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

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OXIDATIVE ASSIMILATION OF GLUCOSE BY AEROBIC BACTERIA by GERALD INE ANN TOMLINSON B.S.A., The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1957. M.A. ( B i o c h e m i s t r y ) , The U n i v e r s i t y o f C a l i f o r n i a ( B e r k e l e y ) , I960. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN AGRICULTURAL MICROBIOLOGY i n t h e D i v i s i o n o f Animal S c i e n c e We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1964. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of • Bri t i sh Columbia, I agree that the Library shall make i t freely available for reference and study» . I further agree that per mission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is 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 /^'c^U^/^t^ The University.of Bri t i sh Columbia, Vancouver 8, Canada Date / ^ ^ y . / 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, at 2:00 P.M. IN ROOM 0, AGRICULTURE BUILDING COMMITTEE IN CHARGE Chairman: F.H. Soward J.J.R. Campbell W.J. Polglase B.A. Eagles J . J . Stock D.P. Ormrod 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 cell components, principally proteinaceous, in. conjunction with the re incorporation 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 incorpora tion into nucleic acids and lipids was increased by the antibiotic, but was only slightly affected by starva tion. A determination of the cytological sites of the assimilated material showed that, in control cell extracts, the soluble proteins of the cytoplasm con tained most of the C^. Starved or antibiotic treated cell 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 synthe tases, but they were rapidly reactivated during oxida tive assimilation. The large amount of heterologous reactions between bacterial soluble ribonucleic acids and synthetases indicated that l i t t l e species speci ficity 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 evi dence indicated that ammonia was assimilated via et- ketoglutarate in P. aeruginosa. GRADUATE STUDIES Field of Study: Agricultural Microbiology Molecular Structure and Biological Function (Proteins and Carbo hydrates) 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 . Bacterid. 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. i i Abstract Oxidative assimilation of glucose-U-C^ by Pseudomonas aerugin osa, P. fluorescens, Achromobacter B81, A. viscosus, Azotobacter ag i l i s , A. v inelandi i , and Acetobacter xylinum was found to involve the assimila tion of radioactivity into nitrogenous cel l components, pr incipal ly 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 ce l ls were starved or treated with chloramphenicol prior to glucose-c'^ oxidation, the amount of assimilation was decreased. The incorporation of radioactivity into protein was severely restricted by both treatments. The amount of labell ing of both l ipids and nucleic acids was increased in the presence of the ant ib iot ic , but was only s l ight ly af fected by starvation. A determination of the cytological sites of the as similated material showed that, in control ce l l extracts, the soluble proteins of the cytoplasm contained most of the c'**. Starved or antibiot ic treated ce l l fractions exhibited a profound decrease in the label of these proteins, whereas the amount of incorporation into the ribosomal r ibonucl eic acid and the "membrane" l ipids 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 cel l extracts were prepared and fractionated in a medium of high ionic strength. Starving the ce l ls resulted in a decrease in the act ivity of the aminoacyl-s-RNA synthetases, but they were rapidly reactivated during oxidative assimilation of glucose. There was found to be l i t t l e i i i species s p e c i f i c i t y between the s-RNA's and synthetases of £_. aeruginosa, P. f luorescens, Achromobacter B81, and E. col i, s ince good cross react ions were obta ined, but the heterologous react ions between bakers' yeast and the bac te r i a were poor, except in the case of the yeast s-RNA and the E. c o l i enzyme. Prel iminary experiments on the route of ammonia ass im i l a t i on in P_. aeruginosa and in P. f luorescens gave no evidence fo r the d i rec t ami na  t i on of pyruvate by a lan ine dehydrogenase, but d id demonstrate a r equ i r e  ment f o r concurrent substrate ox idat ion whi le ammonia was being a s s i m i l  a ted. In con t ras t , several l ines of evidence indicated that ammonia was ass imi la ted v i a 9(*-ketoglutarate in P. aeruginosa. J.J.R. Campbell xi ACKNOWLEDGEMENT I wish to express my appreciation to Dr. J.J.R. Campbell, for his con tinued interest and encouragement throughout my years of study and re search under his direct ion, and also to my husband, without whose help, understanding, and forebearance, the completion of this thesis would not have been possible. In addition, acknowledgement is due to my f e l  low students, for their helpful discussions and assistance during the course of this research. iv TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW 3 I. Oxidative Assimilation 3 II. Nitrogen Assimilation 13 III. Species Specif ic i ty of s-RNA's and Aminoacyl-s-RNA Synthetases 19 MATERIALS AND METHODS I. Oxidative Assimilation into Whole Cells of Aerobic Bacteria 26 A. Bacteriological methods 26 B. Assimilation studies 27 C. Starved cel l experiments 28 1. Starvation procedure 28 2. Assimilation experiments 29 D. Analytical methods 29 1. Analysis of residual fractions 29 2. Analysis of cold tr ichloroacetic acid fractions 30 3. Chemical methods 31 k. Paper chromatography and electrophoresis 31 E. Isotopic methods 32 II. Inorganic Nitrogen Assimilation by Pseudomonas aeruginosa and Pseudomonas fluorescens 32 III. Oxidative Assimilation into the Cytological Fractions of Normal, Starved, or Chloramphenicol Treated Cells of Pseudomonas aeruginosa ATCC 9027 32 A. Assimilation studies 33 B. Preparation of cel l fractions 33 Page C . A n a l y s i s o f c y t o l o g i c a l f r a c t i o n s 34 D. Chemica l f r a c t i o n a t i o n o f t h e c y t o l o g i c a l f r a c t i o n s 34 E. A n a l y s i s o f c h e m i c a l f r a c t i o n s 34 1. "Membrane" r e s i d u a l f r a c t i o n 34 2. C y t o p l a s m i c c o l d 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 f r a c t i o n s 35 F. P r e p a r a t i o n o f P_. a e r u g i n o s a s-RNA 35 G. Assay p r o c e d u r e f o r i n c o r p o r a t i o n o f c'4 amino a c i d s i n t o s-RNA 36 H. I s o t o p i c methods 37 IV. S p e c i e s S p e c i f i c i t y o f s-RNA's and Aminoacy1-s-RNA S y n t h e t a s e s 37 A . P r e p a r a t i o n o f s-RNA's 37 B. P r e p a r a t i o n o f enzymes 38 C . P r e p a r a t i o n o f am inoacy l - s-RNA ' s 38 D. Paper ch romatography 39 RESULTS AND DISCUSSION kO I. O x i d a t i v e A s s i m i l a t i o n i n t o Whole C e l l s o f A e r o b i c B a c t e r i a 40 A . 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 i n t o s t r a i n s o f Pseudotnonas and Ach romobac te r kO 1. Manomet 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 r a d i o a c t i v e p r o d u c t s i n t o t h e s u p e r n a t a n t f l u i d s d u r i n g g lucose-U-C^4 o x i d a t i o n kO 2. I n c o r p o r a t i o n o f C^4 i n t o c e l l s 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 50 3. A s s i m i l a t i o n o f c'4 ; n t o t h e c e l l f r a c t i o n s o l u b l e in c o l d t r i c h l o r o a c e t i c a c i d 55 4. A s s i m i l a t i o n o f C^4 i n t o t h e c e l l f r a c t i o n s i n s o l u b l e in c o l d t r i c h l o r o a c e t i c a c i d 56 v i Page B. 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 i n t o s t r a i n s 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 60 1. Manometric 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 t h e s u p e r n a t a n t f l u i d s d u r i n g g l u c o s e - U - C l ^ o x i d a t i o n 60 2. 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 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 69 3. 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 s o l u b l e i n c o l d t r i c h l o r o a c e t i c a c i d 69 4. I n c o r p o r a t i o n o f i n t o t h e c e l l f r a c t i o n s i n s o l u b l e i n c o l d t r i c h l o r o a c e t i c a c i d 74 C. O x i d a t i v e a s s i m i l a t i o n by s t a r v e d c e l l s o f P. a e r u g i n o s a ATCC 9027 76 1. Manometric o b s e r v a t i o n s 76 2. Ammonia e x c r e t i o n and u p t a k e 77 3. 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 t h e s u p e r n a t a n t f l u i d s 81 4. D i s t r i b u t i o n o f C 1 ^ i n t h e c e l l s 81 5. A n a l y s i s o f t h e c o l d 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 f r a c t i o n s 83 6. The i n f l u e n c e o f v i t a m i n Bg on o x i d a t i v e a s s i m i l a t i o n 86 I I . I n o r g a n i c N i t r o g e n A s s i m i l a t i o n by Pseudomonas a e r u g i n o s a and Pseudomonas f l u o r e s c e n s 87 A. E x p e r i m e n t s w i t h f_. a e r u g i n o s a ATCC 9027 88 1. A s s i m i l a t i o n o f ammonia d u r i n g t h e o x i d a t i o n o f k e t o a c i d s 88 2. A s s i m i l a t i o n o f ammonia i n t h e p r e s e n c e o f i n h i b i t o r s 92 B. E x p e r i m e n t s w i t h P. f l u o r e s c e n s A 3.12 93 I I . O x i d a t i v e A s s i m i l a t i o n i n t o t h e C y t o l o g i c a l F r a c t i o n s o f Normal, C h l o r a m p h e n i c o l 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 ATCC 9027 97 vi i Page A. Chemical composition of cytological fractions 97 B. Incorporation of glucose-U-C^4 into cytological fractions 98 1. "Membrane" fractions 100 2. Ribosomal fractions 105 3. Cytoplasmic fractions 108 C. Experiments with the cytoplasmic proteins 114 1. Amount of radioactivity contained in the "pH 5 enzyme" 114 2. Effect of starvation on the act iv i ty of the aminoacyl-s-RNA synthetases 115 IV. Species Specif ic i ty of s-RNA's and Aminoacyl-s-RNA Synthetases 124 A. Cytological location of aminoacyl-s-RNA synthetases in f_. aeruginosa 124 B. Interspecific reactions between s-RNA's and amino acyl -s-RNA synthetases 127 C. Patterns of amino acid incorporation into homolog ous systems 133 D. Patterns of amino acid incorporation into heterol ogous systems 137 GENERAL DISCUSSION 153 BIBLIOGRAPHY 158 FIGURES 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 Pseudomonas aeruginosa. Time course of NH3 and keto ac id product ion and inco r  porat ion dur ing ox ida t ion of 5 Mmoles of g lucose-U-C^ by washed c e l l suspensions of Pseudomonas aeruginosa. Oxygen uptake with 5 H m o l e s of substrate and disappearance of glucose and fJ4 from supernatant f l u i d s with washed c e l l suspensions of Pseudomonas f l uo rescens . Time course of N H 3 and keto acid product ion and incor  porat ion during ox idat ion of 5 M m ° l e s of g l u c o s e - U - C b y washed c e l l suspensions of Pseudomonas f l uo rescens . 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 Achromobacter B81. Time course of NH3 and keto ac id product ion and incor  porat ion dur ing ox ida t ion of 5 Mmoles of g lucose-U-C^ by washed c e l l suspensions of Achromobacter B81. Oxygen uptake with 5 Mmoles of substrate and disappearance of glucose and from supernatant f l u i d s with washed c e l l suspensions of Achromobacter v i s cosus . Time course ~of NH3 and keto ac id product ion and c '4 incor  porat ion during ox ida t ion of 5 M " 1 0 ! 