pools at 30 min, decreasing in label There was no free glucose present.  thereafter.  In the cold t r i c h l o r o a c e t i c acid soluble  fractions from starved c e l l s , however, the main radioactive component during the entire experiment was glutamate, although it decreased in r a d i o a c t i v i t y  84.  Table 10. Incorporation of C*4 from 5 Hmoles of glucose-U-C^ into washed, starved c e l l suspensions of Pseudomonas aeruginosa  Time (min)  Cold TCA** soluble  Lipid  Alcohol soluble protein  Hot TCA** soluble  total C  Per cent of  Res idual fract ion  Total in fract ions  added to vessel  , / +  5  2.3  0.8  0.3  0.2  0.3  3.9  15  3.0  1.4  0.4  0.3  0.8  5.9  30  4.0  1.5  0.5  0.4  1.1  7.5  120  3.7  1.5  0.6  0.8  2.7  9.1  Per cent of total c'4  incorporated into c e l l  fractions*  62 (36)  20 (26)  7 (3)  4  (11)  7 (26)  100  15  51  (25)  23 (25)  7 (4)  5  (11)  14 (35)  100  30  52 (20)  20 (25)  7 (4)  6 (11)  15 (43)  100  120  40 (15)  16 (21)  6  9  5  (4)  (11)  29 (49)  * Figures in parentheses are values for control c e l l s *" T r i c h l o r o a c e t i c a c i d .  100  (55)%  85.  %  C ^ incorporated 1  into cells  20 Control "O  15  Chloromycetin  •o •o o  10  Starved  O  20  40  60  80  100  Minutes  F I G . 12. I n c o r p o r a t i o n o f C l 4 d u r i n g o x i d a t i o n o f glucose-U-C'^ by w a s h e d , c o n t r o l , c h l o r a m p h e n i c o l (Chloromycetin) t r e a t e d , o r s t a r v e d c e l l s o f Pseudomonas a e r u g i n o s a .  120  86. considerably during this time.  There were also small amounts of radio-  act ive °C ketoglutarate, glucose, gluconic a c i d , and 2-ketogluconic acid _  present in the early stages.  At 15 min, these four compounds had been  replaced by a doubly charged acid whose Rf's Rpicrate '  n  in BFW and EPB, and whose  HCO3 buffer corresponded to those of 6-phosphogluconate or  2-keto-6-phosphogluconate.  Unlike gluconate and 2-ketogluconate, these  phosphorylated derivatives were not separated by EPB.  Pyruvate was not  present at any time in any of the pools. 6.  The influence of vitamin B<s, on oxidative assimilation  It was thought possible that the accumulation of glutamate in the pools of the starved c e l l s might be due to a depletion of the cofactors or enzymes required for transamination.  To test the cofactor theory,  starved c e l l s were allowed to assimilate glucose-U-C^ in the presence of pyridoxal phosphate and pyridoxamine phosphate (8 Mg per f l a s k of each). A p a r a l l e l experiment was run in the absence of the cofactors. there Was no increase in the amount of  However,  incorporated into the c e l l s ,  nor any s i g n i f i c a n t change in the pool when glucose was oxidized in the presence of these cofactors.  Unlike P_. saccharophi l a . which incorporates assimilated carbon into poly-  -hydroxybutyrate, P_. aeruginosa does not form any primary  product during the oxidative assimilation of glucose (55).  This s i t u a t i o n  was emphasized by the results of these experiments with starved c e l l s . Starvation of P. saccharophila c e l l s resulted in a doubling of assimilated material during glucose oxidation (53), but starvation of P. aeruginosa c e l l s led to a k0% decrease.  The reason for this difference seems to be  that oxidative assimilation by f_. aeruginosa involves protein synthesis,  87.  which is a very complex process. lack of protein synthesis  One might postulate two reasons for the  in starved c e l l s .  F i r s t , pyruvate was oxidized  completely, but more slowly by these c e l l s , than by freshly harvested ones, which meant that not as much 0(-ketoglutarate was available for the incorporation of ammonia in the form of glutamate.  In addition, highly  labelled  glutamic acid accumulated in the metabolic p o o l , and so some ammonia was being reincorporated; however, the glutamate thus formed was not used for synthesis.  Therefore, a second reason for the low assimilation could be a  defect in transamination of the glutamate to give other amino acids required for protein synthesis.  Since the addition of vitamin t$6 to starved c e l l s  did not increase a s s i m i l a t i o n , a lack of transaminases could be responsible. The c e l l s apparently have a control mechanism which prevented unlimited assimilation of carbon unless it could be incorporated into c e l l u l a r material.  During glucose oxidation by P. aeruginosa, carbon was not a s -  similated d i r e c t l y , but only after conversion to °C-ketoglutarate, which was,  in t u r n , aminated to y i e l d glutamate (55).  When this glutamate began  to accumulate in the pools of starved c e l l s , perhaps as a result of a low transaminase a c t i v i t y ,  the incorporation of ammonia and carbon was slowed  down, and glucose was oxidized more completely than in freshly harvested cells.  If P. aeruginosa possessed an alternative assimilatory product  such as poly-^-hydroxybutyrate, this sequence of events would not be expected to occur.  11.  Inorganic Nitrogen Assimilation Although the assimilation of ammonia into organic compounds is  known to occur in microorganisms primarily by way of ° f - k e t o g l u t a r a t e and glutamic acid dehydrogenase, recently there has been interest  in a pos-  88. s i b l e additional route v i a pyruvate and alanine dehydrogenase (58,63,122,168). Both pyruvate and °C-ketoglutarate are present in the supernatant f l u i d s during the early stages of oxidative assimilation of glucose by P. aeruginosa, and disappear as the oxidation progresses, with concurrent uptake of ammonia. Since the t r i c a r b o x y l i c acid cycle is functional pyruvate can be converted to of-ketogtutarate  in this microorganism,  (139).  It has been shown that  an NAOPH dependent glutamic acid dehydrogenase is present in c e l l  extracts  of J \ aeruginosa, but direct assays f o r alanine dehydrogenase are d i f f i c u l t , because of the presence of a NADH oxidase.  However, there is some evidence  that, at least under the conditions of the assay used, alanine dehydrogenase levels are much lower than those of glutamic acid dehydrogenase (160). The aspartase a c t i v i t y of extracts of this s t r a i n of P. aeruginosa is also low (160). Resting c e l l experiments were done with f_. aeruginosa and f_. fluorescens to t r y to d i f f e r e n t i a t e between ammonia uptake d i r e c t l y through pyruvate, and that through the o(~-ketoglutarate derived from pyruvate. aspartase route was not investigated.  Two approaches were taken.  The  Firstly,  assimilation of ammonia by P_. aeruginosa was determined during the oxidation of pyruvate or oC-ketoglutarate separately, and in various combinations. Secondly, the effects of inhibitors of keto acid metabolism in P. f l u o r e s cens and P. aeruginosa were determined. A.  Experiments with P. aeruginosa ATCC 9027 1.  Assimilation of ammonia during the oxidation of keto acids  Ammonia, as (NHz^SO^, was added at the concentrations of 5, 7.5, and 15 umoles per Warburg f l a s k , to resting c e l l suspensions oxidizing 5 Hmoles of glucose, pyruvate, or «>C-ketoglutarate, and the uptake of both  89. oxygen and ammonia was followed.  In the cases where glucose or pyruvate  were the substrates, the addition of ammonia decreased oxygen consumption, although the decrease was much greater with glucose than with the keto acid.  The largest reduction was found with 15 Mmoles of ammonia.  The  addition of 5 Hmoles of ammonia to c e l l s o x i d i z i n g °C-ketoglutarate r e s u l t ed in an apparent was terminated 11).  increase in oxygen consumption at the time the experiment  (80 min) perhaps because the lag period was shorter  (Table  However, the f i n a l oxygen uptake, at the time that the oxidation of  the keto acid was complete was not increased.  Table  When 5 Hmoles of ammonia  11.  Oxygen and ammonia uptake during the oxidation of 5 umoles of glucose, pyruvate, or ° ^ - k e t o g l u t a r a t e in the presence and absence of 5 Hmoles of ammonia  Oxygen uptake at 80 min  NH3 uptake at 80 min (5 Hmoles NH3 added)  Per cent theoretical  Hmoles  Substrate  +NH  3  Glucose  67  50  2.95  oC-Ketog1utarate  22*  28*  1.80  Pyruvate  80  59  1.00  Oxygen uptake had not ceased.  were added, a r e l a t i v e l y  small amount of ammonia was consumed during the  oxidation of any of the substrates, and increasing the amount of ammonia did not increase its uptake.  More ammonia was assimilated with glucose  (on a molar basis) than with the keto acids (Table 11).  This may indicate  90. an energy or cofactor requirement ( e . g . , reduced pyridine nucleotides) for ammonia uptake not s a t i s f i e d by the oxidation of the keto acids. In the experiments where the two keto acids were oxidized s i m u l taneously, the second substrate was not added to the f l a s k u n t i l oxidation of the f i r s t had proceeded for 40 min.  This was done because when°T-keto-  glutarate is oxidized by glucose grown c e l l s of P. aeruginosa, there is a lag period before oxygen uptake begins; however, pyruvate is oxidized immediately.  It  is evident that the addition of pyruvate to c e l l s o x i d i z i n g  °C-ketoglutarate neither stimulated nor inhibited ammonia uptake (Table 12).  Table 12. Oxygen and ammonia uptake during the oxidation of pyruvate and °<"-ketogl utarate Initial Substrate  ^Ketoglutarate  Substrate added at 40 min  -  Oxygen uptake % theoret ical at 100 min  NH^ uptake Found Expected 60 min 100 min 100 min  % 28  umoles 1.5  umoles  1.8  o("-Ketoglutarate  pyruvate  34  3.0  2.9 (1.8+1.1)  oC-Ketoglutarate  <**-ketoglut.  37  3.0  3.3  Pyruvate Pyruvate Pyruvate  °C-ketoglut. pyruvate  60  1.1  (1.5+1.8)  0.8  32  3.0  2.3 (1.5+0.8)  65  1.9  1.9  (1.1+0.8)  In f a c t , a l l combinations resulted in approximately the amount of am-  9U monfa uptake predicted, except in the s i t u a t i o n where was added to c e l l s already oxidizing pyruvate, ammonia was incorporated than c a l c u l a t e d .  It  oC-ketoglutarate  in which instance 30% more is possible that  pyruvate  oxidation, l i k e that of glucose, provided cofactors (such as reduced p y r i d ine nucleotides) or energy for the assimilation of ammonia by °C-ketoglutarate. At f i r s t glance, these experiments appear to show that there was no competition between the two keto acids for available routes of ammonia assimilation.  On closer examination, however, one can detect a flaw in  this reasoning, since it was not known whether one system was saturated with substrate when the second was added.  Ammonia was in excess, because  increasing its amount did not increase its uptake.  There did not appear to  be a way of ensuring that reduced cofactors and substrate were in excess without r a d i c a l l y a l t e r i n g in vivo cond it ions.  One must conclude, there-  f o r e , that these experiments did not prove that ammonia was assimilated in the presence of the two keto acids by non competitive routes, even though the data f i t this  interpretation.  If one assumes that the 60% of theoretical oxygen uptake during pyruvate oxidation was consumed in oxidizing the keto acid to completion, then 40%, or 2 umoles was available for a s s i m i l a t i o n .  Free pyruvate was  not found in the metabolic pool of £. aeruginosa, and therefore, pyruvate did not appear to be assimilated without p r i o r amination to alanine, or oxidation to another compound.  If assimilation of the keto acid as alanine  had occurred, 2 umoles of ammonia should have been taken up. actual  However, the  incorporation of ammonia was only 1.1 umoles, indicating that prob-  ably l i t t l e or no direct assimilation of pyruvate occurred.  A calculation  of the amount of ammonia which should be assimilated if this were done only  92. through the^C-ketoglutarate derived from pyruvate is complicated by the fact that we do not know how much of the pyruvate was oxidized to complet i o n , how much only to oC-ketoglutarate, and how much of the o f - k e t o glutarate was, 2.  in turn, o x i d i z e d . Assimilation of ammonia in the presence of  inhibitors  Arsenite, at the concentration of 10~3M, was found to i n h i b i t , by 8 0 - 9 0 % , the uptake of both ammonia and oxygen by resting c e l l s of f_. aeruginosa in the presence of  ° C - k e t o g l u t a r a t e or pyruvate  (Table 1 3 ) . Endog-  enous oxidation was also i n h i b i t e d , although to a lesser extent.  Experi-  ments with c e l l extracts showed that the glutamic acid dehydrogenase of  P.  aeruginosa, and the alanine dehydrogenase of B_. cereus ( 1 2 2 ) are unaffected  Table 1 3 . Inhibition of oxygen and ammonia uptake by 10"3M arsenite during the oxidation of pyruvate and ° C - k e t o g l u t a r a t e by washed c e l l suspensions of f_. aeruginosa  Initial Substrate  Substrate added at hO min  «»C-Ketogl utarate  tm  <K -Ket og1ut a rat e  pyruvate  Inhibition by arsenite Oxygen uptake NH3 uptake %  %  93  85  90  92  Pyruvate  -  80  80  Pyruvate  oC-ketoglutarate  88  76  by this concentration of arsenite, but it  is not known whether the inhib-  itor also prevents oxidative assimilation as a result of the lack of oxidation.  In this connection, Fairhurst et al_. (58) reported that the  93. formation of alanine by B. cereus resting c e l l s from pyruvate and ammonia was inhibited 88% in the presence of 2X10~3M sodium arsenite.  Since the  glutamic dehydrogenase of f_. aeruginosa was not inhibited by arsenite, and since there was almost no assimilation of ammonia even in the presence of OC-ketoglutarate,  it  is probable that,  in the absence of substrate o x i d a -  tion which provides both the reduced cofactors, and the energy necessary, ammonia assimilation does not occur. The second inhibitor used was fluoroacetate, at the concentrations of 10 and 25 Hmoles per f l a s k , tion at c i t r a t e .  in hopes of blocking glucose oxidar  However, glucose d i s s i m i l a t i o n was affected very  by the i n h i b i t o r , and the uptake of ammonia proceeded as usual.  little  It was  then learned that neither acetate nor fluoroacetate are converted to c i t r a t e by acetate grown c e l l s of P_. aeruginosa, although pyruvate (139).  is  This observation would explain these findings with glucose grown  £. aeruginosa c e l l s .  It  is interesting, therefore, that ammonia was a s -  similated during acetate oxidation by P. aeruginosa, and that the endogenous oxygen uptake was inhibited 35%.  The use of fluoroacetate was  abandoned with this s t r a i n of P. aeruginosa, and experiments were done with a second s t r a i n , P. aeruginosa 120 Na, but this too appeared to be unaffected by the i n h i b i t o r , since oxygen uptake with glucose was i n creased 25%, and endogenous oxygen consumption was decreased 36%. B.  Experiments with P. fluorescens A 3.12 Resting c e l l s of P. fluorescens were found to have an unchanged  rate of glucose oxidation in the presence of 10 and 25 umoles of f l u o r o acetate, initial  but the total oxygen uptake was reduced 25%.  There was a s l i g h t  lag in oxygen uptake, and the curve broke at a different point.  94. In a d d i t i o n , the endogenous oxygen consumption was found to be decreased 54%.  When 50 umoles of the inhibitor were used per f l a s k , the rates of  pyruvate and acetate oxidation were reduced by 82% and 71%, respectively, whereas those for glucose and c^-ketoglutarate oxidation were unaffected. The f i n a l oxygen uptake with pyruvate was decreased from 67% of to 45%, but there was no such decrease with acetate.  theoretical  Oxidation of the keto  acid appeared to have ceased at the end of the experiment. A manometric experiment was then done, in which ammonia a s s i m i l a tion was followed during pyruvate oxidation in the presence of 50 umoles of fluoroacetate per f l a s k . 14.  The results are shown in Figure 13 and Table  In the control experiment, ammonia uptake followed oxygen uptake, and  when oxidation ceased, ammonia was evolved.  In the presence of f l u o r o -  acetate, both ammonia and oxygen consumption remained linear u n t i l the experiment was terminated at 80 min; however, the c o n t r o l .  levels were much lower than in  At 20 min, the inhibition of both the uptake of ammonia and  oxygen was 90%, this decreased to 76% and 52%, respectively at 80 min (Table 14). Since the incorporation of ammonia and the uptake of oxygen were inhibited to the same extent by fluoroacetate,  it  is not unlikely that, as  with the experiments in the presence of arsenite, ammonia assimilation does not occur in the absence of substrate oxidation.  This series of experiments, therefore,  represented a preliminary  study on the route of ammonia incorporation by P_. aeruginosa and £. f l u o r e s cens.  It was established that the oxidation of pyruvate by both Pseudomonas  species was accompanied by ammonia uptake, but no evidence was obtained for the direct amination of this keto a c i d .  In contrast, ammonia was c e r t a i n l y  95.  minutes  F I G . 13. Uptake of oxygen and NH3 by washed c e l l s of Pseudomonas fluorescens during the oxidation of 10 umoles of pyruvate with and without 50 umoles of fluoroacetate.  96.  Table 14. Inhibition of uptake of ammonia and oxygen during the oxidation of 10 umoles of pyruvate in the presence of 50 umoles fluoroacetate by washed c e l l s of Pseudomonas fluorescens  Time  Control NH^ uptake  Plus  0  2  uptake  Fluoroacetate  NH^ uptake  0  uptake  2  Inhibit ion NH3  °2  min  umoles  Hi iters  Hmoles  Hi iters  %  %  0  0.000  000  0.000  000  -  -  20  0.675  225  0.067  33  90  87  40  0.837  260  0.120  66  86  81  60  0.812  275  0.176  100  88  64  80  0.470  285  0.210  137  76*  52  * Calculated on basis of 40 min ammonia uptake in c o n t r o l , since oxygen uptake had not stopped at this time.  assimilated v i a °C-ketoglutarate in f_. aeruginosa, since it was taken up during the oxidation of °C-ketoglutarate, and there is known to be an active glutamic dehydrogenase present, with a K acid synthesis.  eq  far  in favour of amino  Supporting data for the primary role of ©C-ketoglutarate  in ammonia assimilation by P. aeruginosa came from the starved c e l l  ex-  periments, where a large amount of labelled glutamate accumulated in the metabolic pool under circumstances where protein synthesis was not occurring.  However, there was l i t t l e radioactive alanine in the pool.  would expect, since pyruvate was present that,  in large quantities  One  in the medium,  i f ammonia were incorporated to any extent via alanine dehydrogenase,  then alanine would contain the highest amount of C ^ and that glutamic 1  97. acid would be of lower s p e c i f i c a c t i v i t y , glutarate for its synthesis.  because of the lack of ©("-keto-  In addition, investigation of the metabolic  pools of freshly harvested c e l l s of JP. aeruginosa after glucose a s s i m i l a t i o n , showed glutamic acid to be the most highly labelled component. Therefore, the data from these experiments were consistent with the oxidation of pyruvate to cf-ketoglutarate, amination to form glutamate, followed by transamination to y i e l d alanine and other amino a c i d s . Although the experiments with P. aeruginosa and P. f1uorescens in the presence of inhibitors gave negative r e s u l t s , they did serve to emphasize the interrelationships between c e l l processes.  Thus, whenever  the oxidation of a substrate was prevented, no assimilation of ammonia, and by inference, of carbon, occurred.  The energy requirement for uptake of  both ammonia and of organic nitrogen by microbial c e l l s has been indicated in a number of experiments (19,34,66,100,110,175), and the cofactor r e quirements of the dehydrogenases are well known. It would seem that any further work on alternate pathways for ammonia assimilation by either of these microorganisms w i l l have to take another d i r e c t i o n .  Alternative approaches would be either the study of  c e l l extracts, or short term experiments, similar to those performed with algae by C a l v i n ' s group, to determine whether alanine is formed d i r e c t l y through pyruvate, or only by transamination with glutamate.  III.  Oxidative Assimilation into the Cytological Fractions of Normal. Chloramphenicol Treated, or Starved Cells of Pseudomonas aeruginosa ATCC 9027 A.  Chemical composition of cytological  fractions  98. Recent work in this laboratory on cytological fractions of f_. aeruginosa c e l l s has been done mainly with lysozyme-versene disrupted cells  (29,30,75).  However,  it was discovered that some commercial lyso-  zyme preparations contained a ribonuclease which resulted in degradation of most of the RNA, so that it appeared in the cytoplasm as small f r a g ments (75).  Accordingly, an alternate method of breaking c e l l s was sought.  The French pressure c e l l , used at 15,000 to 17,000 lbs pressure, was found to give an e f f i c i e n t breakage of small volumes of £_. aeruginosa c e l l suspensions.  Since it was desired to obtain representative "membrane",  ribosomal, and cytoplasmic fractions from these c e l l s  in as pure a state  as p o s s i b l e , several c r i t e r i a were set up to determine the purity of the f r a c t i o n s , as follows oxidizing a c t i v i t y ,  (30):  "membranes"- contain most of the glucose  approximately 5% of the RNA, and l i t t l e DNA; ribosomes-  contain l i t t l e or no glucose oxidizing a c t i v i t y ,  most of the RNA, have an  RNA:protein r a t i o approaching unity, and contain l i t t l e DNA; cytoplasmcontains no glucose oxidizing a c t i v i t y , DNA.  l i t t l e RNA, and almost a l l of the  When the fractionation procedure described in Section  fl B was used,  it was found that these c r i t e r i a were met, and that the results were r e producible.  The data from one of these experiments are shown in Table 15.  The properties of these fractions are similar to those described by Campbell et_ a h fractions  (149).  (30) for P. aeruginosa, and also resemble those of JE. col ? If magnesium was omitted from the buffer in which the  c e l l s were broken, most of the RNA appeared in the cytoplasm.  There is  probably contamination of at least the "membrane" f r a c t i o n with c e l l wall material, and some p a r t i c l e s of "membrane" appear with the ribosomes. B.  Incorporation of glucose-P-C^ into cytological Three experiments were performed:  fractions  99.  Table 15. Distribution of glucose o x i d i z i n g a c t i v i t y , in various cytological  Fract ion  Glucose oxid izing act i v i t y  protein and nucleic acids  fractions  Protein  mg  mg  units*  %total  Cell free extract  675  100  "Membranes" IX  381  62  9.8  15.7  1.2  6.7  "Membranes" 2X  80  20  2.7  4.3  0.3  "Membranes" IX wash  33  6  4.4  7.0  "Membranes" 2X wash  0  0  1.3  40T xg pel let  86  14  3.8  Ri bosomes  20  5  12.5  20  11.7  Cytoplasm  0  0  28  45  0.7  Total Recovery (%)  64  %tota1 100  18  RNA/ Prot  DNA  RNA  %total  %total 100  0.30  0.03  1  0.12  1.7  0  0  0.11  1.1  6.2  0.10  3.4  0.25  2.1  0.7  3.9  0  0  0.54  6.1  2.1  1.2  0  0  0.55  0  0  0.94  96  0.03  600  62.5  17.8  89  97.5  99  100  mg  66 3.9  3  2.8 2.93 97.5  Units- Hi oxygen taken up per mg protein X total protein in fract ion.  100. (a) Cells from a typical assimilation experiment were disrupted, the constituents physically fractionated, and then each of these fractions chemically fractionated.  This w i l l be referred to as the " c o n -  t r o l " experiment, and the c e l l s used w i l l be referred to as " f r e s h l y harvested" or "normal". (b) Cells which had oxidized glucose-C^ in the presence of 200 Hg of chloramphenicol per ml were disrupted, the constituents p h y s i c a l l y fractionated, and then each of these fractions chemically fractionated. (c) C e l l s which had been starved for 3 hr under aseptic conditions, and then allowed to oxidize glucose-c'^ were disrupted, the constituents p h y s i c a l l y fractionated, and each of these fractions chemica l l y fractionated. When the r e l a t i v e incorporation of r a d i o a c t i v i t y  into these  physical fractions was determined, it was found that the "membranes" IX, ribosomes, and cytoplasm accounted for most of the l a b e l l i n g of the c e l l extracts.  Table 16 gives the incorporation of C^** into each of the f r a c -  tions from the three types of experiments, expressed as a percentage of the r a d i o a c t i v i t y 1.  in the c e l l  extracts.  "Membrane" fractions  The percentage of radioactivity which was incorporated into the "membrane" fractions was similar in the three types of c e l l s , and remained quite constant during the experiments, but the actual number of counts i n creased with time in each case. lower percentage of the C ^  Q  The control "membranes" contained a s l i g h t l y  f the extracts than did those from chloramphen-  icol treated or starved c e l l s .  On chemical fractionation of the "membranes",  the d i s t r i b u t i o n of C ^ within each fraction was found to vary greatly (Table 17).  By far the largest part of the label from control "membranes"  101.  Table 16. Incorporation of into cytological fractions during oxidation of 5 Hmoles of glucose-U-Cl4 by resting c e l l s of Pseudomonas aeruginosa (Results expressed as per cent of the c'4 of the c e l l extract)  Time (min)  "Membranes" A* B* C*  Ribosomes A B C  Cytoplasm A B C  5  14  16  17  3.4  5.8  3.1  77  72  68  15  14  18  20  2.7  3.9  3.4  78  70  66  30  14  16  18  2.6  4.4  3.7  79  71  73  120  13  16  18  2.2  4.9  5.3  79  75  72  * A- Freshly harvested c e l l s (3.53xlo6 p dded). B- Chloramphenicol treated c e l l s (3.70xlo6 cpm added). C- Starved c e l l s (3.94x106 cpm added). C  m  a  was in the residual f r a c t i o n , whereas that in chloramphenicol treated or starved c e l l "membranes" during the early stages of glucose assimilation was found in the l i p i d (Figure 14).  However, as the oxidation of glucose  progressed, there appeared to be a marked transfer of r a d i o a c t i v i t y  from  the l i p i d to the protein residue in the starved c e l l "membranes", and a s l i g h t s h i f t from the l i p i d label to the residue label treated c e l l s .  in the a n t i b i o t i c  The l i p i d from the control c e l l "membranes" contained  l i t t l e radioactivity,  although l i p i d was found experimentally to make up  21% of the dry weight of this  fraction.  There seemed to be less inhibition by chloramphenicol of C  14 in-  corporation into the residual f r a c t i o n s , presumably p r o t e i n , of the "membranes" than into the cytoplasm.  At 120 min, based on the percentage of C  102. Table 17. Incorporation of C into chemical fractions of "membranes" during oxidation of glucose-U-C^ by c o n t r o l , chloramphenicol treated, or starved resting c e l l s of Pseudomonas aeruginosa l Z f  Time (min)  Alcoholsoluble protein  Hot soluble*  Lipid  Residue  Control Per cent of Cl4 in c e l l extract 5 30 120  1.4 1.9 1.3  1.2 2.2 1.5  1.5 1.0 0.5  Per cent of C 5 30 120  11.3 13.6 10.0  1 i f  10.6 7.1 4.1  in "membranes" 8.4 15.4 10.7  Chloramphen icol Per cent of C 5 30 120  1.3 1.1 0.9  1 / f  9.8 9.0 9.9  70 64 76  treated  in c e l l extract  6.4 7.7 6.3  1.7 2.2 2.3  6.3 5.0 6.5  Per cent of C l ^ in "membranes" 5 :3o 120  8.5 7.0 5.4  41 48 39  11.0 13.5 14.5  41 31 41  Starved Per cent of C'4 in eel 1 ext ract 5 30 120  0.2 0.2 0.2  12.5 11.5 8.6  1.0 1.6 1.6  3.4 4.7 7.8  Per cent of C'4 in "membranes" 5 30 120  1.0 1.0 1.0  73 64 48  6.0 9.0 9.0  20 26 43  * Hot t r i c h l o r o a c e t i c acid soluble f r a c t i o n .  103.  Chemical  fractionation  of  Residue  membranes  _ >  ®  o I 0)  c o  £ £ o  20  40.  60  80  100  120  Minutes  FIG. 14. Incorporation of into the protein the "membrane" fractions during oxidation of ed c e l l s of Pseudomonas aeruginosa. Control amphenicol treated cel I s B — — — El , starved  residue and l i p i d of glucose-U-Cl4 by washcells 8 — — © , chlorc e l l s A •—•— »A.  104. in each f r a c t i o n , the inhibition was 48% and 86%, cytoplasm, respectively.  for "membranes" and  Accordingly, the 30 and 120 min "membrane  1  samples were hydrolysed, and analysed by column chromatography.  residue  As Table  18 shows, there was a high percentage of non amino acid cJ^ in the water eluates of the Dowex-50 columns after chloramphenicol treatment of the c e l l s , but only about 10-15% in the other two.  Table  18.  Column chromatography of the residual fractions from "membranes" of c o n t r o l , chloramphenicol treated, or starved c e l l s Dowex-50 H0 (neutral and ac idic)  , 2° , (neutral)  Dowex-1 HCl (ac id ic)  cpm*  %  cpm  cpm  Control-30  0.99  16  0.48  8  0.51  Chioram.-30  1.29  55  0.79  33  Starved-30  0.88  25  0.46  13  Control-120  1.46  11  Chloram.-120  1.08  31  -  -  Starved-120  1.35  15  0.63  Fract ion  cpm x  2  Dowex-1 H  %  cpm  %  8  4.68  84  0.51  22  1.12  45  0.42  12  2.64  -  75  -  11.30  89  2.46  69  7.58  85  7  Dowex-50 NH^OH (amino acids)  0.72  %  8  10"3  The non amino acid r a d i o a c t i v i t y was found, on further  fractiona-  tion by column chromatography on Dowex-1 r e s i n , to consist of about equal quantities of neutral and acidic compounds, but there was so l i t t l e r a d i o activity  in these materials that t h e i r  identities could not be established.  105. The neutral compounds may represent c e l l wall carbohydrates, or sugars from the "membranes" themselves.  Studies have shown that,  in some b a c t e r i a ,  hexose (glucose or mannose) makes up to 20% of the dry weight of the membranes, probably in the form of a g l y c o l i p i d (70).  The a c i d i c compound  found in these "membranes" could come from this g l y c o l i p i d a l s o , or from the rhamnolipid which has been found to be formed by P_. aeruginosa c e l l s (80,81,93).  Chloramphenicol treatment of the c e l l s caused l i t t l e or no  inhibition of l a b e l l i n g of these non amino acid components, but did result in a d r a s t i c reduction (75%) of incorporation of c'**  into the "membrane"  p r o t e i n , after correction for non proteinaceous r a d i o a c t i v i t y . harvested c e l l "membrane" f r a c t i o n s , the a c t i v i t y  In freshly  of the protein was high,  while in the starved c e l l "membranes" during the early stages of a s s i m i l a t i o n , protein was of low s p e c i f i c a c t i v i t y ,  although this  increased r a p i d -  ly as the experiment progressed. Radioactive peaks in the NH^OH eluates from the columns were found, by paper chromatography and scanning, to correspond only to ninhydrin p o s i t i v e compounds.  No diaminopimelic acid could be demonstrated.  Table 19 gives the s p e c i f i c a c t i v i t y  of the nucleic acids at 5  min and 120 min in each of the "membrane", ribosomal, and cytoplasmic fractions.  Although the RNA of the "membranes" made up only 6-7% of the  total RNA of the c e l l extract,  it was of high s p e c i f i c a c t i v i t y  in each of  the three types of c e l l s , but especially so in the 5 mini chloramphenicol sample. 2,  Ribosomal  fractions  The incorporatIon of r a d i o a c t i v i t y  into the ribosomes, which con-  tained 20% of the p r o t e i n , and 66% of the total was low throughout each experiment,  RNA of the c e l l  extracts,  indicating a slow turnover of the  106.  Table 19. Specific a c t i v i t y of nucleic acid from cytological fractions of c o n t r o l , chloramphenicol treated, or starved c e l l s of Pseudomonas aeruginosa  mg RNA i h f r a c t i o n  Fract ion  cpm in RNA  Specific activity cpm/mg RNA  120 min  5 min  760  3,600  13,100  50,000  0.076  1,120  3,060  18,700  40,300  0.062  0.062  710  3,080  11,400  49,900  Control  0.552  0.552  500  600  905  1,090  Chloramphen icol  0.517  0.545  1,650  3,400  3,190  6,250  Starved  0.560  0.515  700  4,000  1,250  7,750  Control  0.080  0.085  507  3,120  6,340  36,700  Chloramphen icol  0.088  0.088  1,060  2,930  12,100  33,300  Starved  0.075  0.085  213  3,090  2,840  36,400  5 min  120 min  Control  0.058  0.072  Chloramphenicol  0.060  Starved  5 min  120 min  "Membranes"  R i bosomes  Cytoplasm  protein and nucleic acid (Table 16). of added c'^  In the control c e l l s , the percentage  incorporated into the ribosomes decreased with time, although  the actual amount of r a d i o a c t i v i t y  remained f a i r l y constant  This decrease in apparent l a b e l l i n g was shown, by a chemical  (Figure 15). fractionation,  C  1 4  incorporation into ribosomes  6^ Starved  5. Chloromycetin 4.  S  3-  \  Control 2. l_  20  40  60  80  100  120  Minutes  FIG. 15. Incorporation of Cl4 into the ribosomal fractions d u r i n g oxidation of glucose-U-C.14 by washed, c o n t r o l , chloramphenicol ( C h l o r o m y c e t i n ) treated, or starved c e l l s of Pseudomonas a e r u g i n osa.  108. to be the result of a low Incorporation of c'^  into the nucleic acid of  the hot t r i c h l o r o a c e t i c acid extract, while the amount of c'^  in the p r o t -  ein residue increased during the experiment, and made up most of the r a d i o a c t i v i t y of the f r a c t i o n (Figure 16, and Table 20).  