6 5 of glucose-U-C'4 by washed eel 1 suspensions of Achromobacter v i s cosus . Oxygen uptake with 5 Mmoles of subst ra te and disappearance of glucose and C.14 from supernatant f l u i d s with washed c e l l suspensions of Acetobacter acet i . Time course of NH3 and keto ac id product ion and incor  porat ion dur ing ox ida t ion of 5 Mmoles of glucose-U-Cl4 by washed c e l l suspensions of Acetobacter a c e t i . Oxygen uptake with 5 Mmoles of subst ra te and disappearance of glucose and CJ4 from supernatant f l u i d s with washed c e l l suspensions of Acetobacter xyl inum. Time course of NH3 and keto acid product ion and incor  porat ion dur ing ox idat ion of 5 Mmoles of g lucose-U-C^ by washed c e l l suspensions of Acetobacter xy l inum. ix' Figure Page 7A Oxygen uptake with 5 Mmoles of substrate and disappearance of glucose and c ' ^ from supernatant f luids with washed ce l l suspensions of Azotobacter v inelandi i . 66 7B Time course of NH3 and keto acid production, and C 1^ incor poration during oxidation of 5 Mmoles of glucose-U-C'4 by washed cel l suspensions of Azotobacter v inelandi i . 66 8A Oxygen uptake with 5 Rmoles of substrate and disappearance of glucose and from supernatant f luids with washed ce l l suspensions of Azotobacter ag i l i s . 68 8B Time course of NH3 and keto acid production, and incor poration during the oxidation of 5 Mmoles of glucose-U-c'^ by washed cel l suspensions of Azotobacter ag i l i s . 68 9 Oxygen uptake during oxidation of pyruvate and glucose by washed control and starved cel ls of Pseudomonas aeruginosa. 79 10 Production and uptake of NH3 by washed, starved cel l sus pensions of Pseudomonas aeruginosa. 80 11 Disappearance of and glucose from, and excretion of keto acids into, supernatant f luids during oxidation of glucose- U-Cl4 by washed, starved cel ls of Pseudomonas aeruginosa. 80 12 Incorporation of during oxidation of 5 Mmoles of gluc- ose-U-cJ^ by washed c o n t r o l , chloramphenicol (Chloromycetin) treated, or starved cel ls of Pseudomonas aeruginosa. 85 13 Uptake of oxygen and NH3 by washed cel ls of Pseudomonas f luo  rescens during the oxidation of 10 M m ° les of pyruvate with and without 50 Mmoles of fluoroacetate. 95 14 Incorporation of into the protein residue and the l ip id of the "membrane" fractions during oxidation of glucose-U-C'4 by washed c o n t r o l , chloramphenicol (Chloromycetin) treated, or starved cel ls of Pseudomonas aeruginosa. 103 15 Incorporation of Cl^ into the ribosomal fractions during ox idation of glucose-U-Cl4 by washed control, chloramphenicol (Chloromycetin) treated, or starved ce l ls of Pseudomonas  aeruginosa. 107 16 Incorporationoof C'^ into the RNA and protein residue of the ribosomes during oxidation of glucose-U-C'4 by washed cont 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 ) t r e a t e d , or s t a r v e d cel ls of Pseudomonas aeruginosa. 109 17 Incorporation of jnto the cold tr ichloroacetic acid 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 washed 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 . I n c o r p o r a t i o n o f C l 4 amino a c i d s i n t o s-RNA by c y t o p l a s  m i c f r a c t i o n s o f Pseudomonas a e r u g i n o s a . 1. INTRODUCTION " O x i d a t i v e a s s i m i l a t i o n " i s t h e t e r m used t o d e s c r i b e t h e i n c o r  p o r a t i o n o f c a r b o n i n t o c e l l u l a r components d u r i n g t h e o x i d a t i o n o f a s u b  s t r a t e by washed c e l l s o f m i c r o o r g a n i s m s ( 1 0 0 ) . E n e r g y f o r t h i s p r o c e s s , w h i c h t a k e s p l a c e i n t h e a b s e n c e o f added n i t r o g e n , i s p r o v i d e d by o x i d a  t i o n o f p a r t o f t h e s u b s t r a t e , w h i l e t h e r e m a i n d e r i s a s s i m i l a t e d . S i n c e 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 , i t m i g h t be e x p e c t e d t h a t t h e i n c o r p o r a t e d c a r b o n w o u l d be f o u n d e x c l u s i v e l y i n non n i t r o g e n o u s c e l l c o n s t i t u e n t s , d e s i g n a t e d " p r i m a r y p r o d u c t s " , and i n d e e d , t h i s has been found 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 . R e c e n t l y , h o w e v e r , i t has become e v i d e n t t h a t , 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 i n t o a v a r i  e t y o f c e l l c o m p o n e n t s , i n c l u d i n g n i t r o g e n o u s o n e s ; t h e n i t r o g e n f o r t h i s p r o c e s s b e i n g d e r i v e d f r o m t h e endogenous r e s p i r a t i o n o f t h e c e l l s . S t u d i e s i n t h i s l a b o r a t o r y have shown t h a t t h i s i s t h e s i t u a t i o n found i n r e s t i n g c e l l s o f Pseudomonas a e r u g i n o s a . and m o r e o v e r , t h a t a p r i m a r y p r o d u c t i s no t fo rmed 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 ) . T h i s t h e s i s i s c o n c e r n e d w i t h s e v e r a l a s p e c t s o f o x i d a t i v e a s  s i m i l a t i o n i n a e r o b i c b a c t e r i a . F i r s t l y , t h e e x p e r i m e n t a l a p p r o a c h u sed by Duncan and C a m p b e l l (55) w i t h 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 a e r o b i c b a c t e r i a , t o d e t e r m i n e i f , unde r t h e s e c o n d i t i o n s , t h e y w o u l d f o r m a s p e c i a l r e s e r v e p r o d u c t s u c h as c a r b o h y d r a t e o r l i p i d , o r w h e t h e r a l l c e l l components w o u l d be s y n t h e s i z e d . S e c o n d l y , t h e e f f e c t o f s t a r v a t i o n o f P_. a e r u g i n o s a c e l l s on t h e e x t e n t 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 d e t e r m i n e d , 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 mechan i sm o f ammonia 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 and P . f l u o r e s c e n s . T h i r d l y , t h e c y t o l o g i c a l s i t e s o f o x i d a t i v e a s s i m i l a t i o n w e r e i n v e s t i g a t e d i n P . a e r u g i n o s a , and t h e 2. i n f l u e n c e o f s t a r v a t i o n o r i n h i b i t i o n o f p r o t e i n s y n t h e s i s o n t h i s p r o c e s s was s t u d i e d . F i n a l l y , s i n c e p r o t e i n was one o f t h e ma in p r o d u c t s o f o x i d a  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 (as w e l l as i n t h e o t h e r a e r o b e s s t u d i e d ) , an i n v e s t i g a t i o n was made o f t h e f o r m a t i o n o f a m i n o a c y l s o l u b l e r i b o n u c l e i c a c i d ( a m i n o a c y l - s - R N A ) i n P . a e r u g i n o s a , and t h e s y s t e m p r e s e n t i n t h i s m i c r o o r g a n i s m was compared t o t h o s e i n o t h e r b a c t e r i a , and i n y e a s t . 3. LITERATURE REVIEW I. Oxidat ive Ass im i l a t i on H. A. Barker (11) f i r s t used the term " o x i d a t i v e a s s i m i l a t i o n " to descr ibe the convers ion , by the c o l o r l e s s a lga Prototheca zopf i i , of part of an organic molecule into c e l l u l a r m a t e r i a l , whi le the remainder was o x i d i z e d . This process was manifested by an oxygen uptake which was less than expected, although the subst ra te had completely d isappeared. In a d d i t i o n , the dry weights of the microorganisms had r i sen somewhat. A s i m i l a r phenomenon had been noted prev ious ly in Escher i ch ia c o l i by Cook and Stephenson (41), but no reason f o r i ts occurrence had been pos tu l a t ed . From the molecular quant i t i es of the reac tants , and the fac t that one of the products was carbon d i ox ide , Barker was able to wr i te balanced equa t ions f o r the ox ida t ion of severa l subs t ra tes . Thus, the equation fo r the ox ida t ion of acetate was wr i t ten as f o l l ows : CH3COOH + 0 2 ) C0 2 + H 20 + (CH 20) The formula fo r the product of a s s im i l a t i on of several organic compounds could a l so be wr i t ten as CH 2 0. S ince P. zopf i i was known to synthes ize and s to re g lycogen, Barker (11) concluded that t h i s was the primary p r o  duct of ox ida t i v e a s s im i l a t i on in the a l ga , and that i t might be used as the source of raw mater ia l f o r c e l l u l a r synthes is dur ing prolonged incuba- t ion or growth. The Warburg manometric technique was used in the subsequent years by a success ion of authors to study ox ida t i ve a s s i m i l a t i o n , and t h e i r r e su l t s supported Barker 's hypothes is . Thus, C l i f t o n with Pseudomonas  ca lcoacet? and E. col? (32), Giesberger using Spir?11 urn (70), and Doudoroff with Pseudomonas saccharophi la (52). a l l postu lated primary products of assimilation similar to that suggested by Barker. These results were based on the amount of oxygen consumed, and the carbon dioxide l iberated, 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 (174) on heat production during alcoholic fermentation. From the heats of fo r  mation, and the heat produced during exogenous respiration, they concluded that 70.5% of the glucose was fermented to ethyl alcohol and carbon diox ide, and 29.5% stored. Corresponding figures for aerobic dissimilation were 24.5% and 75.5%. 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 Cl i f ton (134,135) have studied the relationship be tween 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 ce l ls of E. co l i (134). From manometry with resting ce 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 less, and lactose the least. Previous studies on the extent of assimilation in resting and growing ce l ls of JE. co l i had indicated that this occurred to a similar measure in both cases (37). In a second paper, Siegel and Cl i f ton (135) reported on a similar investigation of the energetics involved in the assimilation of succinate, fumarate, lactate, pyruvate and glycerol by short term cultures of E. co l I . The same situation 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 pai 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 cel ls were used in assessing one value, and growing ce l ls for the other. When the amounts of assimilation in resting ce l ls 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 efficiency 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 inter mediates needed for synthesis. That the same situation existed in Baci1 - lus subtil is was suggested by the studies of Wilner and Cl i f ton (172) also with organic acids and glucose as substrates for assimilation. Again, fumarate and pyruvate were assimilated to a greater degree than would have been predicted by the free energy changes from oxidation of the molecule. Cl i f ton and his associates have concluded from these in  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 (65) using resting ce l ls of purple bacteria. As a result of these investiga t ions, 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 photo synthet ical ly on butyrate. However, further investigations of this unique polymeric product was discouraged by van Niel 's report (154) that the gross composition of purple bacteria corresponded closely to the composition of Gaffron's proposed product of assimilation. The use of manometric techniques introduced several limitations which, despite the considerable volume of work which was undertaken, pre vented the identif ication of the nature of the assimilated material. One d i f f i cu l t y was the uncertainty of the actual oxygen uptake, since it was not known whether endogenous respiration continued unabated, was stimulat ed, or was inhibited by, the addition of oxidizable substrate. Qualita t ive tests were usually carried out on supernatant f luids 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 some times 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 prior 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 assimilation. Since some bacteria appeared to assimilate carbon as e f f i c ient ly in the absence of nitrogen as in its presence, it seemed evident that as similation was not necessarily coupled with general ce l l synthesis. One could, therefore, conclude that the assimilated material would be found in a limited number of ce l l constituents, or primary products. In many micro organisms this was found to be the case. 