On the other hand,  with chloramphenicol treated or starved c e l l s , there was an increase both in absolute and in r e l a t i v e incorporation of label that of the c o n t r o l .  into the ribosomes over  The ribosomal RNA of the former c e l l s was much more  highly labelled than that of the control c e l l s , whereas protein contained little C^.  A c a l c u l a t i o n of the s p e c i f i c a c t i v i t y of the ribosomal RNA  showed that it was low in a l l three cases (Table 19). b i o t i c treated c e l l s early  However,  in a n t i -  in the experiment, and in starved c e l l ribosomes  in the later stages, the s p e c i f i c a c t i v i t i e s were much higher than that of the c o n t r o l . 3.  Cytoplasmic fractions  In the c e l l s from each type of experiment, the cytoplasm, which contained 45% of the protein of the extract, also contained the bulk of the C  l i f  (Table 16).  The r e l a t i v e amount of r a d i o a c t i v i t y  incorporated into  this f r a c t i o n did not change s i g n i f i c a n t l y during the course of each experiment, but as with the "membranes" and ribosomes, the absolute number of counts increased.  Changing the conditions of the experiments proved to  cause only a s l i g h t variation (Table 16). activity  in the r e l a t i v e amount of cytoplasmic c l 4  The s i g n i f i c a n t change occurred in the d i s t r i b u t i o n of r a d i o -  among the chemical constituents of the cytoplasm (Figure 17 and  Table 21).  At 120 min, most of the label  in the cytoplasm of the control  experiment was found in the protein residue, the C ^ being incorporated into it at a faster rate than into either the cold t r i c h l o r o a c e t i c acid pool components, or the l i p i d and alcohol soluble p r o t e i n .  This c e l l u l a r compon-  1  109.  Chemical fractionation of  ribosomes  lOO Protein  I  •  20  40  I  I  60 80 Minutes  100  120  FIG. 16. Incorporation of C'4 into the RNA and protein r e s i . due of the ribosomes during oxidation of glucose-U-C'4 by washed c e l l s of Pseudomonas aeruginosa. Control c e l l s 0 — — 0 , chloramphenicol treated c e l l s • • , starved cells & •  110  Table 20. Incorporation of into the protein residue and the RNA of the ribosomal fractions during oxidation of glucose-U-C^ by c o n t r o l , chloramphenicol treated, or starved resting c e l l s of Pseudomonas aeruginosa  Time  (min)  Hot soluble*  Residue  Hot soluble*  Residue  Control Per cent of C ^ in c e l l extract 5 30 120  1.0 0.41 0.29  2.4 2.2 . 1.9  Per cent of c'^  -  in ribosomes  30 16 13  70 84 87  Chloramphenicol treated Per cent of O * 1  5 30 120  2.5 2.4 3.2  in c e l l extract  Per cent of c'^  3.3 2.0 1.7  j  n  43 54 65  ribosomes 57 46 35  Starved Per cent of C ^ in c e l l extract 5 30 120  1.6 2.0 3.2  Per cent of c'^  1.5 1.7 2.0  " Hot t r i c h l o r o a c e t i c acid soluble f r a c t i o n .  52 55 61  i  n  ribosomes 48 45 39  Ill  Chemical fractionation of cytoplasm  100  &—  A A  1  80. I  £  a)  A  Pool  o  6Qj Protein  CL O >» U  »  20.  7  ^  #  __Pool  Protein •vJ.lTrj  I 3  1  20  —  »»  •  „  U  40  Protein .60  80  y  100  120  Minutes  FIG. 17. Incorporation of C*4 into the cold t r i c h l o r o a c e t i c , . acid soluble pools and residual fractions of the cytoplasm during oxidation of glucose-U-Cl^ by washed c e l l s of Pseudomonas aeruginosa. Control c e l l s 6 chloramphenicol treated c e l l s B <- S , starved c e l l s  A •  —  •  —  •A .  Table 21 Incorporation of c'4 into chemical fractions of the cytoplasm dur g1ucose-U-cl4 oxidation by c o n t r o l , chloramphenicol treated, or starved resting c e l l s of Pseudomonas aeruginosa  Time (min)  Cold soluble*  Alcohol soluble protein  Lipid  Hot soluble*  Res idue  Control Per cent of c l ^ in eel 1 extract 5 30 120  38 28 17  2.6 2.9 2.9  1.3 0.6 0.4  0.8 1.3 1.3  22 35 51  Per cent of c l ^ in cytoplasm 5 30 120  49 36 22  2.1 0.7 0.5  4.1 3.8 2.8  1.1 1.6 1.6  29 44 65  Chloramphenicol treated Per cent of Cl4 in c e l l 5 30 120  66 61 60  1.4 1.2 1.1  0.9 1.5 2.0  extract 1.6 1.7 2.2  2.6 8.0 9.3  Per cent of c l ^ in cytoplasm 5 30 120  92 85 81  1.2 2.0 2.6  2.0 1.6 1.4  2.2 2.2 3.0  3.6 10.4 12.4  Starved Per cent of C'4 in c e l l 5 30 120  64.5 68.0 51.5  0.20 0. 15 0.20  extract 0.3 0.5 1.6  3.0 4.5 12.9  Per cent of c'4 in cytoplasm 5 30 120  95 93 78  0.30 0.20 0. 35  0.4 0.65 2.4  4.4 6.2 19.5  * Cold and hot t r i c h l o r o a c e t i c acid soluble f r a c t i o n s .  113. ent was very highly 1abelled, indicating that a rapid turnover of the s o l uble proteins was occurring during glucose oxidation.  As with the "mem-  branes" and ribosomes, synthesis of the cytoplasmic proteins was greatly decreased in the presence of chloramphenicol, and also by p r i o r s t a r v a t i o n of the c e l l s .  However,  in starved c e l l s , the rate of l a b e l l i n g of  the protein continued at a rapid rate u n t i l the end of the experiment. In both the chloramphenicol treated and starved c e l l s , the t r i chloroacetic acid soluble pool made up a very much larger proportion of the r a d i o a c t i v i t y of the cytoplasm than it did in the control c e l l s ure 17 and Table 21).  (Fig-  The pool from starved c e l l s at 5 min contained 95%  of the cytoplasmic l a b e l , and this decreased only 15% during the course of the  experiment, as the label  tion c'4  in the protein residue rose.  The incorpora-  into l i p i d and alcohol soluble proteins was extremely low.  With  chloramphenicol, 92% of the r a d i o a c t i v i t y was in the metabolic pool at 5 min, decreasing to 81% at 2 hr. slightly c'4  The residual protein f r a c t i o n  increased  in r a d i o a c t i v i t y during this period, although there was 86% less  incorporated than in the c o n t r o l .  Ether extraction of the cold t r i -  chloroacetic acid soluble pools, followed by chromatography and e l e c t r o phoresis of the aqueous solutions and scanning to detect the radioactive components, showed that, as with the metabolic pools from whole c e l l s , the c'4 was mainly in the form of amino acids in a l l three experiments.  The  d i s t r i b u t i o n of r a d i o a c t i v i t y was s i m i l a r to that found in pools of the d i f f e r e n t types of whole c e l l s ,  in that glutamate was highly l a b e l l e d , and  accumulated in the starved c e l l  fractions.  Although the actual amount of c'4  incorporated into the hot t r i -  chloroacetic acid soluble f r a c t i o n of the cytoplasm was low, a c a l c u l a t i o n of the s p e c i f i c a c t i v i t y of the nucleic acid in this f r a c t i o n showed that  114 It was highly labelled (Table 19).  The nucleic acid extracted with hot  t r i c h l o r o a c e t i c acid proved to be almost a l l RNA, presumably soluble RNA, which has a higher rate of turnover than does ribosomal RNA.  The c y t o -  plasmic DNA, which was degraded by the DNase treatment of the extracts to reduce t h e i r v i s c o s i t y , was a part of the cold t r i c h l o r o a c e t i c acid p o o l . The i n i t i a l s p e c i f i c a c t i v i t y of the RNA formed in the presence of c h l o r amphenicol was higher than that in the other two experiments. C.  Experiments with the cytoplasmic proteins 1.  Amount of r a d i o a c t i v i t y contained in the "pH 5 enzyme"  To obtain some estimate of the  incorporated into the synth-  e t i c enzymes of the cytoplasm, the following experiment was performed. Each of the cytoplasmic fractions from the three types of c e l l s was adjusted to pH 5.4 with acetic a c i d , allowed to stand in the cold 20 min, and centrifuged.  Counts were made on aliquots of each f r a c t i o n before this  treatment, and on the supernatant solution after a c i d i f i c a t i o n and c e n t r i fugation.  Since the enzymes responsible for the activation of amino acids,  and t h e i r transfer to soluble RNA and ribosomes, are precipitated by a c i d i f i c a t i o n to pH 5.4, the amount of c'4 removed at this pH should be a measure of the synthesis of these enzymes during glucose oxidation.  The  results are given in Table 22. These data indicate that, under conditions of p r i o r  starvation  of f_. aeruginosa c e l l s , there was a d e f i n i t e decrease in the amount of  c'4  incorporated during glucose oxidation into the fractions containing the enzymes responsible for protein synthesis, as compared to freshly harvested cells.  This would help to explain why,  thesis was greatly reduced.  in the starved c e l l s , protein syn-  Chloramphenicol caused a complete i n h i b i t i o n  115. Table 22. Percentage of Cl4 removed by a c i d i f i c a t i o n of the cytoplasmic fractions to pH 5.4  Time  Control  Chloramphenicol  Starved  (min)  %  %  %  5  3  0  1  15  15  0  2  30  18  0  3  120  10  3  10  of the incorporation of r a d i o a c t i v i t y  into these f r a c t i o n s , except at 2  hr, when a s l i g h t l a b e l l i n g was found, and shows that protein synthesis was occurring. 2.  Effect of starvation on the a c t i v i t y of the aminoacyl-s-RNA synthetases  Additional  information on the state of the amino acid activating  enzymes during starvation of P. aeruginosa c e l l s , and during glucose assimi l a t i o n by these c e l l s , was gained by assaying the formation of aminoacyls-RNA by these synthetases.  Preliminary experiments indicated that only the  cytoplasmic enzymes were capable of activating the amino acids.  An e x p e r i -  ment was therefore carried out in which the c e l l s were starved, allowed to oxidize glucose, then disrupted and p h y s i c a l l y fractionated. taken at  Samples were  intervals during the starvation and assimilation periods.  The  amount of enzyme which was l i m i t i n g for the formation of aminoacyl-s-RNA in the zero time sample was determined, and then the r e l a t i v e a c t i v i t i e s the enzymes of the other time intervals were assayed in the same manner.  of  116. Protein determinations were done on each f r a c t i o n , and the s p e c i f i c activity  (cpm per ug protein) was c a l c u l a t e d .  The results are shown in  Figure 18 and Table 23.  Table 23. Incorporation of C'4 amino acids into s-RNA by cytoplasmic enzymes of starved c e l l s of Pseudomonas aeruginosa  Fract ion  Protein Mg/0.003 ml  incorporated  Specific  Activity  cpm/0.003 ml  cpm/ng protein  Period of starvat ion zero  7.0  3710  530  1.5 hr  8.2  3780  460  3.0 hr  7.4  3240  , 440  15 min  6.5  3320  510  30 min  8.0  4130  515  120 min  8.4  4480  535  Period of ass imi1 at ion  Therefore, it appeared that, during starvation of P. aeruginosa, the enzymes responsible for amino acid incorporation were degraded, and that they were resynthesIzed while glucose was assimilated. explain the low incorporation of r a d i o a c t i v i t y  This would  from glucose into protein  by starved c e l l s during the early stages of a s s i m i l a t i o n .  As glucose o x i d a -  tion progressed, the protein synthesizing enzymes were quickly regenerated, and incorporation into protein increased.  Despite the indication that these  117.  starvation  assimilation minutes  FIG. 18. Incorporation of C.1^ amino acids into s-RNA .. by cytoplasmic fractions of Pseudomonas aeruginosa.  113. enzymes were resynthesized while glucose was being oxidized, they accounted for only a small proportion of the cytoplasm of the starved c e l l s , at least during the early stages of the experiment, compared to that for the control c e l l s  (Table 22).  One would think from the reactivation curve in  Figure 18, that the proteins of the pH 5 enzyme would be p r e f e r e n t i a l l y synthesized during a s s i m i l a t i o n , but this did not appear to be the case.  The cytological d i s t r i b u t i o n of the carbon assimilated from glucose was perhaps not s u r p r i s i n g , since it was known from a previous chemical fractionation of whole c e l l s  (55) that the incorporation of label  occurred mainly into proteins, and from preliminary experiments that c y t o plasmic proteins constituted by far the largest chemical f r a c t i o n of the cell.  Therefore, the finding that 60-65% of the  of the c e l l was in  the soluble proteins of the cytoplasm in the control experiment was consistent with these observations  (Table 21).  However, the high s p e c i f i c  a c t i v i t y of the cytoplasmic proteins, which are mainly enzymes, was somewhat unexpected, since the c e l l s were grown on glucose, and one would have thought that most of the necessary enzymes for its oxidation would have a l ready been present.  That most of these newly synthesized enzymes were not  degradative was shown by the experiment with chloramphenicol, where,  al-  though the synthesis of soluble protein was inhibited 86%, glucose oxidation proceeded at a rapid r a t e .  Since nearly 20% of the total  radioactivity  of the control cytoplasm was found in the pH 5 enzyme f r a c t i o n , some of the newly synthesized soluble protein was concerned in protein synthesis. lower incorporation of  The  into this fraction in starved c e l l s corresponded  to the decrease of protein synthesis  in these c e l l s .  During endogenous  r e s p i r a t i o n , the a c t i v i t y of the aminoacyl-s-RNA synthetases also declined,  119.  but was restored by the assimilation of glucose.  The reduction in these  enzymes may be a result of t h e i r endogenous breakdown (75). An effect of chloramphenicol on the metabolic pool components s i m i l a r to that found in these experiments with P_. aeruginosa, has been reported in V i b r i o cholera (78).  In this case a l s o , the amount of amino  acids in the pool was higher, but its q u a l i t a t i v e composition was unchanged. It has been known for some time, that, whereas chloramphenicol inhibits protein synthesis  in bacteria,  it allows RNA synthesis to continue  at an increased rate (67); however, the nature of this "chloramphenicol RNA" is s t i l l  under discussion.  Most reports agree that the RNA is of  high molecular weight, having a sedimentation c o e f f i c i e n t of 14 to 16 S (119), which rises to 30 S if the centrifugation is done in the presence of 10"^ M magnesium ions (47).  Aronson and Spiegelman (9) have found that  after high speed centrifugation of c e l l extracts, the chloramphenicol RNA appeared in the p a r t i c u l a t e f r a c t i o n with the ribosomes.  There are two main  schools of thought as to just what this high molecular weight chloramphenicol RNA represents.  Gros et a l .  (76) showed that in !E. col i. chloramphen-  icol RNA possessed properties s i m i l a r to those of a rapidly labelled RNA, termed "messenger RNA", and that in common with messenger RNA, it s t i m u l ated amino acid incorporation into p r o t e i n .  There is normally a rapid break-  down of messenger RNA, but Gros and his group believe that t h i s does not occur unless protein elongation is completed, and that chloramphenicol s t a b i l i z e s this RNA by stopping protein synthesis.  The a n t i b i o t i c does  not inhibit the continued synthesis of the RNA, however.  The effects of  actinomycin and chloramphenicol on RNA synthesis were investigated by Reich and his associates  (128).  Actinomycin, which prevents DNA-dependent RNA  synthesis, inhibits the synthesis both of the rapidly labelled RNA produced  120. normally, and of chloramphenicol RNA.  Reich et aU (128), and Aronson and  Spiegelman (10) take the stand that messenger and chloramphenicol RNA's are merely precursors of ribosomal RNA, since the pulse labelled f r a c t i o n of RNA enters the ribosomes intact, as does chloramphenicol RNA, providing that the organisms, after removal from the a n t i b i o t i c , are placed in a medium which permits protein synthesis. then, is unstable u n t i l  it  The high molecular weight RNA,  is combined with p r o t e i n .  Thus, there are two  proposed templates for protein synthesis: the ribosomal RNA, or a transient, DNA-like messenger RNA. Despite the p r o l i f e r a t i o n of data indicating that chloramphenicol RNA is of high molecular weight, Yee and Gezon (178), and Yee et aj_.  (177)  have reported that the RNA formed by starved c e l l s of Shigella flexner?  in  the presence of chloramphenicol is a functional s-RNA, with the expected base r a t i o s .  The s-RNA of this organism was found to be increased by 160%  after h hr of incubation in a complete medium with the a n t i b i o t i c .  Extracts  of S_. flexneri were prepared by grinding, however, which Aronson and Spiegelman (10) found resulted in the s o l u b i l i z i n g of ribosomal RNA, as a result of degradation of the ribosomes.  This would give an apparent  in-  crease of s-RNA in these experiments, but it should not give an increase in functional  s-RNA. The results of the experiments with P. aeruginosa, in which the  oxidation of glucose-c'^ by resting c e l l s  in the presence of chloramphenicol  gave r i s e to an increase in the r a d i o a c t i v i t y of the ribosomal RNA over that of the control  (Table 20), are in accordance with previous reports of an  increase in synthesis of high molecular weight RNA when protein synthesis is prevented.  This chloramphenicol RNA would appear mainly in the ribosomal  f r a c t i o n from high speed c e n t r i f u g a t i o n , and result  in a high i n i t i a l  incor-  121.  poration of C turnover  l i f  into the ribosomal RNA.  Normal ribosomal RNA has a slow  (47), as shown by the very low s p e c i f i c a c t i v i t y  of this  in the control c e l l s , whereas chloramphenicol RNA is constantly over  fraction  turning  (88). Cytoplasmic RNA, although only a minor part of the total  was also of high s p e c i f i c a c t i v i t y  in the chloramphenicol treated  during the f i r s t stages of glucose oxidation.  RNA,  cells,  This could be the result of  degradation of high molecular weight RNA, or increased synthesis of s-RNA as suggested by Yee and Gezon (178). The increase in label of the ribosomal RNA in starved c e l l s , on the other hand, occurred late in the experiments  (Table 20), and can be  more readily explained by its resynthesis after depletion during endogenous r e s p i r a t i o n , than as a manifestation of the decrease in protein s y n thesis  (29,75).  that the i n i t i a l be high.  If the l a t t e r s i t u a t i o n were the case, one would expect l a b e l , l i k e that in the a n t i b i o t i c treated c e l l s , should  C l i f t o n (36), and Duncan and Campbell (55) have suggested that  assimilation may serve to replenish the endogenous reserves of microorganisms, and Campbell, Gronlund and Duncan (29) have shown that RNA is a major endogenous substrate for f_. aeruginosa. Hunter et a_U  (89), Butler, Godson and Hunter (28), and Hunter  and Godson (90) have reported that the membrane l i p o p r o t e i n , which contains most of the c e l l in B_. megaterium.  l i p i d , was the i n i t i a l  s i t e of incorporation of  lys?ne-C  Chloramphenicol or phosphol ipase inhibited the uptake  of amino acid by the l i p i d .  Only the phospholipid component was  active,  and after extraction of the lipoprotein with hot a l c o h o l , the amino acid was found to be associated with the l i p i d p o r t i o n .  In this connection,  Silberman and Gaby (137) have shown that the l i p i d of P. aeruginosa contains bound amino acids, but the effect of chloramphenicol on the uptake  lZf  122. of amino acids by this microorganism was not investigated. to the results of Hunter et_ aj_. (28,89,90),  In contrast  in which chloramphenicol was  shown to inhibit the incorporation of c'^ amino acids into B_. megater ium membrane l i p o p r o t e i n , there was a marked increase in the incorporation of from glucose into the alcohol-ether soluble f r a c t i o n of the ''membranes" of P. aeruginosa in the presence of the a n t i b i o t i c  (Table 17).  Duncan and  Campbell (55) have also reported an increased l a b e l l i n g of the total  lipid  of the c e l l when protein synthesis was inhibited in this microorganism. This increase may be the result of more substrate being available for l i p i d synthesis when protein synthesis  is not occurring, or it may be due to the  formation, by a chloramphenicol insensitive route, of lipo-amino acid complexes in P. aerug inosa. but not in B_. megater ium.  These lipid-amino acid  complexes would then accumulate during the i n h i b i t i o n of protein synthesis. Since glucose C ^ was used in the experiments with P. aeruginosa, it  is  not known whether the r a d i o a c t i v i t y was in the l i p i d per se. or whether the c'^ was in the form of amino acids attached to the l i p i d . icol  Chloramphen-  is known to inhibit the processes involved in transfer of the amino  acid residues from s-RNA to the ribosomes (99), and therefore,  if the  postulated lipid-amino acid complexes were formed p r i o r to the production of aminoacyl s-RNA, one could v i s u a l i z e how the a n t i b i o t i c would have no effect on t h e i r formation.  However, Hunter and Godson (90) have proposed  that the amino acids are transferred from the s-RNA to phospholipids, and then to the ribosomes. An even greater  initial  increase in label of the l i p i d component  of the "membranes" was found in starved c e l l s  (Table 17).  As the e x p e r i -  ment progressed, the rate of C ^ incorporation into l i p i d decreased, as 1  protein synthesis  increased (Figure 14).  This phenomenon may be explained  123. by either of the two explanations previously considered. amphenicol treated c e l l s , however, the r e l a t i v e label  Unlike c h l o r -  in the total  of the starved c e l l s was not higher than that of the control I,  lipids  (see Section  C 4). Many reports have appeared concerning the presence of small  amounts of RNA in the membrane f r a c t i o n of microorganisms (1,24,30,43,89, 142), and the high s p e c i f i c a c t i v i t y of this RNA after exposure of E. c e l l s to P3  2  or  has also been demonstrated (43,142,158).  col?  It has been  suggested that this RNA is not merely an a r t i f a c t a r i s i n g as a result of the fractionation procedure, but that  it may be an integral part of r i b o -  somes attached to the membranes, and represent an RNA active in protein synthesis  (43).  Campbell et aj_. (29) were able to remove the RNA from  lysozyme prepared "membranes" of P. aeruginosa by treatment with Versene, and alternate freezing and thawing.  After such treatment, the "membrane"  f r a c t i o n could be separated into two components by centrifugation at 25,000xg, which yielded a p e l l e t  ("membranes")  and the supernatant from  which ribosomes could be sedimented at 100,000xg.  McQuillen et a]_.  (112)  have shown that some ribosomes of JE. c o l i are associated with the membrane. Suit (149),  in P^  2  experiments with E. col? "membranes", was able to find  no function for "membrane" RNA in c e l l wall synthesis, but instead obtained evidence that  later  it might consist of messenger RNA, s i n c e , after  infection of the organism with T  2  bacteriophage, "membrane" RNA had base  ratios s i m i l a r to that of the phage DNA (150). The small amount of RNA associated with washed "membranes" of _P. aeruginosa was also found to have a high s p e c i f i c a c t i v i t y ,  and therefore  appears to be d i f f e r e n t than the ribosomal RNA, whose l a b e l : i s low (Tables 17, 19 and 20).  The high incorporation of C ^ into the "membrane" RNA of 1  124. control c e l l s could be explained by its possible function as a messenger suggested by Suit (150), which would result increased l a b e l .  in a rapid turnover, and an  The same interpretation of results could be used for the  starved c e l l s , with the additional argument that this RNA may also be u t i l i z e d as an endogenous reserve, and thus be resynthesized during glucose oxidation.  The i n i t i a l high s p e c i f i c a c t i v i t y of the membrane RNA in a n t i -  b i o t i c treated c e l l s would be a manifestation of the formation of c h l o r amphenicol RNA, some of which would be attached to the "membrane" ribosomes.  IV.  Species S p e c i f i c i t y of s-RNA's and Aminoacyl-s-RNA Synthetases A.  Cytological location of aminoacyl-s-RNA synthetases  in P. aerug-  inosa When the cytological fractions  ("membranes", ribosomes, and  cytoplasm) from P. aeruginosa were tested for t h e i r aminoacyl-s-RNA synthetase a c t i v i t y with the homologous s-RNA,  it was found that the cytoplasm was  the only fraction which was active (Table 24).  Addition of the "membranes"  or ribosomes to the cytoplasm did not stimulate incorporation of C ^ amino acids into the P. aeruginosa s-RNA, nor did either of these fractions the s-RNA.  replace  Chloramphenicol (200 Mg per ml) produced no i n h i b i t i o n of the  formation of aminoacyl-s-RNA by the cytoplasmic enzymes.  The low blank  values when the s-RNA was omitted showed that none of the c e l l  fractions  contained enough s-RNA to support the incorporation of measurable amounts of amino acids.  If the amount of cytoplasm was reduced by one-half, there was  an increase of 15% in the number of counts incorporated; this could not be duplicated by r a i s i n g the amount of ATP in the assay, and may have been due to the presence of unlabel led amino acids in the cytoplasmic fractions which  125.  Table 24. Incorporation of C ' ^ amino acids into s-RNA by Pseudomonas aeruginosa c e l l fractions  Fraction  s-RNA  Incorporation of C'4 into s-RNA cpm  Cytoplasm  ( 0.025 ml )  Cytoplasm  ( 0.050 ml )  -  Cytoplasm  ( 0.025 ml )  +  24,210  Cytoplasm  ( 0.050 ml )  +  "Membranes"  ( 0.050 ml )  -  21,220  "Membranes"  ( 0.050 ml )  +  1,150  Cytoplasm "Membranes"  ( P.025 ml ) ( 0.025 ml )  -  800  Cytop 1 asm "Membranes"  ( ml ) 0.025 ( 0.025 ml )  +  26,130  Cytoplasm R i bosomes  ( 0.050 ml ) ( 0.050 ml )  -  900  Cytoplasm Ribosomes  ( 0.050 ml ) ( 0.025 ml )  +  21,440  Cytoplasm ( 0.050 ml ) Chloramphen ico ( 80 H9 )  +  19,740  700 910  770  126. would cause a d i l u t i o n of the c'^ amino acids added. Hunter et, a K  (89) have reported that, when B. megater ium ex-  tracts were prepared by lysozyme treatment  in a medium of high ionic  strength, the enzymes responsible for activation and incorporation of the , amino acids were associated with the membrane f r a c t i o n , whereas,  i f the  ionic strength were low, these enzymes appeared in the cytoplasm.  In an  e f f o r t to determine whether the use of a high ionic strength suspending f l u i d would cause the amino acid activating enzymes of P. aeruginosa to be associated with the membranes, extracts were prepared in 0.33 M phosphate buffer as was used for the preparation of membranes of B. megaterium.  Per-  haps because of the deficiency of magnesium ions in the phosphate buffer, it was d i f f i c u l t to obtain an extract of low v i s c o s i t y by the use of DNase treatment.  However, s a t i s f a c t o r y extracts were f i n a l l y prepared, and sep-  aration of the fractions by centrifugation proceeded normally.  These f r a c -  tions were tested for t h e i r a b i l i t y to incorporate amino acids into P. aeruginosa s-RNA.  The amount of s-RNA used was only one-half of that of  the experiments reported in Table 24, and the incorporation of respondingly lower  (Table 25).  It  was c o r -  is evident that, unlike those of B_.  megater ium, the aminoacyl-s-RNA synthetases of f_. aeruginosa were s t i l l found in the cytoplasm, even at a high ionic strength.  However, the B_.  megaterium extracts were prepared by digestion with lysozyme, which is a less severe treatment than the high pressure used in these experiments for breaking P. aeruginosa c e l l s . Fraser (61) has recently shown with rat plasm could be used as a source of s-RNA.  l i v e r that heated c y t o -  Since this would simplify the  experiments considerably, t h i s approach was investigated with P. aeruginosa. The heated cytoplasm was prepared as described by Fraser  (61), from the  127. Table 25. Incorporation of C ^ amino acids into s-RNA by cytological fractions of Pseudomonas aeruginosa prepared at a high ionic strength  Fract ion  s-RNA  Incorporation of C ^ into s-RNA cpm  "Membranes"  +  620  Cytop 1 asm  +  8940  +  9060  Cytoplasm "Membranes"  1  f r a c t i o n used in the experiments reported in Table 24. periment in this case contained 0.5 mg s-RNA (Table 26).  The control exThe data in  Table 26 show that the heated cytoplasm did not cause a stimulation of counts incorporated into the alcohol p r e c i p i t a t e .  There was an increase  of C ^ incorporated in the presence of the heated cytoplasm, but this was due to the occlusion of C ^ by the large amount of p r e c i p i t a t e , as i n d i c ated by the controls in which no enzyme was added.  The heated cytoplasmic  f r a c t i o n did not appear to inhibit the incorporation of c'^ into the system where both enzyme and s-RNA were present. B.  Interspecific  reactions between s-RNA's and aminoacyl-s-RNA  synthetases In preliminary experiments with P. aeruginosa ATCC 9027, it was found that yeast s-RNA reacted very poorly with the bacterial enzyme system. A s i m i l a r s i t u a t i o n with yeast s-RNA and E. c o l i activating enzymes has been reported for a number of amino acids (14,31,51,182), but there has  128. Table 26. Incorporation of c'V amino acids into the s-RNA present in the heated cytoplasm of Pseudomonas aeruginosa  Enzyme  Cytoplasm  s-RNA  -  Cytoplasm Cytop 1 asm Cytoplasm None Cytop1 asm None  +  -  Heated pH 5 f r a c t i o n of cytoplasm  Incorporat ion of Cl4  ml  cpm  -  400  0.25  7250  -  5270  0,10  1460  0.10  1380  0.25  2660  0.25  2530  been 1 i t t l e work done on interbacterial  systems.  Three pseudomonads (iP.  aeruginosa ATCC 9027, P. aeruginosa 120 Na, and P. fluorescens A 3.12), as well as Achromobacter B81, JE. c o l i B, and bakers' yeast were selected for study.  It was thought that,  i f any species s p e c i f i c i t y existed between  s-RNA's and aminoacy1-s-RNA synthetases, this should be shown by greater heterologous reactions among the Pseudomonas species and Achromobacter B81, than between these bacteria and E. col? or yeast. The cross reactions were done in a l l combinations p o s s i b l e , since sometimes an enzyme w i l l show a greater reaction with a heterologous s-RNA than would be indicated by the reverse cross (51,182).  Blank tubes con-  taining enzymes, but no s-RNA, were run to rule out a contribution from the s-RNA in the enzyme f r a c t i o n .  The incorporation of C ^ in these controls  129. was found to be of the order of 1200 cpm.  Table 27 gives the number of  counts per minute incorporated into each of the s-RNA's by the homologous and heterologous enzymes.  Table 28 shows the percentage of incorporation  of C''^ into each s-RNA, based on the amount of incorporation by the homologous enzyme system, and Table 29 gives the per cent of r a d i o a c t i v i t y  in-  corporated as compared to the homologous s-RNA system. These data show that the conditions of the assay were at  least  adequate for each homologous system, s i n c e , with the exception of Achromobacter B81, there was an almost constant amount of c'^ each s-RNA (40,000 cpm).  incorporated into  The poor reaction in the Achromobacter system,  into which only about half as much  was incorporated, appeared to be  due to the s-RNA, because the synthetases from this organism were able to acylate the s-RNA's from the other bacteria (though not the yeast) with a higher e f f i c i e n c y than the homologous s-RNA.  None of the bacterial s-RNA's  reacted well with the yeast enzyme, and only the enzyme from E. c o l i corporated much  into the yeast s-RNA.  The E. col? synthetases  in-  incor-  porated 68% of the counts of the homologous b a c t e r i a l , or 74% of the homologous yeast, system into the yeast s-RNA. A higher amount of incorporation of amino acids into a h e t e r o l ogous s-RNA than into the homologous one was found with two other enzyme systems besides that of the Achromobacter already mentioned.  Thus, the P.  aeruginosa ATCC 9027 and the jE. col? enzymes gave better reactions with the s-RNA's of P. aeruginosa 120 Na and P. fluorescens than they did with t h e i r own.  Doctor and Mudd (51), studying i n t e r s p e c i f i c reactions between  yeast, E. c o l i . and rat l i v e r systems, reported a s i m i l a r phenomenon, esp e c i a l l y with the rat  l i v e r enzymes.  They suggested several  reasons for  these anomalous f i n d i n g s , but none was established as being responsible  Table 27 Incorporation of C amino acids into s-RNA's of various microorganisms by homologous and heterologous enzymes H  Enzyme  P. aeruq. 9027  P. aeruq. 120 Na  P. f l u o r . A 3.12  Achr.'B8l  cpm  cpm  cpm  cpm  E.  coli cpm  Yeast cpm  P. aeruq. 9027  41,060  26,080  30,730  23,200  43,830  8,670  P. aeruq. 