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 bacteria. Polysaccaride is the primary product of assimilation by many bacteria, especially the enteric group, which have been found to contain levels of glycogen as high as 48% of the dry weight of the ce l ls (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 lkal i stable polyglucose, apparently identical to glycogen in its properties, formed from glucose by growing or resting ce l ls of E_. co l i during nitrogen starvation. If the polysaccharide containing ce l ls were transferred to a medium containing inorganic nitrogen, but no carbon, the newly formed polyglucose was broken down, and used for protein syn thesis. The oxidative assimilation of glucose, and other substrates in  to Sajxlna Jutea was investigated by Binnie, Dawes and Holms (21). When the ce l ls were grown on peptone, carbohydrate made up 10% of their dry weight, and after oxidation of glucose by freshly harvested or lyophil- ized endogenous diminished c e l l s , this rose to 28%. The assimilated 8 m a t e r i a l was u t i l i z e d d u r i n g endogenous r e s p i r a t i o n , abou t 50% d i s a p p e a r  i n g i n 3.5 h o u r s , t h e r e s t more s l o w l y . When c e l l s w h i c h had been a l l o w e d t o a s s i m i l a t e u n i f o r m l y l a b e l l e d g l u c o s e w e r e f r a c t i o n a t e d c h e m i c a l l y , t h e b u l k o f t h e r a d i o a c t i v i t y was f o u n d i n t h e a l c o h o l s o l u b l e m a t e r i a l , and y i e l d e d g l u c o s e on h y d r o l y s i s . 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 p o l y - g l u c o s e , t h e a u t h o r s c o n c l u d e d t h a t g l u c o s e had been a s s i m i l a t e d d i r e c t l y . 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 t h a t i t was o f r e l a t i v e l y l ow m o l e c u l a r w e i g h t . The u s e o f a c e t a t e o r p y r u v a t e as s u b  s t r a t e s d i d no t r e s u l t i n an i n c r e a s e i n t h e c a r b o h y d r a t e o f t h e c e l l s , t h e 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 i n t h e c o l d 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 f r a c t i o n s , w i t h o t h e r m e t a b o l i c i n t e r m e d i a t e s . W i t h t h e a i d o f l a b e l l e d s u b s t r a t e s , i t has been shown t h a t b a c t e r i a a r e a b l e t o a s s i m i l a t e c a r b o n i n t o b o t h l i p i d and c a r b o h y d r a t e . 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 ^ i n c o r p o r a t e d 37% o f t h e a s s i m i l a t e d r a d i o a c t i v i t y i n t o t h e l i p i d f r a c t i o n , w h i c h a c c o u n t e d f o r 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 , whereas 52% was i n c o r p o r a t e d i n t o c a r b o h y d r a t e , w h i c h made up l e s s t h a n 8% o f t h e c e l l m a t e r i a l (115). The h i g h e r , s p e c i f i c a c t i v i t y o f t h e c a r b o h y d r a t e s u g g e s t e d t h a t i t was t h e i n i t i a l s i t e o f a s s i m i l a t i o n . W i t h some b a c t e r i a , t h e s u b s t r a t e d e t e r  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 . T h u s , w i t h JE. c o l ? , t h e p r e s e n c e o f a c e t a t e i n c r e a s e d t h e f o r m a t i o n o f l i p i d , and d e c r e a s e d g l y c o g e n f o r m a  t i o n , whe reas 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 (171). Y e a s t s have been t h e s u b j e c t o f a number o f s t u d i e s on a s s i m i l a  t i o n , and t h e r e seems t o be some d i s a g r e e m e n t as t o w h e t h e r t h e i n c o r p o r  a t e d m a t e r i a l t a k e s t h e f o r m o f l i p i d o r c a r b o h y d r a t e . W i n z l e r (173) f o u n d an i n c r e a s e i n t h e r e d u c i n g s u g a r c o n t e n t o f y e a s t w h i c h had o x i d  i z e d a c e t a t e . T h i s i n c r e a s e was enough t o a c c o u n t f o r 80% o f t h e m a t e r i a l 9. theoretical ly assimilated. On the other hand, McLeod and Smedly-Mclean (111) found that l ip id was synthesized from acetate, without the inter mediate formation of carbohydrate. These experiments were generally of a longer duration, and were carried out in the presence of higher concen trations of acetate than employed by Winzler. Perhaps an analysis made earl ier in the course of the oxidation would have revealed the primary synthesis of carbohydrate. Pickett and Cl i f ton (125) established that carbohydrate was the primary product of assimilation by Saccharomyces  cerevisiae during glucose oxidation, since the increase in readily hydro- lysable 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 radioactivity was incor porated into both carbohydrate and l ip id (92). if acetate-c'^ were used alone, however, nearly 50% of the assimilated material was calculated, by difference, to be "prote in , " although there was no net increase of protein in the ce l ls during the experiment. This f inding, by Jackson and Johnson, of assimilation into protein, was one of the f i r s t reports of a nitrogen ous product of assimilation. Although the l ip id formed by assimilation into yeast ce l ls is thought to consist of conventional tr iglycerides (171), the importance of the l ip id 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 ip id 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 intracel lular 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 ce 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 this polymer was soon demonstrated by Forsyth, Hayward and Roberts (60), who found it to be present in many Gram negative bacteria, including Azotobacter species and nonpigmented pseudomonads. The presence of poly-^-hydroxybutyrate in cocci was demonstrated by Sierra and Gibbons (136), who studied the biosynthesis and oxidation of the polymer in Micro coccus halodenitr if icans. Doudoroff and Stanier (53) found that poly- /3 - hydroxybutyrate was the product of photosynthetic assimilation in Rhodo- spir i l ium rubrum. thus confirming the much ear l ier conclusion of Gaffron (65) with purple bacteria. 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) on the oxidative as similation of labelled substrates by f_. saccharophila had shown that, in this microorganism, two carbon fragments were the fundamental building blocks of assimilation. 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. A similar situation occurred with suc cinate, i .e . , 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 sup porting data being given, that "the carbon flowed into many different materials in the c e l l , including prote in . " 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 . f o u n d t h a t f r e s h l y h a r v e s t e d c e l l s , when i n c u b a t e d w i t h g l u c o s e-c '^ , a s s i m i l a t e d o n l y 21% o f t h e added r a d i o a c t i v i t y , and o f t h i s , t w o - t h i r d s a p p e a r e d as p o l y - fi - h y d r o x y b u t y r a t e . Howeve r , i f t h e c e l l s w e r e s t a r v e d b e f o r e t h e e x p e r i m e n t , more t h a n 50% o f t h e g l u c o s e was a s s i m i l a t e d , a g a i n as t h e p o l y m e r . T h i s was a l s o t h e m a i n p r o d u c t fo rmed d u r i n g t h e o x i d a t i o n o f a c e t a t e o r b u t y r a t e . D o u d o r o f f and S t a n i e r r e p o r t e d t h a t 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 t h e s u b s t r a t e f o r endogenous r e s  p i r a t i o n f o r P . s a c c h a r o p h ? l a i n t h e 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 , bu t ne t t r a n s f e r o f p o l y m e r c a r b o n t o o t h e r c e l l c o n s t i t u e n t s c o u l d no t be d e m o n s t r a t e d . Up t o t h i s t i m e , r e p o r t s on a s s i m i l a t i o n by b a c t e r i a had c o n  c e r n e d t h e f o r m a t i o n o f a p r i m a r y c a r b o n a c e o u s r e s e r v e . However , i n d i c a  t i o n s t h a t t h i s m i g h t no t be a u n i v e r s a l s i t u a t i o n w e r e p r o v i d e d by t h e e x p e r i m e n t s o f W a r r e n , E l l s and C a m p b e l l (163), who showed t h a t P . a e r u g  i n o s a c o n s i s t e n t l y p r o d u c e d c o n s i d e r a b l e q u a n t i t i e s o f ammonia d u r i n g endogenous r e s p i r a t i o n , and t h a t i t r e i n c o r p o r a t e d t h i s ammonia when g l u  c o s e was a d d e d . G r o n l u n d and C a m p b e l l (74) e x t e n d e d t h i s f i n d i n g t o a number o f b a c t e r i a , as w e l l as t o S,. c e r e v i s i a e . Bac? 11 us c e r e u s was shown by C l i f t o n and Sobek (38) t o p r o d u c e ammonia endogenous 1y, and l a t e r e x p e r i m e n t s w i t h g l u c o s e-c '^ r e v e a l e d t h a t 50% o f t h e r a d i o a c t i v i t y was a s s i m i l a t e d by r e s t i n g c e l l s u s p e n s i o n s o f t h i s o r g a n i s m (35). I n i t i a l l y , most o f t h e l a b e l was found i n t h e c o l d 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 p o o l c o m p o n e n t s , bu t soon p a s s e d i n t o t h e compounds s o l u b l e and i n s o l u b l e i n ho t t r i c h l o r o a c e t i c a c i d , 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 and p r o  t e i n s . 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 no t appea r t o o c c u r . C l i f t o n (36) has s i n c e r e p o r t e d s i m i l a r r e s u l t s f o r a number o f b a c t e r i a , i n c l u d i n g B a c ?1 l u s m e g a t e r i u m , A z o t o b a c t e r ag?1 i s , 12. B. subti1 i s , and Hydrogenomonas faci1 is (45). Almost simultaneously with C l i f ton 's report on B_. cereus. Duncan and Campbell (55), using P. aeruginosa, demonstrated that this microorgan ism also formed no primary product during oxidative assimilation of g lu  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 tr ichloroacetic acid soluble pool, then passed into compounds soluble and insoluble in hot t r i  chloroacetic acid and alcohol. However, in contrast to the bac i l lus , and to the conclusions suggested by oxygen uptake data, the pseudomonad assimil ated only 16% of the substrate. Duncan and Campbell were able to relate assimilation in P. aeruginosa to the formation of V-ketoglutarate, which was excreted into the medium during the early stages of glucose oxidation, and reincorporated concurrently with ammonia provided by endogenous res p i rat ion. Since this microorganism has an active glutamic acid dehydro genase, Duncan and Campbell believe that "the part ial block of glucose oxidation at of-ketoglutarate represents a control mechanism, ensuring that the method of entry for nitrogen wi l l be present as soon as any be comes available by d i f fus ion, leaching, or from endogenous storage products." Assimilation by P. aeruginosa appeared to be limited by the amount of nitrogen available, since the addition of ammonia greatly increased the amount of C ^ incorporated, and prevented the accumulation of •( -keto- glutarate in the suspending f l u i d . When chloramphenicol was added to resting ce l l suspensions oxidizing glucose, the amount of incorporated radioactivity was decreased, and assimilation into the l i p i d , cold t r i  chloroacetic acid soluble pool, and hot tr ichloroacetic acid soluble fractions was increased, at the expense of the residual f ract ion, 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 Cl i f ton (36), that oxidative assimilation may serve, at least in part, to replenish the nitrogenous endogenous reserves of some microorgan isms. 11. Inorganic Nitrogen Assimilation In the previous section, the l i terature on oxidative assimila tion was reviewed, and it was shown that this process, although by de f in i  tion occurring in the absence of added nitrogen, often involves protein and nucleic acid biosynthesis. For this reason, the assimilation of in  organic nitrogen becomes important in the overall phenomenon of oxidative ass imilation. The major pathway by which organisms incorporate inorganic nitrogen into organic compounds involves the ammonium ion. As early as 1926, Quastel and Woolf (127) reported the formation of aspartate from fumarate and ammonia by cel ls of E. col?, and later this was extended to several anaerobes (42). The equilibrium of this reaction was found to favour aspartate formation, with a K eq of approximately 20. Virtanen and Tarnenen (157) extracted aspartase from cel ls of Pseudomonas fluorescens. and demonstrated that the reaction occurred in cel l free extracts. The enzyme proved to be fumarate spec i f i c , and later work by Ichihara et_ a l . (91) indicated that f o l i c acid, reduced glutathione, and cobalt ions were required by the purif ied enzyme. 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 d e h y d r o g e n a s e , bu t o x i d i z e d g l u t a m a t e . I t was assumed t h a t t r a n s a m i n a  t i o n o f g l u t a m i c a c i d w i t h 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 t h e amino a c i d s o fo rmed was t h e n o x i d i z e d by an enzyme as y e t unknown. Exam i n a t i o n o f c e l l e x t r a c t s r e v e a l e d an a l a n i n e d e h y d r o g e n a s e s p e c i f i c f o r L_- 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 d i n u c l e o t i d e ( N A D ) . P y r u v a t e was t h e o x i d a t i o n p r o d u c t , and a l a n i n e was s y n t h e s i z e d i n t h e p r e s e n c e o f r e d u c e d 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 (NADH), p y r u v a t e and ammonium i o n s . T h i s enzyme has been r e p o r t e d i n o t h e r b a c i l l i , many o f w h i c h l a c k g l u t a m i c d e  h y d r o g e n a s e , bu t some o f w h i c h have b o t h enzymes (58,86,87). A l t h o u g h t h e enzyme was f o r m e r l y t h o u g h t t o be s p e c i f i c f o r L - a l a n i n e , i t has r e c e n t l y been r e p o r t e d t h a t s e v e r a l o t h e r 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 a r e a l s o o x i d i z e d (123). The e q u i l i b r i u m c o n s t a n t o f t h e d e a m i n a t i o n r e a c t i o n a t pH 10 has been r e p o r t e d t o be 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 +18.6 k c a l . T h u s , t h e s y n t h e s i s o f a l a n i n e i s f a v o u r e d , u n l e s s p y r u v  a t e and NADH a r e removed by o x i d a t i o n , as t h e y a r e i n b a c t e r i a l s p o r e s , i n w h i c h t h e d e a m i n a t i o n has been shown t o o c c u r (122). The r e a c t i o n mechan ism has no t y e t been e l u c i d a t e d , but an enzyme-bound i n t e r m e d i a t e has been p o s t u l a t e d (71). G l u t a m i c a c i d d e h y d r o g e n a s e , w h i c h has been e x h a u s t i v e l y s t u d i e d i n y e a s t , b a c t e r i a l , and a n i m a l s y s t e m s , has a l w a y s been t h o u g h t t o be t h e p r i m a r y r o u t e f o r t h e i n c o r p o r a t i o n o f ammonia i n t o amino a c i d s . In y e a s t (3), and i n E . c o l i (2) c e l l f r e e p r e p a r a t i o n s , t h e r e a c t i o n has been shown 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 (NADP) s p e c i f i c , and t o o c c u r i n two s t a g e s . L - g l u t a m i c a c i d + NADP ^ V i m i n o g l u t a r i c a c i d + NADPH i m i n o g l u t a r i c a c i d + H 20 ^ 1 p f - k e t o g l u t a r i c a c i d + NH3 A t pH 6.5, t h e f r e e e n e r g y c h a n g e i s +17.6 k c a l i n t h e d i r e c t i o n o f ° f - k e t o -15. glutaric acid formation, and therefore, l ike alanine dehydrogenase, favours the synthesis of the amino acid (124). NADP specif ic glutamate dehydrogenases have also been found in Neurospora crassa (59), and in many bacteria (2). The animal enzymes can use either NADP or NAD as hydrogen acceptors. Like alanine dehydrogenase, glutamic acid dehydrogenase was thought to be specif ic for one substrate only. However, Struck and Sizer (145) found that, under appropriate conditions, certain other a l iphat ic , monocarboxylic acids were also oxidized by the chicken l iver 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 dithiol groups are involved in the enzymatic reaction. The inhibition of alanine dehydrogenase act iv i ty by the sulfhydral binding reagents can be reversed by L_-cysteine (122). Possibly related to this finding are the reports of the inhibit ion, by shaking, of alanine biosynthesis, from pyruvate and ammonia, by resting ce l ls 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. subtil is ce l ls grown in the presence of pyruvate, there must be some mechanism for-assimilation of ammonia in these 16. ce l ls other than the animation of this 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 in  corporation in microorganisms. A single 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. Amides are known to be pro ducts of nitrogen f ixat ion (6), and the synthesis of amides from ammonia and either aspartate or glutamate is a major ammonia incorporation reaction of plant tissue (143). Cell free preparations of Staphylococcus aureus (57) and Proteus vulgaris (77), have been shown to synthesize glutamine from glutamic acid, ammonia, adenosine triphosphate (ATP), and magnesium. Nothing is known of the mechanism of asparagine synthesis in bacteria; however, in plants, 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 acid, yielding 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 ixation 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 specif ic act ivity of the isotope being found in glutamate. The concentration of i n the glutamic acid was usually two or more times 17. that of the nearest compound. C. pasteurianum cel l free extracts were found to follow the same route of nitrogen f ixat ion, 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 de hydrogenase (116). Virtanen et aJL (156) also found that yeast ce l l s , exposed to ammonium or nitrate salts after a period of nitrogen starvation, synthesized glutamic acid the ear l iest , and the most actively of al 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 cel ls of B_. abortus, but that reductive amination of pyruvate occurred to about one-third the amount of trans amination. Washed ce 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). No amination of of-ketoglutarate or oxalacetate could be demonstrated. Burk and Pateman (26) showed that mutants of N_. crassa lacked glutamic acid dehydrogenase, but possessed an alanine dehydrogenase specif ic 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). The enzyme was found to be able to act in the direction of synthesis in vegetative ce l l s , but it is probable that the function of the enzyme in spores is to deaminate alanine, to yield the pyruvate which has been re 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 their nitrogen f ix ing eff ic iency. To date, however, despite the pro l i ferat ion 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 ut i l iza t ion by microbial cel ls during the assimilation of nitrogenous compounds has been suggested by a number of studies. For instance, Winzler et aj_. (175) reported that the assimila tion of ammonia by yeast was dependent both on the biotin content of the ce l l s , and on glycolysis; requiring the presence of glucose in the environ ment, and being inhibited by azide. Possibly an expenditure of energy is required for active transport across cel l barr iers, or for endergonic u t i l i za t ion 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 ze glutamate only in the presence of glucose, or a similar energy source (66,67). Endogenous uptake of added ammonia occurred in M.  tuberculosis. the oxygen consumption in the presence of added ammonia be ing greater than in its absence (19). Heating prevented the assimilation of the ammonia, although oxygen uptake at the level characteristic of the absence of ammonia continued. Following a recovery period after heating, 19. assimilation of ammonia started once more, and was accompanied by an in  crease in oxygen uptake. Hence, an oxidative process appeared to be coupled to the endogenous assimilation of ammonia in M. tuberculos is . Bernheim (18) has reported that both an oxidizable substrate and potassium were necessary for the ut i l iza t ion of ammonia by f_. aeruginosa under normal conditions, but in the absence of the metal, assimilation could be restored to a s ignif icant degree by surface active agents, such as polymyxin B, or behzalkonium chloride. 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 bac i l l i (168), it would seem that alanine dehydrogenase must play the major role. 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 germina t ion . 