120 Na  54,480  43,230  36,730  37,550  57,480  13,650  P. f l u o r . A 3.12  51,230  35,980  38,250  48,250  55,130  17,030  Achr.  17,370  13,830  13,750  20,980  24,330  6,320  32,930  28,180  26,030  33,400  39,930  10,850  15,130  8,000  13,380  13,700  26,980  36,150  E.  B81  coli  Yeast  OJ  o  Table 28 Percentage of C'4 amino acids incorporated into s-RNA's based on the amount found in the system homologous for the enzyme  EnzymeX^^  P. aerug. 9027  P. aeruq. 120 Na  P. f l u o r . A 3.12  P. aeruq. 9027  100  60  82  110  110  24  P. aeruq. 120 Na  132  100  96  179  144  38  P. f l u o r . A 3.12  125  83  100  230  138  47  Achr. B81  42  31  37  100  61  18  E.  79  65  70  160  100  30  37  19  36  66  68  100  5-RNA  coli  Yeast  Achr. B81  . E.  coli  Yeast  Table 29 Percentage of C amino acids incorporated into s-RNA's based on the amount found in the system homologous for the s-RNA 1 4  Enzymex^ ^x"s-RNA  P. aerug. 9027  P. aeruq. 120 Na  P. f l u o r . A 3.12  Achr. B81  P. aeruq. 9027  100  63  75  54  106  21  P. aeruq. 120 Na  125  100  87  87  132  33  P. f l u o r . A 3.12  134  94  100  125  144  44  Achr. B81  83  64  66  100  116  30  E.  83  71  65  81  100  27  43  22  37  38  74  100  coli  Yeast  E.  coli  Yeast  133. for t h e i r observations. ments, it  In the case of Achromobacter B81 in these e x p e r i -  is probable that the s-RNA was damaged, because neither the enz-  ymes from this microorganism, nor those from any of the others reacted well with it  (Tables 27 and 28).  As for the increase in some of the h e t e r o l -  ogous reactions with the E. col? and Pseudomonas species, one of the other explanations advanced by Doctor and Mudd (51) may be invoked:  there may be  d i f f e r e n t amounts of s-RNA's in d i f f e r e n t species, o r , where multicomponent s-RNA's e x i s t , the heterologous enzyme may recognize more than one s-RNA. C.  Patterns of amino acid incorporation into homologous systems Table 30 gives the percentage of each amino a c i d , or group of  amino acids, incorporated into the s-RNA's of the s i x microorganisms by the homologous enzyme systems.  The incorporation of some of the amino acids  was found to be very low, especially that of p r o l i n e , glutamic acid and alanine. A comparison of the average amino acid composition of several bacteria (species of Enterobacteriaceae,  Pseudomonas, and Baci1lus)  (144,  146), and that of the C h l o r e l l a hydrolysate (Merck, Sharpe and Dohme Ltd.) is given in Tab1e 31. The pattern of incorporation of the amino acids by the bacteria does r e f l e c t , to some degree, their a v a i l a b i l i t y , of the c e l l s .  and also the composition  The leucine group was incorporated to the largest extent  a l l of the homologous reactions, accounting for from 31% (E. c o l i ) (Achromobacter) of the total r a d i o a c t i v i t y  in the s-RNA's.  in  to 52%  The basic amino  acids were the next highest, making up nearly one-quarter of the counts in P_. aeruginosa ATCC 9027, £. fluorescens, and E. col i.  However, large amounts  of the valine group (valine and methionine) were incorporated into the s-RNA's  134.  Table 30. The percentage of Incorporation of amino acids into s-RNA's by homologous enzymes  Organism.—• Amino acid  •- p. aeruq. 9027  %  P.. aerug. 120 Na  %  P. f l u o r . A 3.12  %  Achromo. B81  %  E. col ? Yeast B  %  %  19*4  10.6  18.5  14.0  22.3  Asp. group*  9.5  9.9  9.0  18.4  11.9  Glu. group*  9.3  2.0  3.2  10.6  3.0  3.5  Alan ine  5.8  0.0  0.0  1.6  1.2  1.1  Proline  1.1  0.0  5.1  0.0  3.6  6.4  Tyros ine  5.6  14.4  9.9  0.0  10.1  12.7  V a l . group*  12.1  21.1  16.5  19.2  16.2  20.3  Leu. group*  37.2  42.1  37.8  52.4  31.6  24.2  Basic group*  * Basic group - l y s , h i s , arg. Aspartic acid group - asp, g l y , s e r . Glutamic acid group - g l u , t h r . Valine group - v a l , met. Leucine group - leu, i l e u , phe.  25.2 6.9 .  Table 31. A comparison of the amino acid composition Of C h l o r e l l a hydrolysate* and of that of several bacteria  Amino acid  Bacteria % amino acid  (144,146)  C h l o r e l l a hydrolysate % amino ac id  % C 14  13.7  7.4) 2.5 8.9 )  18.8  18.5  10.2 4.6 9.2  24.0  8.8 ) 2.9 \ 4.4  16.1  12.9  Glutamic acid Threon ine  11.2 5.5  16.7  I J  13.3  13.0  Alanine  10.4  7.4  5.8  Proline  4.2  7.4  5.8  Tyros ine  2.7  5.9  6.1  Val ine Meth ion ine  7.2 3.0  10.2  5.9  14.2  Leucine Isoleuc ine Phenylalan ine  8.7 5.4 3.5  17.6  Lys ine Hist idine Arginine  6.4 2.0 5.9  Aspartic acid Ser ine Glyc ine  1  10.4 2.9  *  13.3 ) 5,9 5.9  25.1  21.8  From the data supplied by Merck Sharpe and Dohme, methionine is not present in this hydrolysate. However, if this is so, then the v a l i n e is of higher s p e c i f i c a c t i v i t y than the other amino acids.  136.  of most of the b a c t e r i a .  This was somewhat s u r p r i s i n g , for the amounts  of these amino acids in the bacteria as shown by analysis (144,146), and t h e i r s p e c i f i c a c t i v i t i e s  (Table 31)  in the C h l o r e l l a extract were  about the same as the corresponding values for the aspartate and glutamate groups, and yet  incorporation of these amino acids into the s-RNA's was  much lower than that of valine and methionine.  The incorporation of t y r o s -  ine was also higher than one would have expected from the data in Table 3 1 , whereas that of alanine was lower.  The only enzyme systems which catalysed  the formation of prolyl-s-RNA were those of E. c o l ? , £. fluorescens. and yeast. The lack of incorporation of glutamate, g l y c i n e , and p r o l i n e has been reported previously.  Glutamyl-s-RNA synthetase was found by  Alford et a h  inactivated by mild procedures during its  (4) to be e a s i l y  i s o l a t i o n , and Zubay (183) was unable to detect any incorporation of glutamate into s-RNA by crude preparations.  Extracts of various tissues and  organisms which have been examined for glycine activation with ATP have shown l i t t l e or no ATP-PP exchange (104,118).  Although many aminoacyl"  s-RNA synthetases are protected by 2-mercaptoethanol  (5),  it has been report-  ed that alanyl s-RNA synthetase is inhibited by t h i s substance (164).  Since  2-mercaptoethanol was added routinely both to enzyme preparations and to assay mixes, the low incorporation of alanine by most of the enzymes might have been due to inactivation of the alanyl synthetase by the sulfhydryl compound.  Moreover, it has also been found that alanyl-s-RNA synthetase  from pig l i v e r can be preserved in an active form only at the temperature of l i q u i d nitrogen (164), a method of storage not available  in this labora-  tory. On the other hand, the rather high incorporation of the hydro-  137. phobic group of amino acids (leucine, methionine, and tyrosine)  isoleucine, phenylalanine,  valine,  is common, and could be the result either of the  presence of multicomponent s-RNA's for these amino acids, or the ent selection of conditions favouring t h e i r reaction.  It  inadvert-  is known that in  £. c o l i . there is more than one s-RNA for leucine, isoleucine, valine and methionine, but t h i s  is also true for s e r i n e , threonine and glutamic acid  (8,17,147). The pattern of amino acid incorporation into yeast s-RNA was somewhat different than that of the bacteria.  Instead of the leucine group  making up one-third to one-half of the r a d i o a c t i v i t y of the s-RNA, it  ac-  counted, as did the basic amino acids, and valine and methionine, for about 25% of the el**.  The remaining 25% was divided between tyrosine (13%) and  the aspartic acid group and p r o l i n e (6% each). D.  Patterns of amino acid incorporation into heterologous systems The amino acids incorporated by the heterologous s-RNA - enzyme  systems followed the same general scheme as that of the homologous combinations  (Tables 32 - 37).  Inspection of these tables revealed that each  enzyme system, and each s-RNA mixture, contained a l l of the amino acid a c t i v i t i e s which were tested f o r , although in some cases, the incorporation was very low.  Even with the two Pseudomonas s t r a i n s , there was no "perfect  f i t " between heterologous s-RNA's and enzymes.  Moreover, the E.  col?  enzymes appeared to react as well with P. aeruginosa s-RNA as did the enzymes of Achromobacter or P. fluorescens. interesting findings especially  in the cases where the heterologous reac-  t i o n was greater than the homologous one. increase in  There were,'nevertheless, some  In. some of these instances, the  incorporation was due to a corresponding increase in uptake  Table 32a Incorporation of C amino acids by P_. aeruginosa ATCC 9027 enzyme into heterologous s-RNA's, and by heterologous enzymes into P. aeruginosa ATCC 9027 s-RNA 1 4  Microorganism Amino a c i d :  P. aeruginosa ATCC 9027 s-RNA cpm x 10~3  P. aeruginosa 120 Na s-RNA  Enz.  cpm x 10-3  P.  fluorescens A 3.12  s-RNA  Enz.  cpm x 10-3  Achromobacter B81 s-RNA  Enz.  cpm x 10-3  E. c o l i B s-RNA  Yeast  Enz.  cpm x 10-3  s-RNA  Enz.  cpm x 10-3  1.7  5.3  3.6  6,0  0.8  2.0  1.9  3.0  2.4  4.9  4.7  1.7  1.3  2.3  1.2  1.7  1.3  1.6  2.5  0.9  0.4  0  1.4  0  0  1.9  0.3  0  0  0  0.5  0  1.1  0  0  0  1.0  3.8  0  0.5  2.3  5.5  3.2  3.5  3.9  1.5  1.0  2.9  3.1  0  0.9  Val.*  5.0  10.3  5.0  4.6  5.9  3.3  3.2  5H  7.0  7.7  1.2  Leu."  15.2  27.2  10.1  27.4  10.7  6.3  8.1  13.3  16.6  4,0  2.4  k].0  54.5  26.0  51.2  30.7  17.4  23.2  32.9  43.8  15.1  8.7  Bas i c *  8.0  4.0  3.3  4.3  Asp.*  3.9  3.8  3.4  6.5  Glu.*  3.8  2.0  1.1  Ala.  2.4  1.2  Pro.  0.5  Tyr.  Total:  * Basic group - h i s , arg, l y s . Aspartic group - asp, gly, ser. Glutamic group - g l u , thr. Leucine group - leu, i l e u , p h i .  6.9  *Valine group - v a l , met.  Table 32b. Percent of each amino acid incorporated by f_. aeruginosa ATCC 9027 enzymes into heterologous s-RNA's, and by heterologous enzymes into P. aeruginosa ATCC 9027 s-RNA Microorgan ism:  P. aeruginosa ATCC 9027  P. aeruginosa 120 Na  P.  fluorescens A 3.12  Achromobacter B81  E.  coli B  Yeast  Amino a c i d :  s-RNA  s-RNA  Enz.  s-RNA  Enz.  s-RNA  Enz.  s-RNA  Enz.  s-RNA  Enz.  Bas ic  19.4  7.4  12.8  8.5  22.5  9.6  22.9  10.9  13.8  5.1  23.6  Asp.  9.5  7.0  12.9  12.7  6.3  17.3  10.3  14.7  10.8  11.4  15.2  Glu.  9.3  3.6  4.3  4.5  3.9  9.8  5.6  4.9  5.8  6.1  4,3  Ala.  5.8  2.2  0  2.7  0  0  8.1  0.8  0  0  0  Pro.  1.1  1.0  0  2.2  0  0  0  3.0  8.7  0  5.4  Tyr.  5.6  10.1  12.2  6.9  8.4  4.5  9.1  7.2  0  10.4  Val.  12.1  18.9  19  8.9  19.3  19.1  13.8  15.5  16  51  14  Leu.  37.2  49.7  38.8  35  35.8  35  41.3  37.9  26.4  27.2  53.5  12.9  Table 33a Incorporation of C amino acids by P. aeruginosa 120 Na enzyme into heterologous s-RNA's, and by heterologous enzymes into P_. aeruginosa 120 Na s-RNA Microorgan ism: Amino a c i d :  P. aeruginosa 120 Na s-RNA cpm x 10~3  P. aeruginosa ATCC 9027 s-RNA  Enz.  cpm x 10-3  P.  fluorescens A 3.12  s-RNA  Enz.  cpm x 10-3  Achromobacter B81 s-RNA  Enz.  cpm x 10-3  E. c o l i B  s-RNA  Yeast  Enz.  cpm x 10-3  s-RNA  Enz.  cpm x 10-3  Basic  4.5  3.3  4  3.1  4.2  1.2  9.7  4.6  8.5  0.4  2.9  Asp.  4.3  3.4  3.8  5.8  2.4  2.3  2.8  3.8  6.4  1.6  1.7  Glu.  0.9  1.1  2.0  0  0.6  0.8  1.3  0  1.5  0  0.8  Ala.  0  0  1.2  0  0.7  0.6  2.3  0  0  0  0.2  Pro.  0  0  0.5  0  0  0  1.8  1.2  1.5  0  0.6  Tyr.  6.3  3.2  5.5  4.7  3.9  2.5  3.4  3.2  6.2  0  0.8  Val.  9.1  5.0  10.3  5.4  7.8  2.6  5.0  5.8  8.0  5.9  1.4  Leu.  18.1  10.1  27.2  17.2  17.1  3.8  11.2  9.6  25.5  0  5.4  43.2  26.1  54.5  36.0  36.7  13.8  37.5  28.2  57.5  8.0  13.6  Total:  Table 33b Percent of each amino acid incorporated by P. aeruginosa 120 Na enzymes into heterologous s-RNA's, and by heterologous enzymes into P. aeruginosa 120 Na s-RNA Microorganism:  1P. aeruginosa 120 Na  P. aeruginosa ATCC 9027  P.  fluorescens A 3.12  Achromobacter B81  E.  coli B  Yeast  s-RNA  Enz.  s-RNA  Enz.  s-RNA  Enz.  s-RNA  Enz.  8.5  11.5  8.4  25.7  16.4  14.7  5.5  21.4  6.4  16.6  7.5  13.6  11.1  20.4  12.7  0  1.6  5.5  3.4  0  2.6  0  6.1  2.2  0  1.9  4.5  6.2  0  0  0  1.6  1.0  0  0  0  4.7  4.1  2.6  0  4.7  18.3  9.1  11.3  10.8  0  6.0  21.1  18.7  13.3  20.4  13.9  46.7  27.5  29.9  34.2  44.5  Enz.  Amino a c i d :  s-RNA  s-RNA  Bas ic  10.4  12.8  7.4  Asp.  9.9  12.9  7.0  Glu.  2.0  4.3  3.6  Ala.  0  0  Pro.  0  0  Tyr.  14.4  12.2  10.1  13  10.7  Val.  21.1  19  17.9  14.9  Leu.  42.1  38.8  48.7  47.6  16  74 0  9.9 39.6  Table 34a. Incorporation of amino acids by P. fluorescens enzymes into heterologous s-RNA's, and by heterologous enzymes into JP. fluorescens s-RNA  Microorganism: Amino a c i d :  P.  fluorescens A 3.12 s-RNA cpm x 10~3  P. aeruginosa ATCC 9027 s-RNA  Enz.  cpm x 10-3  P. aeruqinosa 120 Na s-RNA  Enz.  cpm x 10-3  Achromobacter B81 s-RNA  Enz.  cpm x 10-3  E. c o l i  s-RNA  B.  Enz.  cpm x 10-3  Yeast  s-RNA  Enz.  cpm x 10-3  Bas ic  7.1  6.9  4.3  4.2  3.1  1.3  7.9  4.0  7.5  0.9  3.4  Asp.  3.5  1.9  6.5  2.4  5.8  1.6  5.6  2.5  8.8  1.2  2.3  Glu.  1.3  1.2  2.3  0.6  0  0.6  2.0  0  3.1  0  0  Ala.  0  0  1.4  0.7  0  0  1.9  0  0.3  0.4  0  Pro.  2.0  0  1.1  0  0  0  1.0  1.1  6.1  0  1.6  Tyr.  3.8  4.0  3.5  3.9  4.7  0.7  2.7  2.5  5.5  0.5  0.9  Val.  6.3  5.9  4.5  7.8  5.4  2.4  5.8  5.6  7.3  7.1  2.4  Leu.  14.5  10.8  27.3  17.1  17.2  7.1  21.5  10.4  17.7  3.3  6.5  38.2  30.7  51.2  36.7  36.0  13.7  48.2  26.0  55.1  13.4  17.0  Total:  Table 34b. Percent of each amino acid incorporated by P.. fluorescens enzymes into heterologous s-RNA's, and by heterologous enzymes into f_. fluorescens s-RNA  Microorgan ism:  1P.  fluorescens A 3.12  P. aeruginosa ATCC 9027  P. aeruqinosa 120 Na  Achromobacter B81  E.  coli B  Yeast  Enz.  s.RNA  Enz.  s-RNA  Enz.  s-RNA  Enz.  s-RNA  Enz.  22.5  8.5  11.5  8.5  9.7  16.4  15.3  13.6  6.6  20.3  9.0  6.3  12.6  6.4  11.6  11.6  9.8  16  8.6  13.2  Glu.  3.2  3.9  4.5  ;i.6  0  4.2  4.2  0  5.6  0  0  Ala.  0  0  2.7  1.9  0  0  4.0  0  0.6  2.5  0  Pro.  5.1  0  2.2  0  0  0  2.0  4.3  11.1  0  9.1  Tyr.  9.9  12.9  6.9  10.7  13  5.4  5.5  9.4  9.9  4.0  5.0  Val.  16.5  19.3  8.8  21.1  14.9  18.3  11.9  21.4  13.2  52.9  13.8  Leu.  37.8  35  53.2  46.7  47.6  51.6  44.5  39.9  32.0  25.4  39  s-RNA  s-RNA  18.5  Asp.  Amino a c i d :  Bas ic  16  Table 35a. Incorporation of C amino acids by Achromobacter B81 enzymes into heterologous s - R N A ' s , and by heterologous enzymes into Achromobacter B81  Microorganism: Amino A c i d :  Achromobacter B81 s-RNA cpm x 10"3  P. aeruginosa ATCC 9027 s-RNA  Enz.  cpm x 10"3  S-RNA  P. aeruginosa 120 Na s-RNA  Enz.  cpm x 10-3  P.  fluorescens A 3.12  s-RNA  Enz.  cpm x 10-3  E. c o l i B  s-RNA  Enz.  cpm x 10-3  Yeast  s-RNA  Enz.  cpm x 10-3  Bas ic  2.9  5.3  1.7  9.7  1.2  7.9  1.4  6.4  3.6  3.0  1.0  Asp.  3.8  2.4  3.0  2.8  2.4  5.6  1.6  3.4  3.1  0.8  1.2  Glu.  2.2  1.3  1.7  1.3  0.8  2.0  0.6  1.1  1.3  0.6  0.3  Ala.  0.3  1.9  0  2.3  0.6  1.9  0  1.2  0.3  0.2  0  Pro.  0  0  0  1.8  0  1,0  0  1.9  1.1  1.4  0.2  Tyr.  0  1.0  1.5  3.4  2.6  2.7  0.8  3.0  1.0  0.4  0.6  Val.  4  3.2  3.3  5.0  2.7  5.8  2.5  6.0  3.5  5.4  1.1  8.1  6.3  11.2  4.1  21.5  7.3  10.4  11.4  2.1  2.0  23.2  17.4  37.5  13.8  48.5  13.7  33.4  24.3  13.7  6.3  Leu. Total:  11 21.0  Table 35b Percent of each amino acid incorporated by Achromobacter B81 enzymes into heterologous s-RNA's, and by heterologous enzymes into Achromobacter B81 s-RNA Microorgan ism:  Achromobacter B81  P.  aeruqinosa ATCC 9027  P. aeruqinosa 120 Na  P.  fluorescens A 3.12  Enz.  s-RNA  25.7  8.4  16.4  17.3  7.5  16.6  5.6  9.8  3.4  1.6  8.1  0  Pro.  0  0  Tyr.  0  Val. Leu.  Amino a c i d :  Enz.  Enz.  E.  coli B  Yeast  s-RNA  Enz.  s-RNA  Enz.  9.9  19.1  14.7  22.2  16.2  11.6  11.9  10.3  12.9  5.7  18.1  5.5  4.2  4.3  3.2  5.2  4.5  4.9  6.2  4.5  4.0  0  3.7  1.1  1.1  0  0  4.7  0  2.0  0  5.8  4.6  10.3  3.3  4.5  8.4  9.1  18.3  5.5  5.5  8.9  4.1  2.7  9.1  19.2  13.8  19.1  13.3  18.7  11.9  18.7  17.8  14.2  39.2  17.6  52.4  35  35.8  29.9  27.5  44.5  57.9  3K2  46.7  15.1  30.8  s-RNA  s-RNA  Bas ic  14  22.9  9.6  Asp.  18.4  10.3  Glu.  10.6  Ala.  s-RNA  Table 36a. Incorporation of C ' amino acids by E. col? enzymes into heterologous s-RNA's, and by heterologous enzymes into E_. col ? s-RNA 4  Microorganism: Amino A c i d :  E. c o l i B  s-RNA cpm x 10~3  P. aeruginosa ATC 9027 s-RNA  Enz.  cpm x 10-3  P. aeruginosa 120 Na s-RNA  Enz.  cpm x 10-3  P.  fluorescens A 3.12  s-RNA  Enz.  cpm x 10-3  Achromobacter B81 s-RNA  Enz.  cpm x 10-3  Yeast  s-RNA  Enz.  cpm x 10-3  Bas ic  8.9  6.0  3.6  8.5  4.6  7.5  4  3.6  6.4  6.6  1.7  Asp.  4.8  4.7  4.9  6.4  3.8  8.8  2.5  3.1  3.4  1.2  1.7  Glu.  !-  2.5  1.6  1.5  0  3.1  0  1.3  1.1  1.5  0.4  Ala.  0.5  0  0.3  0  0  0.3  0  0.3  1.2  1.7  0  Pro.  1.4  3.8  1.0  1.5  1.2  6.1  1.1  1.1  1.9  2.2  0.2  Tyr.  4.0  3.1  2.9  6.2  3.2  5.5  2.5  1.0  3.0  0  1.2  Val.  6.5  5.1  8.0  5.8  7.3  5.6  3.5  6.0  9.3  1.8  Leu.  12.5  16.6  13.3  25.5  9.6  17.7  10.4  11.4  10.4  4.6  3.8  39.9  43.8  32.9  57.5  28.2  55.1  26.0  24.3  33.4  27.0  10.8  Total:  2  7.0  Table 36b Percent of each amino acid incorporated by JE_. col ? enzymes into heterologous s-RNA's, and by heterologous enzymes into E. c o l i s-RNA  Microorganism:  E. c o l i B  P. aeruqinosa ATCC 9027  P. aeruqinosa 120 Na  P.  fluorescens A 3.12  Achromobacter B81  Yeast  s-RNA  s-RNA  Enz.  s-RNA  Enz.  s-RNA  Enz.  s-RNA  Enz.  s-RNA  Enz.  Bas ic  22.3  13.8  10.9  14.7  16.4  13.6  15.3  14.7  19.1  24.2  15.4  Asp.  11.9  10.8  14.7  11.1  13.6  16  9.8  12.9  10.3  4.5  15.4  Glu.  3.0  5.8  4.9  2.6  0  5.6  0  5.2  3.2  5.6  3.5  Ala.  1.2  0  0.8  0  0  0.6  0  1.1  3.7  6.2  0  Pro.  3.6  8.7  3.0  2.6  4.1  11.1  4.3  4.6  5.8  8.0  2.0  Tyr.  10.1  7.2  9.1  10.8  11.3  9.9  9.4  8.9  0  10.6  Val.  16.2  16  15.5  13.9  20.4  13.2  21.4  14.2  17.8  34.6  16.2  Leu.  31.6  37.9  41.3  44.5  34.2  32  39.9  46.7  31.2  16.9  36.5  Amino a c i d :  4.1  Table 37a Incorporation of C amino acids by yeast enzymes into heterologous s-RNA's, and by heterologous enzymes into yeast s-RNA H  Microorganism:  Yeast  Amino a c i d :  s-RNA cpm x 10"3  P. aeruginosa ATCC 9027 s-RNA  Enz.  cpm x 10-3  P. aeruginosa 120 Na s-RNA  Enz.  cpm x 10-3  P.  fluorescens A 3.12  s-RNA  Enz.  cpm x 10-3  Achromobacter B81 s-RNA  Enz.  cpm x 10-3  E. c o l i B  s-RNA  Enz.  cpm x 10-3  0.8  2.9  0.4  3.5  0.9  1.0  3.0  1.7  6.0  1.7  1.7  1.6  2.3  1.2  1.2  0.8  1.7  1.2  0.4  0.9  0.8  0  0  0  0.3  0.6  0.4  1.5  0.4  0  0  0.2  0  0  0.4  0  0.2  0  1.7  Pro.  2.3  0.5  0  0.6  0  1.6  0  0.2  1.4  0.2  2.2  Tyr.  4.6  0.9  0  0.8  0  0.9  0.6  0.6  0.4  1.2  0  Val.  7.3  1.2  7.7  1.4  5.9  2.5  7.4  1.1  5.4  1.8  9.3  Leu.  8.8  2.4  4  5.4  0  6.7  3.4  2.0  2.1  3.8  4.6  36.1  8.7  15.1  13.6  8.0  17.0  13.4  6.3  13.7  10.8  27.0  Bas ic  9.1  Asp.  2.5  Glu.  1.3  Ala.  Total:  2.0  1.3  Table 37b Percent of each amino acid incorporated by yeast enzymes into heterologous s-RNA's, and by heterologous enzymes into yeast S-RNA  P. aeruqinosa 120 Na  fluorescens A 3.12  Yeast  Amino a c i d :  s-RNA  s-RNA  25.2  23.6  5.1  21.4  5.5  20.3  Asp.  6.9  15.2  11.4  12.7  20.4  Glu.  3.5  11.3  6.1  6.1  Ala.  1.1  0  0  Pro.  6.4  5.4  Tyr.  12.7  10.4  Val.  20.3  14  51  Leu.  24.7  27.2  26.4  Bas ic  P. aeruqinosa ATCC 9027  P.  Microorgan ism:  E. c o l i B  s-RNA  Enz.  s-RNA  Enz.  6.9  16.2  22.2  17.4  24.2  13.2  8.9  18.1  5.7  18.4  0  0  0  4.9  1.6  0  0  2.6  0  0  4.7  0  9.1  0  0  6.0  0  5  4.2  Enz.  s-RNA  9.9 39.6  Enz.  74 0  s-RNA  Enz.  Achromobacter B81  4.1  5.6  1.1  0  6.2  3.3  10.4  2.3  8.0  9.1  2.7  12.3  0  13.8  55  17.6  39.2  18.7  34.6  39  26.4  30.8  15.1  42.5  16.9  150. of the leucine group.  For example, the P. aeruginosa ATCC 9027 enzyme  formed nearly double the amount of the P. aeruginosa 120 Na and P. f l u o r e s cens leucyl-, i s o l e u c y l - , and phenylalanyl- s-RNA's as that of its own. In the case of the second £. aeruginosa s t r a i n , valine - methionine incorporation was also doubled, but neither of the other two pseudomonads r e acted as well with the basic amino acids as did P. aeruginosa ATCC 9027. The increase in heterologous reaction between E. c o l i enzyme, and P. aeruginosa 120 Na s-RNA, could also be attributed mainly to the leucine group, and p a r t l y to these amino acids in the s-RNA's of the other strains of £. aeruginosa and of £. fluorescens.  The incorporation of p r o l i n e , for which  E. c o l i has an active enzyme, was nearly t r i p l e d into £. aeruginosa 120 Na s-RNA, and quadrupled into the s-RNA of £. fluorescens. None of the amino acids (except for valine and methionine) was well incorporated into the yeast s-RNA by the bacterial enzymes.  Compared  to the amount incorporated into the s-RNA's in the interbacterial  heterol-  ogous systems, the uptake of the leucine group was e s p e c i a l l y poor in most cases.  From the pattern of amino acid incorporation into yeast s-RNA by  the homologous enzymes however,  it would appear that there may have been  less of the s-RNA's for these amino acids in the yeast preparation than in s-RNA's from the bacteria.  The lack of incorporation of tyrosine into yeast  s-RNA by E. c o l i enzyme has also been reported by Clark and Ezyaguirre and Benzer and Weisblum (14).  (31),  However, the results with valine in this  heterologous system are in contrast to those of L o f t f i e l d and Eigner  (105),  who, using an assay procedure s i m i l a r to that employed here, and valine as the sole amino acid present, found that there was only 18% as much incorporation into yeast s-RNA by the E. c o l i enzyme as into the homologous E. c o l i s-RNA.  The yeast s-RNA and the E. c o l i s-RNA were shown to be in com-  151. p e t i t i o n for the v a l i n e .  The presence in the;.Chlorel la hydrolysate of  methionine, which is not separated paper chromatographically from v a l i n e , would explain the differences between these results and those of L o f t f i e l d and Eigner  (105). The yeast enzymes proved to be much more v e r s a t i l e than the  yeast s-RNA, not from the standpoint of the amount of C ^ incorporated, but from that of the number of amino acids transferred to the bacterial  s-RNA's.  The yeast enzymes for the leucine and basic amino acid groups reacted f a i r l y well with a l l of the bacterial s-RNA's, and there was generally a f u l l complement of amino acids incorporated, although the incorporation was low in each case.  Despite the high cross reaction exhibited between  the E. c o l i enzyme, and the yeast s-RNA, the reverse cross was no better than that with the other b a c t e r i a .  The poor incorporation of  radioactivity  by the yeast enzyme into bacterial s-RNA's may be a problem of slow reaction rates, as previously demonstrated with E_. col i enzyme and yeast s-RNA in the case of valine  (105).  This study was undertaken in the hope that differences  in the  species s p e c i f i c i t y of microbial s-RNA's and amino acyl-s-RNA synthetases could be demonstrated.  However, there proved to be a rather large amount  of heterologous reaction even between those microorganisms which were not closely r e l a t e d .  In the months since this  investigation was  initiated,  there have been several extensive experiments reported in which s i m i l a r results were obtained to those presented here.  The latest demonstration  of the lack of s p e c i f i c i t y between s-RNA's and aminoacyl-s-RNA synthetases has been in the work of Yamane and Sueoka (176), who showed that the enzymes of many microorganism w i l l  interact to a greater or lesser extent  152. with E. c o l i s-RNA.  The one case in which there does appear to be s p e c i f -  i c i t y for some amino acids is that of yeast, since many reports have i n dicated that bacterial enzymes incorporate only poorly a number of amino acids into yeast s-RNA, but that animal enzymes react f a i r l y w e l l . s i t u a t i o n has been v e r i f i e d  in these experiments, although the yeast enz-  yme appeared to show a greater vice versa (Table 37).  This  interaction with the bacterial s-RNA than  Since the experiments done in this study were a l l  performed under the same conditions, the results obtained represent a minimum amount of interaction, which very possibly could be increased if optimum conditions were employed for each system. The experiments of McCarthy and Bolton (107), Giacomoni and Spiegelman (69),  and Goodman and Rich (72)  on hybrid formation between  messenger RNA's and s-RNA's with DNA, are in contrast to these amino acid incorporation s t u d i e s , since hybrid formation appears to occur only between c l o s e l y related b a c t e r i a , there being, for example, very low hybridization between E_. col i and Pseudomonas.  The lack of s p e c i f i c i t y  in the  amino acid incorporation reactions may be due, then, to the enzymes, rather than to the s-RNA's.  153.  GENERAL DISCUSSION The underlying theme of the data from the experiments described in this thesis proved to be the importance of protein synthesis assimilation by the aerobic bacteria studied.  in oxidative  Thus, in both strains of  Pseudomonas aeruginosa, and in Pseudomonas fluorescens. Achromobacter B81, Achromobacter viscosus. Azotobacter a g i l i s . Azotobacter vinelandii and Acetobacter xylinum, the major part of the r a d i o a c t i v i t y  incorporated into  the c e l l s during gl ucose-U-C^ oxidation was found in the proteinaceous fractions.  When more detailed studies were done with P. aeruginosa, under  circumstances  in which protein synthesis was greatly reduced, oxidative as-  s i m i l a t i o n was also markedly decreased.  The addition of chloramphenicol  to c e l l s o x i d i z i n g glucose-C^ resulted in a reduction of about 25% in the total  amount of oxidative a s s i m i l a t i o n , and a large part of the assimilated  material was found in the metabolic pool as free amino acids. c e l l s p r i o r to glucose oxidation, i . e . ,  aerating the c e l l s  Starving the  in a non nutrient  medium, proved to decrease oxidative assimilation to a greater extent  (40%)  than did the presence of the a n t i b i o t i c .  There are several possible ex-  planations for t h i s phenomenon which are,  in each instance, based on the  use of protein and nucleic acid as major endogenous reserves by P. aeruginosa (29,75).  One result of the starvation period was a greatly diminished  a c t i v i t y of the aminoacyl s-RNA synthetases, which are necessary for protein synthesis.  A second was the accumulation of radioactive glutamate in the  metabolic p o o l , perhaps due to a lack of transaminases. the decrease in the rate of pyruvate oxidation.  A t h i r d result was  This led to a slower forma-  tion of © f - k e t o g l u t a r a t e , which Duncan and Campbell (55) showed to be a key compound in oxidative assimilation by P. aeruginosa.  Therefore, the over-  154. a l l effect of the period of starvation was the s a c r i f i c e of v i t a l  cellular  constituents, which were resynthesized as soon as a substrate became a v a i l able. The studies of oxidative assimilation in P_. aeruginosa were extended to an investigation of the cytological s i t e s of the assimilated material.  It was found that the soluble proteins of the cytoplasm, which  are mainly enzymes, contained the major portion of the c'^* of the c e l l extracts.  This indicated that these enzymes were turning over rapidly  during glucose oxidation.  Experiments with chloramphenicol revealed, that  although t h i s turnover was inhibited by 87%, glucose oxidation proceeded at almost the usual rate.  Therefore, it was tentatively  concluded that,  under normal circumstances, most of the enzymes synthesized were synthetic, rather than degradative in function.  Support for this conclusion was found  in the incorporation of c'^ during glucose d i s s i m i l a t i o n , either by freshly harvested or by starved c e l l s ,  into the f r a c t i o n  t e i n which includes aminoacyl s-RNA synthetases.  of the cytoplasmic p r o Further experiments with  starved c e l l s showed that the a c t i v i t y of these enzymes was decreased during the starvation period, but Was rapidly restored during oxidative ass imi1 at ion. Since protein synthesis seemed to be of such great  importance in  oxidative assimilation of glucose by JP. aeruginosa, some investigations were made on the aminoacyl s-RNA synthetases  in this microorganism.  As  mentioned above, these enzymes were found in the cytoplasm, and further experiments with the ribosomes and "membranes" showed that these fractions were inactive  in the formation of aminoacyl s-RNA.  Because preliminary ex-  periments had revealed that yeast s-RNA was not charged with amino acids by P. aeruginosa synthetases, the studies were extended to a determination of  155. species s p e c i f i c i t y between the enzymes and s-RNA's of other microorganisms.  Three of the bacteria which had been found to have a pattern of  oxidative assimilation s i m i l a r to that of P.. aeruginosa were selected - a second s t r a i n of JP. aeruginosa, as well as P. fluorescens. and Achromobacter 681.  For comparative purposes, a more d i s t a n t l y related organism, E. c o l i .  which is known to assimilate glucose into a carbonaceous reserve product, was chosen.  Yeast was also included because of i t s poor cross reaction with  IP. aeruginosa.  It was found that there was l i t t l e s p e c i f i c i t y exhibited  between bacterial enzymes and s-RNA's since good heterologous reactions were obtained, but that the yeast gave a poor cross reaction with each of the bacteria except the E. c o l i enzyme system.  In the past few months, the same  general conclusion as to the lack of species s p e c i f i c i t y has been reached by a number of authors  (45,52,176).  Oxidative assimilation also occurred into the nucleic acids, primarily  into the RNA.  Under normal conditions, the ribosomal RNA showed  a very slow turnover, but i f starved c e l l s were used, the into the RNA was greatly  incorporated  increased, supporting the contention of Duncan and  Campbell (55) and of C l i f t o n (36) that oxidative assimilation takes place to replace endogenous reserves.  Chloramphenicol also increased the incor-  poration of C l ^ into the ribosomal RNA, in agreement with previous reports on i t s action  (9,66,67).  The nitrogen f o r synthesis of these components in JP. aeruginosa was found by Duncan and Campbell (55) to be derived from the ammonia p r o duced during endogenous respiration by this microorganism.  A similar con-  clusion was reached in the cases of the other aerobic bacteria studied here, where the uptake of c'^ was p a r a l l e l e d by the uptake of endogenously produced ammonia.  Like the s t r a i n of f_. aeruginosa used by Duncan and  156. Campbell (55),  oxidative assimilation by P, aeruginosa. P, f luorescens.  Achromobacter B81, A. viscosus and Acetobacter xylinum involved the excretion of pacemaker compounds, whose presence in the surrounding medium during glucose d i s s i m i l a t i o n increased the extent of oxidative a s s i m i l a t i o n . Not a l l of these compounds were keto acids.  They served, under the condi-  tions of these experiments, to slow glucose oxidation so that carbon would still  be present to permit assimilation of ammonia, as it became available  from the oxidation of endogenous substrates.  The bacteria which formed  these pacemaker compounds assimilated more C ^ than those, such as the two Azotobacter species, which did not excrete such intermediates rounding medium.  into the s u r -  These organisms, by v i r t u e of t h e i r a b i l i t y to f i x atmos-  pheric nitrogen, should not have lacked nitrogen for the synthesis of c e l lular material, yet they assimilated the least c'^ of any of the bacteria studied.  From these r e s u l t s ,  it would seem that an investigation of o x i d a -  t i v e assimilation should include a determination of the  extracellular  products appearing during the course of substrate oxidation. Preliminary  experiments carried out on ammonia assimilation in f_.  aeruginosa and in P. fluorescens showed that there was a requirement for concurrent substrate oxidation during ammonia a s s i m i l a t i o n .  This oxidation  would provide cofactors, such as reduced pyrimidine nucleotides, as well as energy for transport of the aminated compound across c e l l b a r r i e r s .  No  evidence was found for the direct amination of pyruvate through alanine dehydrogenase.  In contrast, the presence of labelled glutamate in the metab-  o l i c pool, the p a r a l l e l uptake of ammonia and disappearance of ©C-ketoglutarate from the surrounding medium (55),  and the presence of an active glutamic  acid dehydrogenase in P_. aeruginosa (55),  a l l supported the assimilation of  ammonia through the conversion of ©C-ketoglutarate to glutamate.  157. Despite the emphasis on nitrogenous c e l l components as products of oxidative assimilation  in P, aeruginosa and the other aerobes studied,  from glucose was also incorporated into non nitrogenous constituents. In Achromobacter B81 a polymeric carbohydrate was formed, and l i p i d s were generally highly labelled in most of the bacteria.  