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 participation of glutamate in transamina tion reactions led Braunstein, in 1957 (23), to attribute to glutamic acid dehydrogenase and ° f-ketog lutara te , the key positions in ammonia assimila t ion . A l l 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 situations. III. Species Specif ic i ty of s-RNA's and Aminoacyl-s-RNA Synthetases Recently, it has become evident that the process of oxidative assimilation, rather than yielding only a carbonaceous product within the c e l l , often results in the synthesis of a number of ce 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 ive years. Within this f i e l d , the elucidation of the role played by s-RNA, and how it is played, is receiving much atten t ion . The participation of s-RNA in protein biosynthesis was f i r s t i n  dicated, in 1958, by its amino acid accepting act iv i ty , and its ab i l i t y to transfer the attached amino acid to a ribosomal fraction ( 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). At the present time, it is generally believed that the biosynthesis of protein proceeds through the following steps: AAj + Enzj + ATP s \ Enzj(AAj— AMP) + PP (1) Enzj (AMP~AA) + sRNAj * \ AMP + Enz f + AA;~sRNA (2) AAj~sRNAi + AAj~sRNAj ^ AA;-AAj + sRNAj + sRNAj (3) where AAj is a particular natural amino acid, Enzj is an enzyme specif ic for the activation of AAj, s-RNA is a molecule of soluble RNA specif ic for AAj, and AAj-AAj represents the growing peptide chain. Reaction (3) is catalysed by guanosine triphosphate and ribosomes. It is now agreed that Reactions (1) and (2) are catalysed by the same enzyme, and specif ic en zymes have been found which activate particular amino acids tor form amino- acyl-s-RNA's (16). Data have also accumulated which indicate that there are different s-RNA's for different amino acids (132). In the last three years, interest has been aroused in the inter specif ic reactions between s-RNA's and transfer enzymes. Most of the reports concern comparisons of the heterologous reactions between yeast, mammalian and E. co 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. co l i systems generally react poorly, i f at a l l . The reaction between the bacterial and mammalian systems is variable. Thus, yeast and hog enzymes yielded either yeast or hog tyrosyl-s-RNA, whereas the E. co 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. co l i in the formation of tyrosyl- 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 ver , 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 £. col i with eff ic iencies 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 ver . There was no cross reaction between the E_. col ? enzyme system and either the yeast or rat l iver s-RNA's or vice versa. According to Doctor and Mudd (51), however, yeast enzyme reacts as well with JE. co l i and rat l iver leucine s-RNA as it does with its own, suggest ing to these authors that the E. co l i s-RNA used by Rend? and Ochoa might have lost some of its act iv i ty during isolat ion. This is supported by the findings of Keller and Anthony (94), who demonstrated that rat l iver en zyme incorporated leucine into E. co l i s-RNA quite readily. One must, therefore, interpret results, especially those obtained with one amino acid only, with caution, since either the enzyme or the s-RNA may have 22. been damaged during isolat ion. Doctor and Mudd (51) have attempted to overcome this problem by a comprehensive study on the interspecif ic reactions between the s-RNA's and enzymes from yeast, E. col? and rat l i ver , for 14 amino acids. In most of the cases studied, there was some reaction, indicating that there seems to be no absolute spec i f i c i ty between the enzymes and s-RNA's of a system. In addition, in some of the cross reactions, the heterologous system was more active than the homologous one, especially with the rat l iver enzymes. The authors have several suggestions to explain this anom alous f inding: damage to s-RNA's or enzymes of the homologous system during isolat ion; incorporation of an amino acid into a different acceptor RNA ( i . e . , the spec i f ic i ty between amino acid and s-RNA is not complete); different amounts of s-RNA's in different species; or, f i na l l y , recogni tion of more than one component of a particular amino acid specif ic s-RNA by the heterologous system, whereas only one is recognized by the homolog ous system. That there is more than one component in several s-RNA's corres ponding to a single amino acid is well established. Apgar, Hoi ley and Merr i l l (8) have used counter current distr ibution to achieve some separa tion of some acceptor s-RNA's, while Sueoka and Yamane (147) fractionated the aminoacyl-s-RNA's on methylated albumin columns. By these methods, leucine, isoleucine, serine, 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 . (17) have demonstrated by enzymatic means that E. col i methionine s-RNA has at least two components, since yeast enzyme re  acted to only one-third the extent with E. co l i s-RNA as did the homolog ous 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 repro ducible, and that the relat ive amount of each component was rather constant under different growth conditions. Moreover, in different microorganisms (E. col i, Micrococcus lysodeikt icus. B_. subt i 1 is . and yeast), the elution prof i les of aminoacyl-s-RNA's were dif ferent, although the elution prof 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 prof i les of the two leucyl-s-RNA's proved to be entirely dif ferent, although there was a small amount of over lap. However, the significance of this anomalous reaction is not clear, since, 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 leucyl-s-RNA. It is not yet known whether there is a corresponding enzyme for each component s-RNA, but this is undoubtedly being investigated. The ex periments of Berg et a l . (17) reported above, would tend to support mult iple enzyme systems, since one would consider that E. col i contains two enzymes, one for each methionine specif ic 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. 2 Z*. The existence of several s-RNA's for one amino acid becomes more interesting when this is considered together with the coding problem. Speyer_et_ a_l_. (141), and Nirenberg and coworkers (114,117), have independ ent evidence for degeneracy in the amino acid code for asparagine, leucine, threonine, and serine. Sueoka and Yamane (148) have reported multiple components for a l l these amino acids except asparagine, which has not yet been investigated by their methods. Using the counter current d is t r ibu  tion 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 poly 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. Loft f ie ld and Eigner (105), in a recently published paper, take the opposite view of the spec i f i c i ty problem. Their idea of the s i tua  tion is not a multicomponent system with species differences governing the components present, but a system depending on kinetics for spec i f i c i t y . In kinetic experiments with s-RNA's and enzymes from yeast and E. c o l i . using valine incorporation as the test , Loft f ie ld and Eigner reported that, although the rate of the heterologous reaction between the E. co l i enzyme, and the yeast s-RNA was much slower than that of the homologous system, the Michael is constants were very s imi lar . The lower incorporation at a given time, then, was due to the slower rate of reaction of the hetero logous enzyme-substrate complex to yield valyl-s-RNA. These authors ad mit that this is a consequence of structural differences, but take the stand that these are not necessarily, or even l ikely to be in the enzyme recognition area. 25. A n o t h e r a p p r o a c h t o s t u d i e s o f s p e c i e s d i f f e r e n c e s i n v a r i o u s s - R N A ' s i s t h r o u g h h y b r i d f o r m a t i o n w i t h h e t e r o l o g o u s , hea t d e n a t u r e d d e o x y r i b o n u c l e i c a c i d ( D N A ) . G i a c o m o n i and S p i e g e l m a n (68) and Goodman and R i c h (72) have r e p o r t e d t h a t t h e r e was a s m a l l , bu t r e p r o d u c i b l e amount o f h y b r i d f o r m a t i o n when Ej_ c o l ? s - R N A and DNA w e r e combined and h e a t e d . The r e s u l t a n t h y b r i d was r i b o n u c l e a s e (RNase) r e s i s t a n t , whe reas t h e f r e e s - R N A was s e n s i t i v e t o t h i s enzyme . A s i m i l a r r e s u l t was o b t a i n e d w i t h t h e s - R N A and DNA o f B a c i l l u s megater?urn (68). W i t h t h e E . c o l i s y s t e m , 0 .024% o f t h e 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 c o m p l e m e n t a r y t o t h e s - R N A . Howeve r , t h e E . c o l ? s - R N A h y b r i d i z e d t o d i f f e r e n t , l e s s e r e x t e n t s w i t h t h e DNA' s o f o t h e r m i c r o o r g a n i s m s . T h u s , Goodman and R i c h (72) f o u n d t h a t 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 D N A ' s o f t h e o t h e r E n t e r o b a c t e r i a c e a e as w i t h E . c o l i , bu t l e s s t h a n 15% as much w i t h t h e D N A ' s o f u n r e l a t e d o r g a n i s m s s u c h as B_. m e g a t e r i u m , B_. c e r e u s . B_. a b o r t u s , £ . f l u o r e s c e n s , and M . l y s o d e i k t i c u s . M c C a r t h y and B o l t o n (107) have r e p o r t e d 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 h y b r i d i z a t i o n o f h e t e r o l o g  ous DNA and messenger RNA w i t h t h e DNA o f E . c o l i . The s -RNA-DNA h y b r i d  i z a t i o n e x p e r i m e n t s w e r e e x t e n d e d t o o t h e r 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 S p i e g e l m a n (68), who f o u n d t h a t when t h e DNA' s o f P . a e r u g i n o s a and B_. m e g a t e r i u m w e r e h e a t e d w i t h B . m e g a t e r i u m s - R N A , h y b r i d i z a t i o n o c c u r r e d o n l y i n t h e homologous B . m e g a t e r i u m s y s t e m . T h e r e f o r e , d e s p i t e t h e f i n d  i n g s t h a t t h e E . c o l ? s - R N A c a n t r a n s m i t t h e g e n e t i c message o f a r a b b i t i n t o h e m o g l o b i n (159), and r e a c t w i t h t h e enzyme s y s t e m s o f a number o f m i c r o o r g a n i s m s , i t c an s t i l l be i d e n t i f i e d w i t h t h e genome o f i t s o r i g i n . 26. MATERIALS AND METHODS I. Oxidative Assimilation Into Whole Cells of Aerobic Bacteria A. Bacteriological 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 cel ls were harvested by centrifugation in the cold after growth for 20 hr at 30 C, washed twice in cold 0.05 M t r is (hydroxy- methyl) aminomethane (tris) buffer (pH 7.2), and resuspended to ten times the growth concentration. Unless otherwise noted, this buffer was used throughout the thesis. This procedure was found to y ie ld a ce l l suspen sion containing approximately 5 mg (dry weight) of ce l ls 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 di lut ion 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 vessel. Dry weights were established by drying 5 ml of the ce l l suspensions to constant weight at 100 C. Achromobacter B8l was grown and harvested as above. The eells were resuspended to 40 times the growth concentration, and 1 ml was used per vessel. Achromobacter viscosus ATCC 12448 fai led to grow in the glucose- mineral salts medium used for the other species, but this situation was remedied by the addition of 0.2% yeast extract (Difco). The cel ls were harvested after 17 hr of growth, washed as previously described, and re suspended to 25 times the growth concentration; 2 ml of this suspension 27. were used per vessel. Azotobacter ag i l i s and Azotobacter vinelandi i were obtained through the courtesy of Or. J . Basaraba, Dept. of Soil Science, The Univ ersity of Br i t ish Columbia, and originated from the University of Wisconsin stock culture co l lect ion. They were grown in the medium of Warren et a h (163), except that 0.5% glucose was used as a carbon source. The cel ls were harvested and washed in the same manner as has already been describ ed. Incubation time for A. agi1 is was 20 hr at 30 C, and for A. vineland i i hO hr. A. agi1 is ce l ls 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 (Difco), with salts added as above, f inal pH 6.0. The medium was dispensed in 100 ml portions in 500 ml Florence f lasks, and the cultures were incubated for 17 hr on a rotary shaker. The cel ls were harvested and washed as before, except that the buffer used was 0.05 M t r i s , pH 6.5. The cel ls were resuspended to 60 times their growth concentration, and 2 ml were used per Warburg vessel. 