From a cytological  investigation of JP, aeruginosa, most of the l i p i d was found to be in the "membrane" f r a c t i o n ,  its label being increased by treatment of the c e l l s  with chloramphenicol, or by p r i o r starvation.  The reason for this  increase  was not established, but it may be the result of the channeling of more substrate into l i p i d when protein synthesis usually taking precedence.  is decreased, the l a t t e r process  This increase, although s u b s t a n t i a l ,  especially  in the case of the a n t i b i o t i c treated c e l l s , did not compensate for the loss of protein synthesis,  indicating that P. aeruginosa does not form a  carbonaceous reserve, even in the absence of protein synthesis.  158.  BIBLIOGRAPHY 1.  . Abrams, A . , and P. McNatnara.  1962.  in isolated bacterial membranes.  Polynucleotide phosphorylase J . B i o l . Chem.  237: 170-175.  2.  Adler, E., V. Hellstrom, G. Gunther, and H. von Euler. 1938. Uber den enzymatischen abbau und aufbau der glutaminsaure in Bacterium c o l i . Z. Physiol. Chem. Hoppe-Seyler's 255: 14r26.  3.  Adler, E., G. Gunther, and J . E . Everett. 1938. Uber den enzymatischen abbau und aufbau der glutaminsaure in hefe. Z. Physiol. Chem. Hoppe-Seyler's 255: 27-35.  4.  A l f o r d , M.A., M. Brotman, M.A. Chudy, and M.J. Fraser. 1963. The activation of glutamic acid and glutamine in mammalian t i s s u e . Can. J . Biochem. Physiol. 4l_: 1135-1145.  5.  A l l e n , E.H., E. Glassmann, and R.S. Schweet. I960. Incorporation of amino acids into ribonucleic a c i d . I. The role of activating enzymes. J . B i o l . Chem. 2J5_: 1061 -1068.  6.  A l l i s o n , R.M., and R.H. B u r r i s .  7.  Altenbern, R.A., and R.D. Housewright. 1951. Alanine synthesis and carbohydrate oxidation by smooth Brucella abortus. J . Bacterid. 62: 97-105.  8.  Apgar, J . , R.W. Hoiley, and S.H. M e r r i l l . I960. Countercurrent d i s t r i b u t i o n of yeast ' s o l u b l e ' ribonucleic acids in a modification of the Kirby system.. Biochim. Biophys. Acta 5_3_: 220-221.  9.  Aronson, A . I . , and S. Spiegelman. 1961. Protein and ribonucleic acid synthesis in a chloramphenicol inhibited system. Biochim. Biophys. Acta 5_3_: 70-84.  1957.  rogen by Azotobacter v i n e l a n d i i .  Kinetics of f i x a t i o n of n i t -  J . B i o l . Chem.  224: 351-364.  10.  Aronson, A . I . , and S. Spiegelman. 1961. On the nature of the r i b o nucleic acid synthesized in the presence of chloramphenicol. Biochim. Biophys. Acta 5_3_: 84-95.  11.  Barker, H.A.  12.  Bautz, E.K.F., and B.D. H a l l . 1962. The isolation of T-4 s p e c i f i c RNA on a DNA-cellulose column. Proc. N a t l . Acad. S c i . U.S. 48: 400-408.  13.  B e l l , D., R.V. Tomlinson, and G.M. Tener. 1964. Chemical studies on mixed soluble ribonucleic acids from yeast. Biochemistry 3_: in press.  1936.  The oxidative metabolism of the colorless, alga,  Prototheca zopfi i.  J . C e l l u l a r Comp. Physiol.  /  8: 231-250.  159. 14.  Benzer, J . , and B. Weisblum. 1961. On the species s p e c i f i c i t y of acceptor RNA and attachment enzymes. Proc. Natl. Acad. S c i . U.S. kj_: 1149-1154.  15.  Berezorskaya, N.N. 1962. Some properties of a p u r i f i e d enzyme system catalysing the direct amination of pyruvic a c i d . Akual'ne. Vopr. Sovrem. Biokhim. 2: 130-137.  16.  Berg, P. 1961. S p e c i f i c i t y of protein synthesis, p. 293 324. In J.M. Luck, F.W. A l l e n , and G. McKinney (eds.), Annual review of biochemistry, V o l . 30. Annual Reviews, Inc., Palo A l t o , C a l i f orn i a .  17.  Berg, P., F.H. Bergmann, E.J. Ofengand, and M. Dieckmann. 1961. The enzymic synthesis of amino acyl derivatives of ribonucleic a c i d . The mechanism of leucyl-, v a l y l - , i s o l e u c y l - , and methionyl ribonucleic acid formation. J . B i o l . Chem. 236: 1726-1734.  18.  Bernheim, F. 1963. Effect of some surface active drugs on assimi l a t i o n of ammonium ions by a s t r a i n of Pseudomonas aeruginosa. Experientia _lj9: 8-9.  19.  Bernheim, F., and W.E. DeTurk. 1957. The effect of temperature on ammonia assimilation in a Mycobacterium. Arch. Biochem. Biophys. 21: 451-457.  20.  Bezborodov, A.M. 1963. The presence of alanine dehydrogenase and fumarase in c e l l free extracts of Streptomyces species. Mikrobiologiya 3J2: 20-26.  21.  Blnnie, B., E.A. Dawes, and W.H. Holmes. 1959. Metabolism of Sarcina lutea. IV. Patterns of oxidative a s s i m i l a t i o n . Biochim. Biophys. Acta 40: 237-251.  -  22.  Bolton, E.T., and B.J. McCarthy. 1962. A general method for the i s o l a t i o n of RNA complementary to DNA. Proc. N a t l . Acad. S c i . U.S. 48: 1390-1397.  23.  Braunstein, A.E. 1957. Les roies principales de 1 assimilation et d i s s i m i l a t i o n de l'azote chez les animaux, p. 339-389. _[n F.F. Nord (ed.), Advances in enzymology, V o l . 19. Interscience Publishers, L t d . , New York.  24.  Brooks, P., A.R. Crathorn, and G.D. Hunter. 1959. S i t e of synthesis of the peptide component of the c e l l wall of Baci1lus megaterium. Biochem. J . 23_: 396-401.  25.  Brouvers, L. 1961. Teneur en alanine-deshydrogenase de diverses souches de Rhizobium t r l f o l l i . B u l l . soc. chem. b i o l . 43: 593-599.  26.  Burk, R.R., and J . A . Pateman. 1962. Glutamic acid and alanine dehydrogenases determined by one gene in Neurospora crassa. Nature  1  160. 196: 450-451. 27.  Burris, R.H.  1942.  Distribution of isotopic nitrogen in Azoto-  28.  Butler, J . A . V . , on the s i t e megaterium, synthesis.  29.  Campbell, J . J . R . , . A . F . Gronlund, and M.G. Duncan.  30.  Campbell, J . J . R . , L.A. Hogg, and G.A. Strasdine. 1962. Enzyme d i s t r i b u t i o n in Pseudomonas aeruginosa. J . B a c t e r i o l . 83: 1155-1160.  31.  Clark, J . M . , and J . P . Eyzaguirre. 1962. Tyrosine activation and transfer to soluble ribonucleic a c i d . P u r i f i c a t i o n and study of the enzyme of hog pancreas. J . B i o l . Chem. 237: 3698-3702.  32.  C l i f t o n , C.E.  33.  C l i f t o n , C.E. 1946. Microbial assimilations, p. 269-308. Jn, F.F. Nord (ed.), Advances in enzymology, V o l . 6. Interscience Publishers, Inc., New York.  34.  C l i f t o n , C.E. 1951. Assimilation by bacteria, p. 531-547. Jn. C H . Werkman and P.W. Wilson (eds.), Bacterial physiology. Academic Press, Inc., New York.  35.  C l i f t o n , C.E.  36.  C l i f t o n , C.E. 1963. Endogenous metabolism and oxidative a s s i m i l a tion of typical bacterial species. Symposium on endogenous metabolism with special reference to bacteria. Ann. N.Y. Acad. Sci. 102: 655-668.  37.  C l i f t o n , C . E . , and W.A. Logan. 1939. On the relation between ass i m i l a t i o n and respiration in suspensions and in cultures of Escherichia col ?. J . Bacteriol. 3_7_: 523~540.  38.  C l i f t o n , C . E . , and J.M. Sobek.  39.  Cohen, P.P., and G.W. Brown. I960. Ammonia metabolism and urea b i o synthesis, p. 161-245. Jn. M. Florkin and H.S. Mason (eds.), Comparative biochemistry, V o l . II. Academic Press, Inc., New York.  bacter v i n e l a n d i i .  J . B i o l . Chem.  143: 509-517.  G.N. Godson, and G.D. Hunter. 1961. Observations and mechanism of protein biosynthesis in Baci11 us p. 349"362. Jhn R.J. Harris (ed.), Protein b i o Academic Press, New York.  ous metabolism of Pseudomonas. 677.  1937.  in respiring c e l l s .  1962.  Endogen-  102; 669-  On the p o s s i b i l i t y of preventing assimilation Enzymologia  4: 246-253.  Oxidative assimilation and d i s t r i b u t i o n of  glucose in Bacillus cereus.  Bacillus cereus.  1963.  Ann. N.Y. Acad. S c i .  J . Bacteriol.  1961.  J . Bacteriol.  83: 66-69.  Endogenous respiration of  82: 252-256.  161. 40.  Conway, E.J.  41.  Cook, R.P., and M. Stephenson. 1928. Bacterial oxidations by molecular oxygen. I. The aerobic oxidation of glucose and its fermentation products in its relation to the v i a b i l i t y of the organism. Biochem. J . 2j2: 1368-1386.  42.  Cook, R.P., and B. Woolf. 1928. The deamination and synthesis of 1 -aspartic acid in the presence of bacteria. Biochem. J . 22; 474-481.  43.  Countryman, J . L . , and E. V o l k i n . 1959. Nucleic acid metabolism and ribonucleic acid heterogeneity in Escherichia c o l i . J, Bacteriol. 2§.: 41-48.  44.  C r i c k , F.H.C. 1957. Discussion - Deoxyribosenucleic acid and p r o tein synthesis, p. 26. _hn The structure of nucleic acids and t h e i r role in protein synthesis. Cambridge University Press, Cambridge.  45.  Crouch, D. 1963. Oxidative metabolism of Hydrogenomonas f a c i l i s . Doctoral d i s s e r t a t i o n . Stanford University. Stanford, C a l i f ornia.  46.  Dagley, S.,  and A.R. Johnson.  47.  Dagley, S.,  A. White, D.G. Wild, and J . Sykes.  48.  De Ley, J . ,  and J . S c h e l l .  49.  DeTurk, W.E.  50.  Doctor, B.P., and C M . Connelly. 1961. Separation of yeast amino acid-acceptor ribonucleic acids by counter current d i s t r i b u t i o n in modified Kirby's system. Biochem. Biophys. Res. Comm. 6: 201-204.  51.  Doctor, B.P., and J . A . Mudd. 1963. Species s p e c i f i c i t y of amino acid acceptor ribonucleic acid and aminoacyl soluble ribonucleic acid synthetases. J . B i o l . Chem. 238: 3677-3681.  52.  Doudoroff, M. 1940. The oxidative assimilation of sugars and r e lated substances by Pseudomonas saccharophi l a . Enzymologia 9_: 59-72.  53.  Doudoroff, M., and R.Y. Stanier. 1959. Role of poly-yff-hydroxybutyric acid in the assimilation of organic carbon by bacteria. Nature 183: 1440-1442.  3rd ed.  1950.  MicrodIffus ion analysis and volumetric e r r o r .  Crosby, Lockwood and Sons L t d . , London.  1953.  The relation between l i p i d and  polysaccharide content of Bacterium c o l i . Acta JJ_: 158-159. proteins in ribosomes by bacteria. by Acetobacter acet i. 1957.  by P. aeruq inosa.  1959.  Biochim. Biophys.  Nature  1962.  194; 25-27.  Oxidation of several  J . Bacteriol.  Synthesis of  27_: 445-451.  substrates  Effect of streptomycin on ammonia assimilation Abstracts Fed. Proc.  j_6: 292.  162. 54.  Duncan, M. 1962. Oxidative assimilation of glucose by Pseudomonas aeruginosa. M.Sc. Thesis, The University of B r i t i s h Columbia. Vancouver, B.C.  55.  Duncan, M., and J . J . R . Campbell.  56.  El Hawari, M.F.S., and R.H.S. Thompson.  1962.  Oxidative assimilation of  glucose by Pseudomonas aeruginosa.  J . Bacteriol. 1953.  84: 784-792.  Separation and e s t -  imation of blood keto acids by paper chromatography.  J.  5J.: 341-347.  Biochem.  57.  E l l i o t , W.H., and E.F. Gale. 1958. Glutamine-synthesizing system of Staphylococcus aureus: its i n h i b i t i o n by crystal v i o l e t and methionine sulphoxide. Nature 161: 129-130.  58.  Fairhurst, A . S . , H.K. King, and C.E. Sewel1. 1956. Studies in amino acid biogenesis: the synthesis of alanine from pyruvate and ammonia. J . Gen. M i c r o b i o l . Jj>,: 106-120..  59.  Fincham, J . R . S . 1951. The occurrence of glutamic dehydrogenase in Neurospora and its apparent absence in certain mutant s t r a i n s . J . Gen. M i c r o b i o l . £: 793-806.  60.  Forsyth, W.G.C., A.C. Hayward, and J.B. Roberts. 1958. Occurrence of poly-/?-hydroxybutyr ic acid in aerobic Gram-negative bacteri a . Nature 182: 800-801.  61.  Fraser, M.J. 1962. A s e n s i t i v e method for measurement of aminoacylribonucleic acid synthetase a c t i v i t i e s . Can. J . Biochem. Physiol. 40: 653-666.  62.  Fraser, J . J . , and D.B. Klass. 1963. Partial p u r i f i c a t i o n and properties of prolyl-RNA synthetase of rat l i v e r . Can. J . Biochem. Physiol. 4]_: 2123-2139.  63.  Freeze, E.,  64.  Friedemann, T . E . 1957. Determination of *C-keto acids, p. 414-418. In S.P. Colowick and N.O. Kaplan (eds.), Methods in enzymology, V o l . III. Academic Press, Inc., New York.  65.  Gaffron, H.  66.  Gale, E.F. 1947. The assimilation of amino acids by bacteria. I. The passage of certain amino acids across the c e l l wall and t h e i r concentration in the internal environment of Streptococcus faecal i s . J . Gen. M i c r o b i o l . J_: 53"76.  67.  Gale, E.F. 1953. Assimilation of amino acids by Gram p o s i t i v e bacte r i a , and some actions of a n t i b i o t i c s thereon, p. 287-371. Jn. M.L. Anson, K. Bailey, and J . Y . Edsall (eds.), Advances in p r o tein chemistry, V o l . VIII. Academic Press, New York.  and J . Oosterwyck.  hydrogenase.  chem. Z.  1935.  Biochemistry  1963.  The induction of alanine de-  2_: 1212-1216.  Uber den stoffwechsel der Purpurbakterein.  27_5_: 301-319.  Bio-  163. 68.  Giacomoni, D., and S. Spiegelman. 1962. Origin and biologic i n d i v i d u a l i t y of the genetic dictionary. Science 138: 1328-  1331.  69.  Giesberger, G. 1936. Beitrage zur Kenntis der Gattung Sp?r?11 urn Ehbg. D i s s e r t a t i o n , University of Utrecht. Quoted from / C l i f t o n , C.E. 1946. Microbial assimilations, p. 269-308. _ln_ F.F. Nord (ed.), Advances in enzymology, V o l . 6. Interscience Publishers, L t d . , New York.  70.  Gilby, A.R., A.V. Few, and K. McQuillen. 1958. The chemical composition of the protoplast membrane of Micrococcus lysodeckticus. Biochim. Biophys. Acta 29_: 21-29.  71.  Goldman, D.S. 1959. Enzyme systems in mycobacteria. VII. Purifi c a t i o n , properties and mechanism of action of the alanine d e hydrogenase. Biochim. Biophys. Acta 3.4: 527-539.  72.  Goodman, H.M., and A. Rich. 1962. Formation of a DNA-soluble RNA hybrid and i t s relation to the o r i g i n , evolution and degeneracy of soluble RNA. Proc. N a t l . Acad. S c i . U.S. 48: 2101-2109.  73.  Grado, C , and C . E . Ballou. 1961. Myo-inositol phosphates obtained by alkaline hydrolysis of beef brain phosphoinosit ides. J . B i o l . Chem. 2j$6: 54-60.  74.  Gronlund, A . F . , and J . J . R . Campbell. 1961. Nitrogenous compounds as substrates f o r endogenous respiration in microorganisms. J . B a c t e r i o l . . 8l_: 721-724.  75.  Gronlund, A . F . , and J.J.R. Campbell. 1963. Nitrogenous substrates of endogenous respiration in Pseudomonas aeruginosa. J . Bacte r i o l . 86: 58-66.  76.  Gros, F., S. Naono, C. Woese, C. Willson, and G. A t t a l d i . 1963. Studies on the general properties of Escherichia c o l i messenger ribonucleic a c i d , p. 387-408. Jji H.I. Vogel, V. Bryson, and J.0. Lampen (eds.), Information macromolecules: A symposium. Academic Press, New York.  77.  Grossowicz, N., E. Wainfan, E. Borak, and H. Waelsch. 1950. The enzymatic formation of hydroxamic acids from glutamine and a s paragine. J . B i o l . Chem. 187: 111-125.  78.  Haldar, D., H. B a l , and A.N. Chatterjee. 1962. Action of some a n t i b i o t i c s in V i b r i o cholera. Ann. Biochem. E x p t l . Med. 22: 191-  196.  79.  Hartmann, G . , and V. Coy. 1961. Fraktionierung der aminosaure spez i f i s c h e n lossichen ribonukleinsaure. Biochim. Biophys. Acta  47_: 612-613.  80.  Hauser, G . , and M.L. Karnovsky.  1954. Studies on the production of  164. g l y c o l i p i d e by Pseudomonas aeruginosa. 654.  68: 645-  81.  Hauser, G., and M.L. Karnovsky.  82.  Hestrin, S. 1948. The reaction of acetyl chloride and other carboxyl i c acid derivatives with hydroxylamine and its analytical a p p l i c a t i o n . J . B i o l . Chem. ]80: 249-253.  83.  Hoagland, M.B., M.L. Stephenson, J . F . Scott, L.I. Hecht, and P.C. Zamecnik. 1958. A soluble ribonucleic acid intermediate in p r o t e i n synthesis. J . B i o l . Chem. 231: 241-257.  84.  Hoi ley, R.W., J . Apgar, B.P. Doctor, J . Farrow, M.A. Marini, and S.H. Merrill. 1961. A s i m p l i f i e d procedure for the preparation of tyrosine and valine acceptor fractions of yeast " s o l u b l e r i b o nucleic a c i d " . J . B i o l . Chem. 236: 200-202.  85.  Holme, T . , and H. Palmstierna. 1955. On the glycogen of Escherichia col? B; its synthesis and breakdown and its s p e c i f i c l a b e l ling with 1%. Acta Chem. Scand. LP_: 1557-1562.  86.  Hong, M.M., S.C.. Schen, and A.E. Braunstein. 1959. Distribution of L_-alanine dehydrogenase and ^-glutamic dehydrogenase in b a c i l l i . . Biochim. Biophys. Acta 3j6: 288-289.  87.  Hong, M.M., S.C. Schen, and A.E. Braunstein. 1959. The main path of nitrogen assimilation in Baci1lus subt?1 i s . Biochim. Biophys. Acta 36: 290-291.  88.  Horowitz, J . , A. Lombard, and E. Chargaff. 1958. Aspects of the s t a b i l i t y of a bacterial ribonucleic a c i d . J . B i o l . Chem. 233: 1517-1522.  89.  Hunter, G.D., P. Brookes, A.R. Crathorn, and J . A . V . Butler. 1959. Intermediate reactions in protein synthesis by the isolated cytoplasmic-membrane f r a c t i o n of Bacillus megaterium. Biochem. J . Z l : 369-376.  90.  Hunter, G.D., and G.N. Godson. 1962. The f i n a l stages of protein synthesis and the role of l i p i d s in the process. J . Gen. Microb i o l . .29.: 65-75.  91.  Ichihara, H., H. Kanagawa, and M. Uschida.  92.  Jackson, W.T., and M.J.  93.  J a r v i s , F.G., and Johnson, M.J.  of L-rhamnose.  ase.  1958.  J . Bacteriol.  J . B i o l . Chem.  J . Biochem. (Tokyo)  Pseudomonas aeruginosa.  233: 287-291.  42: 439-447.  Johnson.  in Torulopsis ut M Is.  Studies on the biosynthesis  1961.  J . Bacteriol. 1949.  1955.  Studies on aspart-  Pathway of sucrose oxidation 81.: 182-188.  A g l y c o l i p i d e produced by  J . Am. Chem. Soc.  21 4124-4126. :  165 94.  K e l l e r , E.B.,  and R.S.  95.  Kretovich, V.L. 1958. The biosynthesis of dicarboxylic amino acids and enzymic transformation of amides in p l a n t s , p. 319~340. .In. F.F. Nord (ed.), Advances in enzymology, V o l . 20. Interscience Publishers Inc., New York.  96.  Kretovich, V . L . , and M. Kasperek.  RNA's from E. c o l i .  Anthony.  1963.  The two leucine acceptor  Abstracts Fed. Proc.  pyruvate in different plants.  1961.  22: 231.  Amino acid synthesis from  Biokhlmiya  2jS: 592-596.  97.  Kretovich, V . L . , and E. Krause. 1961. Biosynthesis of amino acids in bakers' yeast in the presence of ammonia. Mikrobiologiya 2p_: 881-886.  98.  Kritzmann, M.G. 1947. The mechanism of formation of amino acids in surviving animal tissues from pyruvate and ammonia. J . B i o l . Chem. Jj6_7_: 77-100.  99.  Lacks, S.,  and F. Gros.  I960.  A metabolic study of the RNA-amino  acid complexes in Escherichia c o l ? .  J . Mol. B i o l .  ]_: 301-320.  100.  Lamanna, C , and M.F. Mallette. 1959. Basic bacteriology: Its bio, logical and chemical background, p. 608-624. The Williams and Wilkins Company, Baltimore.  101.  Lemoigne, M. 1927. Etudes sur l'autolyse microbiene, origine de 1' acide #-oxybutyrique forme par autolyse. Annal. Inst. Pasteur 4J.: 148-165.  102.  Lemoigne, Ii.  103.  Levine, S., H.J.R. Stevenson, E.C. Tabor, R.H. Bordner, and L.A. Chambers. 1953. Glycogen of enteric bacteria. J . B a c t e r i o l . 66: 664-670.  104.  Lipmann, F.  105.  L o f t f i e l d , R.B.,  106.  Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. 1951. Protein measurement with the Folin-phenol reagent. J . B i o l . Chem. 19J.: 265-275.  107.  McCarthy, B.J., and E.T. Bolton. 1963. An approach to the measurement of genetic relatedness among organisms. Proc. N a t l . Acad. S c i . U.S. 5.0: 156-164.  108.  Macrae, R.M., and J . F . Wilkinson.  Chem. Acta  1946.  Fermentation of acide /ff-hydroxybutyrique.  2j9_: 1303-1310.  1958.  Introduction, Symposium on amino acid a c t i v a t i o n .  Proc. Natl. Acad. S c i . U.S.  transfer RNA.  Helv,  and E.A. Eigner.  44: 67~73. 1963.  Acta Chem. Scand.  Species s p e c i f i c i t y of  T7_: S117-S122.  1958.  Poly-/?-hydroxybutyrate  metabolism in washed suspensions of Bacillus cereus and Bac? 1 lus  166.  megaterium.  J.  Gen. M i c r o b i o l .  J9_: 210-222.  109.  McCormick, N.G., and H.O. Halvorson. 1964. P u r i f i c a t i o n and properties of L- alanine dehydrogenase from vegetative c e l l s of Bacillus cereus. J . B a c t e r i o l . 8_7_: 68-74.  110.  McLean, D.C., and K.C. Fisher. 1947. The relationship between oxygen consumption and the u t i l i z a t i o n of ammonia for growth in Serratia marcescens. J . B a c t e r i o l . j>4: 599-607.  111.  Macleod, L.D., and I. Smedley-Maclean. 1938. The carbohydrate and fat metabolism of yeast. V. The synthesis of fat from acetic a c i d : the influence of metallic ions on carbohydrate and fat storage. Biochem. J . 3_2_: 1571-1582.  112.  McQuillen, K., R.B. Roberts, and R.J. B r i t t e n . 1959. nascent protein by ribosomes in Escherichia c o l i . Acad. S c i . U.S. 45_: 1437-1444.  113.  Marmur, J . , S. Falkow, and M. Mandel. 1963. New approaches to bacte r i a l taxonomy, p. 329-377. Jn. C.E. C l i f t o n (ed.), Annual r e view of microbiology. Annual Reviews, Inc., Palo A l t o , C a l i f o r n i a .  114.  Matthaie, J . H . , O.W. Jones, R.G. Martin, and M.W. Nirenberg. 1962. Characteristics and composition of RNA coding u n i t s . Proc. N a t l . Acad. S c i . U.S. 48: 666-676.  115.  Midwinter, G.G., and R.D. Batt. I960. Endogenous respiration and oxidative assim?lation in Nocardia c o r a l l l n a . J . Bacteriol. 79: 9-17.  116.  Mortenson, L.E. 1962. incorporation. Jn. bacteria, V o l . III.  117.  Nirenberg, M.W., and Matthaie, J . H . 1961. The dependence of c e l l free protein synthesis in JE. col? upon naturally occurring or synthetic polyribonucleotides. Proc. Natl. Acad. S c i . U.S. 47: 1588-1601.  118.  Nismann, B., F.H. Bergmann, and P. Berg. 1957. Observations on amino acid-dependent exchanges of inorganic pyrophosphate and ATP. Biochim. Biophys. Acta 26: 639-640.  119.  Nomura, M., and J . D . Watson. 1959. Ribonucleoprotein protein p a r t icles within chloromycetin-inhibited Escherichia c o l ? . J . Mol. Biol. U 204-217.  120.  N o r r i s , F.C., and J . J . R . Campbell. 1949. The intermediate metabolism of Pseudomonas aeruginosa. III. The applicat ion of paper chromatography to the i d e n t i f i c a t i o n of gluconic and 2-ketogluconic acids, intermediates in glucose oxidation. Can. J . Res.  C.  27_: 253-261.  Synthesis of Proc. N a t l .  Inorganic nitrogen assimilation and ammonia I.C. Gunsalus and R.Y. Stanier (eds.), The Academic Press, Inc., New York.  167. 121.  Norris, F.C., J.J.R. Campbell, and P.W. Ney. 1949. The intermediate metabolism of Pseudomonas aeruginosa. I. The status of endogenous r e s p i r a t i o n . Can. J . Res. p_. 27_: 157-164.  122.  O'Connor, R.J., and H.O. Halvorson. I960. Intermediate metabolism of aerobic spores. V. The p u r i f i c a t i o n and properties of ].alanine dehydrogenase. Arch. Biochem. Biophys. 9J.: 290-299.  123.  O'Connor, R.J.,  124.  Olson, J . A . , and C.B. Anfinson. 1953. Kinetic and equilibrium studies on c r y s t a l l i n e {.-glutamic acid dehydrogenase. J . B i o l . Chem. 202: 841-856.  125.  P i c k e t t , M.J., and C.E. C l i f t o n . 1943. The effect of s e l e c t i v e poisons on the u t i l i z a t i o n of glucose and intermediate compounds by microorganisms. J . C e l l u l a r Comp. Physiol. 22: 147-165.  126.  Pollak, J . K . , and D. F a i r b a i r n . 1955. The metabolism of Ascaris lumbricoides ovaries. II. Amino acid metabolism. Can. J . Biochem. Physiol. 3J.: 307-316.  127.  Quastel, J . H . , and B. Woolf. 1926. The equilibrium between 1aspartic a c i d , fumaric a c i d , and ammonia in presence of resting b a c t e r i a . Biochem. J . 20: 545~555.  128.  Reich, E., G. Acs, B. Mach, and E.L. Tatum. 1963. Some properties of ribonucleic acid metabolism in mammalian and bacterial c e l l s , p. 317-333. i n H.I. Vogel, V. Bryson, and J . O . Lampen (eds.), Informational macromolecules: A symposium. Academic Press, New York.  129.  Rendi, R., and S. Ochoa. 1962. Species s p e c i f i c i t y in activation and transfer of leucine from c a r r i e r ribonucleic acid to r i b o somes. J . B i o l . Chem. 237: 3707-3713.  130.  Roberts, R.B., P.H. Abelson, D.B. Cowie, E.T. Bolton, and R.J. Britten. 1955. Studies of biosynthesis in Escherichia c o l i . Carnegie Institute Wash. Publ. 607.  131.  Schneider, W.C. 1955. Determination of nucleic acids by pentose a n a l y s i s , p. 680-684. In S.P. Colowick, and N.0. Kaplan (eds.), Methods in enzymology, V o l . III. Academic Press, Inc., New York.  132.  Schweet, R.S., F.C. Bovard, E. A l l e n , and E. Glassman. 1958. The incorporation of amino acids into ribonucleic a c i d . Proc. N a t l . Acad. S c i . U.S. 44: 173-177.  133.  Sguros, P.L., and S.E. H a r t s e l l . 1952. Aerobic glucose d i s s i m i l a tion by Achromobactier species. I. Fate of the carbon substrate. J . Bacteriol. 64: 811-819.  and H. Halvorson.  of L-alanine dehydrogenase.  1961.  The substrate s p e c i f i c i t y  Biochim. Biophys. Acta  48: 47-55.  168. 134.  S i e g e l , B.V., and C.E. C l i f t o n . 1950. Energy relationships in carbohydrate assimilation by Escherichia c o l i . J . Bacteriol.  60: 573-583.  135.  S i e g e l , B.V., and C.E. C l i f t o n . 1950. Energetics and assimilation in the combustion of carbon compounds by Escherichia c o l i . J. B a c t e r i o l . 60: 583-593.  136.  S i e r r a , G., and N.E. Gibbons. 1962. Production of poly-/?-hydroxybutyrate granules in Micrococcus ha1oden?tr?f ?cans. Can. J . Micro. 8: 249-253.  137.  Silberman, R.,  and W.L. Gaby.  1961.  l i p i d s of Pseudomonas aeruginosa.  138.  Smith,  I.  Vol.  i960. I,  The uptake of amino acids by J . L i p i d Res.  2: 172-176.  Chromatographic and electrophoretic techniques,  2nd ed.  Interscience Publishers,  Inc.,  New York, N.Y.  139.  Smith, R.A. 1953. Terminal respiration in Pseudomonas aeruginosa. M.Sc. Thesis, The University of B r i t i s h Columbia. Vancouver, B.C.  140.  Sobek. J . M . , and C.E. C l i f t o n . 1962. Oxidative assimilation and C'4 d i s t r i b u t i o n in Azotobacter a g i l i s . Proc. Soc. E x p t l . B i o l . Med. 109: 408-411.  141.  Speyer, J . F . , A. Lengyel, C. B a s i l i o , and S. Ochoa. 1962. Synthetic polynucleotides and the amino acid code. IV. Proc. Natl. Acad. S c i . U.S. 48: 441-448. .  142.  Spiegelman, S. 1959. Protein and nucleic acid synthesis in subcellular fractions of bacterial c e l l s , p. 81-103. Jn, Recent progress in microbiology, Symposium V l l t h International Congress for Microbiology, Stockholm. Charles C. Thomas Publishing C o . , Springfield, Illinois.  143.  Steward, F.C., and J . F . Thompson. 1954. Proteins and protein metabolism in p l a n t s , p. 513~594. _ln_ H. Neurath, and K. Bailey (eds.), The proteins, V o l . II, Part A. Academic Press Inc., New York.  144.  Stewart, J . E . , P.L. Hoogland, H.C. Freeman, and A . E . J . Waddel1. 1963. Amino acid composition of representatives of eight bacte r i a l genera with reference to aquatic p r o d u c t i v i t y . J . Fish. Res. Bd. Canada 20: 729~734.  145.  Struck, J . Jr.,  and I.W.  Sizer.  glutamic dehydrogenase.  1959.  The substrate s p e c i f i c i t y of  Arch. Biochem. Biophys.  86: 260-266.  146.  Sueoka, N. 1961. Correlation between base composition of deoxyribonucleic acid and amino acid composition of p r o t e i n . Proc. N a t l . Acad. S c i . U.S. 47_: 1141-1149.  147.  Sueoka, N., and T. Yamane.  1962.  Fractionation of aminoacyl-  169. acceptor RNA on a methylated albumin column.  S c i . U.S.  48: 1454-1461.  Proc. N a t l . Acad.  148.  Sueoka, N., and T. Yamane. 1963. Fractionation of aminoacyl-acceptor RNA and the coding problem, p. 205-227. _lin H.J. Vogel, V. Bryson, and J . O . Lampen (eds.), Informational macromolecules: A symposium. Academic Press, New York.  149.  Suit, J.C. 1962. Ribonucleic acid in a membrane f r a c t i o n of Escherichia col? and its relation to cell-wall synthesis. J . Bacte r i o l . 84: 1061-1070.  150.  Suit, J.C. 1963. Location of deoxyribonucleic acid l i k e ribonucleic acid in a membrane f r a c t i o n of Escherichia c o l i . Biochim. B i o phys. Acta 72: 488-490.  151.  T i s s i e r e s , A.  1959.  Some properties of soluble ribonucleic acid  from Escherichia c o l i .  J . Mol. B i o l .  J_: 365-374.  152.  Tomlinson, G.A., and J.J.R. Campbell. 1963. Patterns of oxidative assimilation in strains of Pseudomonas and Achromobacter. J. B a c t e r i o l . 86: 434-444.  153.  Trevelyn, W.E., and J.S. Harrison. 1952. Studies on yeast metabolism. I. Fractionation and microdetermination of c e l l carbohydrates. Biochem. J . 5jO: 298-303.  154.  van N i e l , C.B. 1936. Arch. M i c r o b i o l . J: 323-327. Quoted from Doudoroff, M., and R.Y. Stanier. 1959. Role of poly-j0-hydroxybutyrate in the assimilation of organic carbon by bacteria. Nature J83_: 1440-1442.  155.  van N i e l , C.B., and E.H. Anderson. mentative a s s i m i l a t i o n .  On the occurrence of f e r -  1941.  J . C e l l u l a r Comp. Physiol.  JJ7:  49"56.  156.  Virtanen, A. I., T..Z. Csaky, and N. Rautamem. 1949. On the formation of amino acids and proteins in Torula ut ?1 is on n i t r a t e n u t r i t i o n . Biochim. Biophys. Acta 3_: 208-214.  157.  Virtanen, A . I . ,  and J . Tarnanen.  1932.  und synthese der asparaginsaure.  Die enzymatische spaltung  Biochem. Z.  250: 193-211.  158.  V o l k i n , E., and L. Astrachan. 1956. Intracellular d i s t r i b u t i o n of labeled ribonucleic acid after phage infection of Escherichia coli. Virology 2: 433-437.  159.  von Ehrenstein, G., and F. Lipmann. synthesis.  1961.  Proc. N a t l . Acad. S c i . U.S.  160.  von Tigerstrom, M.D.  161.  Wall, J . S . ,  1963.  Experiments on hemoglobin 47_: 941-950.  Unpublished r e s u l t s .  A.C. Wagenknecht, J.W.  Newton, and R.H. B u r r i s .  1952.  Comparison of the metabolism of ammonia and molecular nitrogen  170.  in photosynthesizing b a c t e r i a . 162.  J.  Bacteriol.  6j_: 563"573.  Warner, A . C . I . 1956. The actual nitrogen sources for growth of heterotrophic bacteria in non-limiting media. Biochem. J . 6 4 : 1-6.  163.  Warren, R.A.J., A . F . E l l s , and J.J.R. Campbell.  1959.  respiration of Pseudomonas aeruginosa.  Bacteriol.  879.  164.  Webster, pig  165.  Webster,  G.C.  1961.  liver.  29_ 8 7 5 " :  Isolation of an alanine-activating enzyme from  Biochem. Biophys.. Acta  G . C , and J . E .  asparagine synthesis 99.  166.  J.  Endogenous  Varner,  49_: 141-152.  1955. Aspartate metabol ism and  in plant systems.  J . B i o l . Chem.  215; 91-  Weisblum, G., S. Benzer, and R.W. Holley. 1962. A physical basis for degeneracy in the amino acid code. Proc. N a t l . Acad. S c i . U.S.  4 8 : 1449-1453.  167.  Wiame, J . F . , and M. Doudoroff. 1951. Oxidative assimilation by Pseudomonas saccharophila with C^4 labelled substrates. J . Bacteriol. 6 2 : 187-193.  168.  Wiame, J . M . , and A. Pierard.  1955. Occurrence of an L_(-)-alanine  dehydrogenase in Bacillus s u b t i l i s . 169.  Nature  176: T073-1075.  Williams, A . E . , and R.H. B u r r i s . 1952. Nitrogen f i x a t i o n by blue green algae and t h e i r nitrogenous composition. Am. J . Botany 2£: 340-342.  170.  Wilkinson, J . F .  1958. The e x t r a c e l l u l a r polysaccharides of bacteria.  B a c t e r i o l . Rev. 171.  Wilkinson, J . F . bacteria.  172.  Wilner, B.,  1959. The problem of energy storage compounds in E x p t l . Cell Res., Suppl.  and C.E. C l i f t o n .  lus s u b t i l i s . 173.  Winzler, R.J.  22: 46-73.  1954.  J . Bacteriol.  7_: 111-130.  Oxidative assimilation by B a c i l -  6_7_: 571-575.  1940. The oxidation and assimilation of acetate by  baker's yeast.  J . C e l l u l a r Comp. P h y s i o l .  15_: 343-354.  174.  Winzler, R.J., and J . P . Baumberger. 1938. The degradation of energy y i e l d i n g compounds in the metabolism of yeast c e l l s . J . Cellular Comp. Physiol. . 1 2 : 183-211.  175.  Winzler, R.G., D. Bink, and V. du Vigneaud. 1944. Biotin in ferment a t i o n , r e s p i r a t i o n , growth and nitrogen assimilation by yeast. Arch. Biochem. Biophys. 5j, 25-40.  176.  Yamane, T . , and N. Sueoka.  1963. Conservation of s p e c i f i c i t y  between  171. amino acid acceptor RNA and aminoacyl-s-RNA synthetases. N a t l . Acad. S c i . U.S. %0: 1093-1100.  Proc.  177.  Yee, R.B., S. Fan, and H.M. Gezon. 1962. Effect of chloramphenicol on protein and nucleic acid synthesis by Shi gel la f l e x n e r i . J. Gen. M i c r o b i o l . 2J: 521-527.  178.  Yee, R.B., and H.M. Gezon. 1963. Ribonucleic acid of chloramphenicol-treated Shigel la f l e x n e r i . J . Gen. M i c r o b i o l . 32.: 299" 306.  179.  Zachau, H.G., M. Tada, V/.B. Lawson, and M. Schweiger. 1961. Fraktionierung der loslichen ribonucleinsaure. Biochim. Biophys. Acta 5_3_: 221-223.  180.  Z e l i t c h , I., E.D. Rosenblum, R.H. B u r r i s , and P.W. Wilson. 1951. Comparison of the metabolism of ammonia and molecular nitrogen in Clostridium. J . B a c t e r i o l . 62: 747~752.  181.  Z e l i t c h , I., P.W. Wilson, and R.H. B u r r i s . 1952. The amino acid composition and d i s t r i b u t i o n of 5 in soybean root nodules supp l i e d N'5 enriched N2. Plant Physiol. ZJ: 1-8.  182.  Z i l l i g , W., D. Schachtschnabel, and W.Z. Krone, i960. Untersuchungen zur biosynthese der p r o t e i n . IV. Zusammensetzung, Funktion und Spezifitat der loslichen ribonucleinsaure aus Escherichia col i. Z. Physiol. Chem. 3J8: 100-114.  183.  Zubay, G. is.  1962.  A theory on the mechanism of messenger-RNA synthes-  Proc. Natl. Acad. S c i . U.S.  48: 456-460.  

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