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 cel lu lose. The culture was freed of cel lulose by f i l t r a  tion through several layers of cheesecloth, followed by washing with pH 6.5 t r i s . The cel ls 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 vessel. B. Assimilation studies Manometric experiments were carried out in a Warburg apparatus at 30 C, using conventional techniques for measuring oxygen consumption. 28. E a c h v e s s e l c o n t a i n e d 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 o f g l u c o s e - U-Cl4 i n r a d i o a c t i v e e x p e r i m e n t s ) , t h e a p p r o p r i a t e vo lume o f c e l l s u s p e n s i o n , and 0.05 M t r i s b u f f e r (pH 7.2), t o a f i n a l vo lume o f 3 m l . In t h e c a s e o f t h e two A c e t o b a c t e r s p e c i e s , 0.05 M t r i s , pH 6.5 was u sed i n p l a c e o f t h e pH 7.2 b u f f e r . The v o l u m e o f c e l l s u s p e n s i o n was c h o s e n s o t h a t oxygen u p t a k e was c o m p l e t e i n 120 m i n . A t a p p r o p r i a t e i n t e r v a l s , t o o b t a i n s a m p l e s 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 cup c o n  t e n t s w e r e p i p e t t e d i n t o 1 ml o f t r i s b u f f e r i n c o l d c e n t r i f u g e t u b e s and c e n t r i f u g e d i m m e d i a t e l y . 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 (55), e x c e p t t h a t t h e e x t r a c t i o n w i t h hot t r i c h l o r o a c e t i c 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 . T h i s p r o c e d u r e y i e l d e d f i v e f r a c t i o n s : c o l d and ho 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 , l i p i d , a l c o h o l s o l u b l e , and r e s i d u a l f r a c t i o n s . C . S t a r v e d c e l l e x p e r i m e n t s 1. S t a r v a t i o n p r o c e d u r e Twenty hour o l d c e l l s w e r e h a r v e s t e d unde r s t e r i l e c o n d i t i o n s , washed t w i c e w i t h s t e r i l e t r i s , and r e s u s p e n d e d t o t e n t i m e s t h e i r g r o w t h c o n c e n t r a t i o n i n f r e s h b u f f e r . T w e n t y - f i v e ml o f t h i s s u s p e n s i o n were p l a c e d 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 i n t h e c e n t r e w e l l , and s h a k e n 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 w a t e r b a t h h a v i n g a speed o f 110-120 s t r o k e s p e r m i n . A t t h e end o f t h i s p e r  i o d , t h e c e l l s u s p e n s i o n was removed by p i p e t t e , c e n t r i f u g e d a t 6000xg f o r 15 min i n t h e c o l d , washed o n c e w i t h t r i s b u f f e r , and r e s u s p e n d e d t o 25 m l . T h i s c e l l s u s p e n s i o n was t h e n used f o r a s s i m i l a t i o n s t u d i e s . C e l l s w e r e a l s o s t a r v e d 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 , w a s h e d , and r e s u s p e n d 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 . 29. 2. A s s i m i l a t i o n e x p e r i m e n t s T h e s e w e r e p e r f o r m e d i n t h e same t y p e o f f l a s k and i n c u b a t e d i n t h e w a t e r b a t h as o u t l i n e d a b o v e . A t o t a l o f 25 ml w e r e p l a c e d i n e a c h o f two f l a s k s , c o n s i s t i n g o f 8.3 ml o f c e l l s u s p e n s i o n , 0.83 ml o f g l u c o s e - U-c '4 (50 umoles 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 , and t r i s b u f f e r t o v o l u m e . Two ml o f 20% KOH w e r e added t o t h e c e n t r e w e l l . The s u b s t r a t e was added by p i p e t t e a f t e r 10 min e q u i l i b r a t i o n . P a r a l l e l e x p e r i m e n t s , w i t h n o n r a d i o a c t i v e g l u c o s e o r o t h e r s u b s t r a t e s , w e r e r un by t h e u s u a l Warbu rg t e c h n i q u e , u s i n g 1 ml o f c e l l s u s p e n s i o n , and 5 Mmoles o f s u b  s t r a t e i n a t o t a l o f 3 n i l . When c o f a c t o r s were u s e d , p r e i n c u b a t i o n t i m e was 20 m i n . A t i n t e r v a l s d u r i n g t h e e x p e r i m e n t s w i t h r a d i o a c t i v e g l u c o s e , 2 ml o f t h e f l a s k c o n t e n t s w e r e r emoved , added t o 1 ml o f c o l d t r i s i n a c h i l l e d c e n t r i f u g e t u b e , and c e n t r i f u g e d i m m e d i a t e l y f o r 15 min a t 6000xg a t k C . 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 i n S e c t i o n I B . D. A n a l y t i c a l methods 1• A n a l y s i s o f r e s i d u a l f r a c t i o n s A p o r t i o n (2 m l ) o f e a c h o f t h e 120-min r e s i d u a l f r a c t i o n s was h y d r o l y s e d w i t h 1 N HC1 f o r h h r a t 108 C i n a s e a l e d a m p o u l e . The h y d r b - l y s a t e was t a k e n t o d r y n e s s i n a vacuum d e s i c c a t o r o v e r NaOH and CaSO^; t h e n , 1 ml o f w a t e r was a d d e d , an a l i q u o t t a k e n f o r t h e d e t e r m i n a t i o n o f r a d i o a c t i v i t y , and t h e r e m a i n d e r a p p l i e d t o a Dowex-50 (H) co lumn (50 t o 100 mesh; 0.8x10cm). The co lumn was washed w i t h 50 ml o f w a t e r , f o l l o w e d by 75 ml o f 1 N NH^OH. B o t h e l u a t e s w e r e c o n c e n t r a t e d t o d r y n e s s w i t h a f l a s h e v a p o r a t o r ; 1 ml o f w a t e r was added t o e a c h , and a s a m p l e was t a k e n f o r d e t e r m i n a t i o n o f r a d i o a c t i v i t y . C l o s e t o 100% o f t h e a p p l i e d c o u n t s w e r e r e c o v e r e d i n t h e two f r a c t i o n s . B o t h f r a c t i o n s o f e a c h r e s i d u e w e r e 30. s u b j e c t e d t o a n a l y s i s by p a p e r c h r o m a t o g r a p h y and e l e c t r o p h o r e s i s , and 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 as d e s c r i b e d i n t h e s e c t i o n on p a p e r c h r o m a t o g r a p h y . C o n t r o l e x p e r i m e n t s c a r r i e d o u t w i t h g l u c o s e and 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 s e s u b s t a n c e s w e r e no t d e s t r o y e d by t h e h y d r o l y s i s , and t h a t b o t h compounds a p p e a r e d i n t h e w a t e r e l u a t e f r o m t h e Dowex-50 c o l u m n . 2. A n a l y s i s o f c o l d t r i c h l o r o a c e t i c a c i d f r a c t i o n s T h r e e ml p o r t i o n s o f t h e c o l d t r i c h l o r o a c e t i c a c i d f r a c t i o n s o f 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 15 m i n w e r e e x t r a c t e d w i t h f o u r s u c c e s s i v e 3 ml p o r t i o n s o f e t h e r t o remove t h e t r i c h l o r o a c e t i c a c i d . The e x t r a c t s , w h i c h w e r e s t i l l a c i d i c , w e r e s u b j e c t e d t o p a p e r c h r o m a t o  g r a p h y 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 l o c a t e d by s c a n n i n g . In an e f f o r t t o i s o l a t e t h e p o l y m e r f r o m t h e c o l d 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 f r a c t i o n o f A c h r o m o b a c t e r B 8 1 , t h e c o n t e n t s f r o m n i n e l a r g e W a r b u r g v e s s e l s , c o n t a i n i n g a t o t a l o f 162 m l , w e r e removed a f t e r 15 min on t h e W a r b u r g a p p a r a t u s a t 30 C , p i p e t t e d i n t o c o l d b u f f e r as f o r t h e a s s i m i l a t i o n e x p e r i m e n t s , and t h e p r o c e d u r e c a r r i e d t h r o u g h t h e p o i n t o f t h e t r i c h l o r o a c e t i c a c i d e x t r a c t i o n . The s u p e r n a t a n t f r a c t i o n (330 m l ) f r o m t h i s e x t r a c t i o n was e x t r a c t e d w i t h e t h e r i n a l i q u i d - l i q u i d e x t r a c t o r o v e r n i g h t , c o n c e n t r a t e d t e n f o l d by e v a p o r a t i o n a t 40 C , and t h e e t h e r e x t r a c t i o n r e p e a t e d . The r e s i d u a l s o l u t i o n (30 t o 35 m l ) w h i c h was s t i l l a c i d i c (pH 5 . 4 ) , was c o n c e n t r a t e d t o 8 m l , and a s a m p l e t e s t e d f o r t h e p r e s e n c e o f c a r b o h y d r a t e by t h e a n t h r o n e method (153). E t h y l a l c o h o l was added t o t h e 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 ) . The 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 formed on s t a n d i n g was c o l l e c t e d by c e n t r i f u g a t i o n , w a s h e d , and r e d i s s o l v e d i n w a t e r . T h i s o p a l e s c e n t s o l u -31. tion was used for the following tests: reaction with iodine, periodate (by paper chromatography) s tab i l i t y to alkal i after hydrolysis at 100 C, composition as revealed by acid hydrolysis, and su i tab i l i t y as a substrate for phosphorylase. 3. Chemical methods Glucose in the supernatant f lu id was determined by the "gluco- stat" 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 ° ( -ke tog lu ta ra te , pyruvate, oxalacetate and glyoxylate, the reaction mixtures were allowed to stand for 4 hr at room temperature; for 2-keto- gluconate, the incubation time was 16 hr. Ammonia was determined by the Conway microdiffus ion technique (40). 4. Paper chromatography and electrophoresis Paper chromatography of the fractions obtained from the super natant f luids was carried out on Whatman no. 1 paper, by use of secondary butanol-formic acid-water (BFW; 70:10:20, v/v; 130) or ethanol-methanol- water (EMW; 45:45:10, v/v; 120). EMW separates glucose and gluconic acid, whereas BFW distinguishes between Krebs cycle acids. Gluconic and 2-keto- gluconic acids were differentiated by chromatography in ethyl acetate- pyridine-sat. aq. boric acid (EPB; 60:25:20, v/v; 73). For chromatography of the 2,4-dinitrophenylhydrazone derivatives of keto acids, 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 acids: s i lver 3?. nitrate-sodium hydroxide-thiosulfate dip; organic acids: ani l ine xylose dip; reducing substances: ani l ine phosphoric acid dip; and carbohydrates: periodate-benzidine spray. Radioactive areas on paper chromatograms or electrophoretograms were determined by running str ips through a Nuclear-Chicago Model C 100 B Actigraph II, with a gas-flow counter, a Model 1620 B Analytical Count Ratemeter, and a Chart Recorder. 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 glucose-c '^ was diluted so that it had a specif ic act iv i ty of 0.6 to 0.7 He per "mole, and 5 "moles were added per reaction vessel. II. Inorganic Nitrogen Assimilation by Pseudomonas aeruginosa and Pseudo monas fluorescens Nitrogen assimilation studies were performed in the conventional Warburg apparatus, using 1 ml of cel l suspension (prepared as described in Section I A from 20 hr cel ls ) per Warburg f lask. Ammonium sulphate was used as the source of ammonia. The keto acids, inhibitors, 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 f lu ids from the Warburg f lasks. III. Oxidative Assimilation into the Cytological Fractions of Normal. Starved, or Chloramphenicol Treated Cells of £. aeruginosa ATCC 9027 33. A. Assimilation studies Cells were grown for 20 hr at 30 C, harvested, and resuspended as before (Section I A). The starved cel ls were ^prepared as previously described (Section I C 1), by shaking a 10 times growth suspension under s te r i l e conditions for 3 hr at 30 C, washing, and resuspending the ce l ls to the same volume in fresh buffer. For the assimilation studies, the procedure outlined in Section I C 2 was followed. Chloramphenicol (200 Hg per ml) was in contact with the ce l ls for 30 min prior 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 lask, added to 2.5 ml of cold t r i s buffer in a chi l led centrifuge tube, and then centrifuged immediately at 6000xg in the cold. The ce l ls 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 cel l fractions The ce l ls removed at each time interval were broken separately in a chi l led French pressure cel l at 15,000 to 17,000 lbs pressure, the extracts collected in cold centrifuge tubes, and 0.1 ml of deoxyribonucle ase (ONase) (1 mg per ml) added to reduce their v iscosity. The extracts were st i rred intermittently for f ive 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 prec ip itate wash with .05 M t r i s buffer, pH 7.2 resuspend in buffer I precip itate ("Membranes' IX) ("Membranes' 2X) 1 , „ supernatants (Washes) 1 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 ultra- centrifuge r r " — prec ip itate (R i bosomes) 1 supernatant (Cytoplasm) C. Analysis of cytological fractions Large scale preliminary experiments were done, in which the fractions were analysed for RNA (orcinol method) (131), DMA (diphenylamine method) (131), glucose oxidizing act iv i ty (manometrically), and for pro tein (Lowry method) (106), as a means of establishing the purity of va r i  ous fractions (30). D. Chemical fractionation of the cytological fractions The "membranes," ribosomes, and cytoplasm were chemically f rac  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 ce l l s , it was found that there was so l i t t l e radioactivity present that it was impossible to determine whether these materials were acidic or neutral. The water eluates from the 30 min fractions of control and an t i  biot ic 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 acidic compounds). Each of these e l  uates was taken to dryness in a flash 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 tr ichloroacetic acid soluble fractions The cold tr ichloroacetic acid soluble extracts from the cyto plasmic fractions were ether extracted, and subjected to paper chromato graphy 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 air from a pump, the air being passed through a s te r i l e cotton f i l t e r . In addition, the culture was agitated by means of an overhead s t i r r e r . The medium used consisted of : enzymatic casein hydrolysate (Difco) 3%, glucose 0.3%, I^PO^ 0.2%, 36. K2HP04.3H20 0.3%, FeS04 0.0005%, pH 7.2. After autoclaving, 10 ml of s te r i l e MgS04.7H20 per 1 of medium was added aseptical ly. Ster i le "G.E. Antifoam" was added at intervals as needed. 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 plat ing. Harvesting was done with a Sharpies centrifuge at top speed at room temperature. The ce l ls were collected from the cyl inder, washed twice with cold t r i s buffer in a re frigerated Servall centrifuge, the wet paste weighed, and stored frozen. After being thawed, the bacterial ce l ls (about 150 g) were mixed with enough alumina to give a doughy consistency, and were then broken by grinding in a chi l led mortar until liquefaction occurred. Gen era l ly , this process required about 10 to 15 min. The mixture was ex- tracted with suff ic ient t r i s buffer, containing 10 _ i l M Mg' to give 400 ml of suspension. The viscous suspension was poured into a beaker, 3 mg DNase added, and the mixture was st irred at room temperature for 10 min. By this time the viscosity was greatly reduced. Alumina and unbroken ce l ls were removed by centrifugation twice at 3000xg for 15 min in a refrigerated Servall centrifuge. The supernatant from this centrifugation, amounting to between 170 and 200 ml, was then centrifuged for 2 hr. batchwise, at 105,000xg in a Spinco Model L preparative ultracentrifuge. The upper two- thirds of each tube was removed, and stored frozen overnight. After thaw ing, this fraction was used for the preparation of s-RNA by the phenol method of Tissleres (151). The y ie ld 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: t r i s acetate buffer 0.5M pH 7.4, 1.5 ml; ATP 0.1M, 0.3 ml; MgCl2 1M, 0.075 ml; KC1 2M, 0.075 ml; ethyl mercaptan 5%, 0.15 ml; Chlorel la c'^ amino acid hydrolysate (Merck, Sharpe and Dohme Co. L td . , Montreal), 0.03 ml (3 He). This mixture was stored frozen. The incuba tion 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. Incuba tion was for 30 min at 35 C. 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 ing after each addition. After 10 min in an ice bath, the samples were centrifuged, the supernatants decanted, 1 ml of alcohol-salt solution added, the mix ture st i rred vigorously, allowed to stand 10 min in an ice bath, and then centrifuged. This washing procedure was repeated twice, the third wash consisting of cold, 95% ethanol. The f inal precipitate was dissolved in 0.2 ml of IN NH^OH, and two 0.02 ml portions were plated at inf in i te thinness for determination of the c'^ incorporation into the s-RNA. H. Isotopic methods These were as previously described (Section I E). IV. Species Specif ic i ty 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, Achromo- bacter B81, and Saccharomyces cerevis iae (bakers' yeast). A. Preparation of s-RNA's The bacteria were grown, harvested, and cel l extracts prepared 38. as outlined in Section IV A. For E. col i and Achromobacter B81, the con centration of glucose in the medium was raised to 1%. Soluble RNA1s were prepared from the 105,000xg supernatant fractions of the ce l l extracts by the phenol extraction method of Tissieres (151). Yeast s-RNA was a g i f t of Drs. G.M. Tener and R.V. Tomlinson (13). B. Preparation of enzymes The bacteria were grown for 20 hr at 30 C in a glucose-ammonium salts medium (163). For JE. col ?, 1% glucose was used, instead of the 0.2% normally employed. The cel ls were harvested, washed, resuspended in buffer, and disrupted by passage through a French pressure c e l l , and the ce l l extracts fractionated as before (Section III B). The I05,000xg supernatant fract ions, which were stored at 0 C, were used as the synthet ase systems. The yeast enzyme was obtained from Dr. R.V. Tomlinson, and was prepared in essential ly the same way as described for the bacterial en zymes, the starting material being Fleischmann yeast cakes. A l l the enzymes were tested for act iv i ty on the same day that they were prepared. As long as they were not frozen, the enzymes retained their act iv i ty for several days in the cold, C. 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 re act iv i ty of the systems being tested, but in order to obtain suff ic ient 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. Two volumes of cold ethanol were then added to each sample, and after 10 min in an ice bath, the tubes were centrifuged, decanted, stored at -15 C, and later used for paper chromatography. A determination was made of the radioactivity in each of these alcoholic solutions, and it was found that there was complete recovery of the c'^ present prior to the reprecipitat ion. Chromatograms were run, in the descending manner, on Whatman no. 1 f i l t e r paper, for 30 hr, with nrbutanol: glacial acetic acid: water (120:20:50, v/v) as the solvent. About one-third of each sample of c'^ amino acid was applied to the paper, dried, and then co- chromatogrammed with a set of amino acid standards. After development of the chromatograms, they were cut into s t r ips , and analysed by an Act i- graph Model 1032B 4-pi scanner. The str ips were dipped into ninhydrin (0.2% in acetone), heated at 60 C for 2 min, and the radioactive peaks were identified by comparison with the standards. The percentage incor poration of each amino acid, 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 luids 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 until 67% of the theoretical value was reached. Gluconate was oxidized in similar fashion, whereas °<-ketoglutarate was oxidized slowly and after a period of induction. The relative values for the oxida tion of glucose, gluconate, and ° ( -ketoglutarate were 131, 110, and 7.2, respectively. Glucose had disappeared from the supernatant f lu id 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 partly correct, since gluconate, pyruvate, and °C -keto- glutarate were present in the supernatant f lu id at 15 min and gradually decreased in concentration after this 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 inter mediates were v i r tua l ly exhausted. No 2-ketogluconate was detected by treatment of the supernatant f lu id with 2,4-dinitrophenylhydrazine, 41. Table 1. Radioactive compounds in the supernatant f lu id during glucose oxidation Microorganism Compounds present at 15-45 min 60 min' 120 min* pseudomonas aeruginosa 120 Na P. fluorescens A 3.12 Achromobacter B81 A. viscosus Gluconate Pyruvate ++ •"C-Ketog1ut arat e+ Glucose++ Gluconate*++ Neutral compound A+++ Glucose++ Succinate++ ''C-Ketogl utarate++ Fumaratet+ Succinate+ Fumarate+ Glucose+++ Neutral compounds B++ Succinate++ Fumarate++ Glucose+ Fumarate (trace) D i carboxylie ac i ds (trace) Neutral Neutral compound B+++ compound B+ Radioactive UV-absorbing material was present in a l l cases at 60 and 120 min. hi. Minutes . Minutes FIG. 1 A. Oxygen uptake with 5 Fmoles of substrate and disappearance of glucose and C14 from supernatant f luids during manometric experiments with washed-cell suspensions of Pseudomonas aeruginosa. Oxygen uptake with glucose, P ; with ° f-ketoglutarate ,A ; endogenously, 0 . Dis appearance from supernatant f luids of glucose, • , and jC.14, • . Endogenous oxygen uptake values have been subtracted from the values reported for substrate oxidation. FIG. 1 B. Time course of NH3 production, keto acid formation, and cJ4 in  corporation into ce l ls 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 produc t ion , Q , and C'4 incorporation into ce l l s , A . 43. followed by extraction, chromatography, and scanning. Endogenously produced ammonia was incorporated into the cel ls on the addition of glucose, and none could be detected in the supernatant f lu id 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 in 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 con sumed. Oxygen uptake ceased at 68% of the theoretical to ta l . °< -Keto- glutarate was oxidized at an in i t i a l rate identical to that of glucose, whereas gluconate was attacked more slowly. The values for glucose, gluconate, and -ketoglutarate were 124, 65, and 124, respectively. As one might conclude from the manometric data, analyses of the super natant f luids 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 until 45 min and then disappeared f a i r l y rapidly. This second product, designated as neutral compound A in Table 1, had an Rf of 0.14-0.16 in BFW (Rf of glucose = 0.22), gave a posit ive s i lver nitrate reaction, and did not migrate electrophoretical ly. Similar results were obtained with disaccharides, but attempts to isolate and characterize the unknown compound fa i l ed . 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 cel ls on the addition of glucose; however, some remained in FIG. 2 A. Oxygen uptake with 5 H m ° J e s ° f substrate and disappearance of glucose and from supernatant f luids during manometric experiments with washed-cell suspensions of Pseudomonas fluorescens. Oxygen uptake with glucose, © ; with of-ketoglutarate, 4k ; endogenously, 0 . Dis appearance from supernatant f lu ids of glucose, • , and C l 4 > 0 Endogenous oxygen uptake values have been subtracted from the values reported for substrate oxidation. FIG. 2 B. Time course of NH3 production and cJ4 incorporation into ce l ls 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 ce l l s , A . 45. the supernatant f lu id at a l l times (Figure 2B). Achromobacter B81 oxidized glucose at a rapid in 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 to ta l . After a 5 to 7 min period of induction, gluconate was oxidized rapidly, whereas <K -ketoglutarate oxidation was characterized by a very slow rate of induction and subsequent oxidation. The QQ^ values for glucose, gluconate, and -ketoglutarate were 101, 86, and 6.7 re spect ively. The main product which accumulated during glucose oxidation was found to be °f-ketog1utarate, which disappeared slowly from the super natant f lu id during the interval between 30 and 120 min (Table 1). Pyruvate could not be detected in the supernatant f luids obtained at 15 and 30 min. Succinate, fumarate, and other doubly charged acids identi f  ied by cochromatography and scanning, were present but did not disappear from the supernatant f lu ids . Support for the accumulation of the acids was given by the ultraviolet (UV) spectra, which showed a high end absorption characteristic of dicarboxylic acids. Endogenously produced ammonia was incorporated into the cel ls on the addition of glucose, and none could be detected in the supernatant f lu id 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 oxidation, and leveling of the curve occurred at 54% of theoret ical oxygen uptake. Gluconate and ° (-ketoglutarate were oxidized after an induction period, 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 luids during manometric experiments with washed-cel1 suspensions of Achromobacter B81. Oxygen uptake with glucose, 9 , with ° (-ketoglutarate , & ; endogenously, 0 . Disappearance from supernatant f luids 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 in  corporation into cel ls of Achromobacter B81 during oxidation of 5 Hmoles of glucose-U-C^ by washed-cel I suspensions. Production of NH3 endog enously, 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 luids 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 lu ids 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 cel ls 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 ce 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 lu id for 60 min, and there was no keto acid or gluconate present at any time. Comparison of the rates of disappearance of radioactivity and glucose from the supernatant f lu id revealed that the level of glucose f e l l more rapidly, 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 until 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 ed . 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 lu id 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 radioactivity remained in the supernatant f lu id 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 poss ib i l i ty of some ce l l lysis cannot be eliminated. 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 lu id 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. This would suggest that, at least in this instance, the increase in UV-absorbing material was due to the secretion of RNA and not to cel l l ys i s . A comparison of the results of the oxidation of glucose-U-C^ by resting ce l ls 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 imi lar i t ies between the bacteria. With a l l the strains, the two stage glucose oxidation corres ponded to the accumulation of intermediate compounds, whose rates of oxidation were l imiting for the conversion of glucose to C0£ and water. With P. aeruginosa these were pyruvate, gluconate, and ° f -ke tog lu ta ra te , with Achromobacter B81 it was °C-ketoglutarate, whereas with P. fluorescens it was gluconate. Both P. fluorescens and A. viscosus also accumulated, and later oxidized, unknown neutral compounds. Assuming that a l l four bacteria studied continued to oxidize their endogenous reserves in the presence of exogenous substrate, (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 intermed iates 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. P_. fluorescens did not accumulate °f-keto- glutarate; 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 fai led to accumulate °<-ketoglutarate, 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 lu id during the experiment. However, the high ut i l iza t ion of endogenously produced ammonia indicated that the rate of removal of °C-ketoglutarate by amination would be suff ic ient ly rapid to prevent the extracellular accumulation of this keto acid. 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 lu ids occurred in the instances where the supply of ammonia from endogenous respiration was low, thus permitting a more eff ic ient ut i l iza t ion of ammonia and glucose for synthetic purposes. Although there were s imi lar i t ies 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 lu ids , and, in each case, the compounds were ut i l ized when the parent substrate had disappeared, but unlike f_. aeruginosa and Achromobacter B81 they did not excrete keto acids. 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 ratio of carbon to nitrogen was high, as it was in the Warburg cup during glucose oxidation. 2. Incorporation of j n to cel ls during oxidative assimilation The amount and patterns of incorporation of radioactivity into cel ls of the bacteria are shown in Tables 2a, 2b, 3a and 3b. In each case, the amount of assimilated material was much less than would be expected from the oxygen uptake, a result similar to that found in P. aeruginosa 51. Table 2a. Incorporation of from 5 "moles of glucose-U-C^ into washed-cel1 suspensions of P. aeruginosa Alcohol- Unfrac- Time Cold- soluble Hot Total in tionated (min) soluble* Lipid protein soluble*" Residue fractions ce l ls Per cent of total C l Z f 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 tr ichloroacetic acid soluble fractions. 52. Table 2b. Incorporation of c'^ from 5 Fmoles of glucose-U-c'^ into washed-cell suspensions of P. fluorescens Alcohol- Unfrac- Time Cold- soluble Hot Total in tionated (min) soluble* Lipid protein soluble* Residue fractions cel ls 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]l* incorporated into eel 1 fract ions 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 tr ichloroacetic acid soluble fractions. 53. Table 3a. from 5 Hm ( into washed-cell suspensions of Achromobacter B81 Incorporation of C ^ fro  5 "moles of glucose-U-C1^ Alcohol- Unfrac- Time Cold- soluble Hot Total in tionated (min) soluble" Lipid protein soluble' Residue fractions cel ls Per cent of total C , Z f 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 fract 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 tr ichloroacetic acid soluble fractions 54. Table 3b. Incorporat ion of C ^ from 5 pmoles of glucose-U-C'H into washed-cel1 suspensions of A. viscosus Time (min) Cold- rf soluble" Alcohol- soluble Hot Lipid protein soluble* Residue fractions cel ls Unfrac- Total in tionated Per cent of total c'^ added to vessel 15 6.8 0.29 0.25 0.80 2.0 10.1 9.5 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 1 4 incorporated into ce l l fractions 15 67 . 2.9 2.5 7.9 20 100 30 59 3.1 3.2 12.5 22 100 120 27 4.2 2.3 19 48 100 * Cold and hot tr ichloroacetic acid soluble fract ions. 5 5 . ATCC 9027 ( 5 5 ) . The products of glucose oxidation which were excreted into the supernatant f luids appeared to be important in determining the extent of assimilation, 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 correlation 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 rapidly, 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 imilation. 3. Assimilation of C ^ into the cel l fraction soluble in  cold tr ichloroacetic acid The compounds removed from the cel ls by treatment with t r i ch loro  acetic acid in the cold, and therefore considered to be "pool " constituents, made up a rather large proportion of the total radioactivity present in the c e l l s . As might be expected, if oxidative assimilation 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 ab i l i t y to oxidize ° f-ketoglutarate , and therefore did not accumulate this keto acid during glucose oxidation. P. fluorescens d id , however, contain a permease which brought about the passage of a high concentration of glucose into the tr ichloroacetic acid soluble pool. 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 tr ichloroacetic 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, in this case, the polymer was carbohydrate in nature, but not glycogen. Treatment of whole ce l ls with the anthrone reagent before and after assimilation of glucose, showed that 5% of the dry weight of the cel ls was made up of carbohydrate material which did not increase during the experiment. . A similar analysis on the cold tr ichloroacetic acid extract revealed that 20% of the carbohydrate was present in this fract ion. The remaining carbohydrate was distributed between the hot tr ichloroacetic acid extract and residue. The alcohol precipitable material from the cold t r i ch loro  acetic acid extract reacted with periodate, did not react with iodine, was stable to alkaline hydrolysis, and was not hydrolysed by phosphorylase. The unit compound of the polymer behaved l ike glucose when analysed by paper chromatography. However, other hexoses, notably galactose, would react s imi lar ly . In addition to the polymeric carbohydrate, there was present, before acid hydrolysis, as well as after, a periodate-oxidizable compound, having an Rf in BFW similar to that of glycerol . k. Assimilation of C1** into the ce l l fractions insoluble in  cold tr ichloroacetic acid The assumption that a l l four organisms reincorporated some ammonia during glucose oxidation would seem to be va l id , for analysis of the ce l ls 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 ip id containing ones, (Tables 2 and 3). Moreover, the residual fraction containing the protein was the one which continued to increase in radioactivity with time, and which, in a l l cases, accounted for almost half the radioactivity of the ce l l at 120 min. When these residual protein-containing fractions from cel ls which had respired for 2 hr were hydrolysed and applied to Dowex-50 (H) columns, some of the radioactivity was removed by elution with water (Table 4). The small amount of radioactive material in the Table k. Acid hydrolysis and separation by ion exchange of res idual*f ract ion Microorgan ism "Glucose 1 1 content** Eluted by water (neutral compounds) Eluted by NH4OH i(ninhydrin • compounds) % % Pseudomonas aerugi nosa 0.6 10 90 P. fluorescens 0.3 7 93 Achromobacter B81 1.0 30 70 A. viscosus 0.5 7.5 92.5 insoluble in hot 5% tr ichloroacetic acid. Glucose content was measured by the anthrone test. 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 cel 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 tr ichloroacetic acid, as is free glycogen. The compounds in the water eluates should be either neutral or acidic in nature; however, paper chromatography and electrophoresis of these eluates never revealed any acidic components. The sole radioactive peak in each of the water eluates reacted similar ly 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 -Hydroxy- butyrate, 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 fract ions. Paper chromatography and scanning of the ammonia eluates showed that a l l radioactive areas were ninhydrin posit ive. The individual amino acids were not ident i f ied. To a lesser extent than the protein fract ion, the fraction soluble in hot tr ichloroacetic acid, which contained primarily nucleic acids, also increased with time. A. viscosus assimilated an unusually high percentage of the of glucose, a considerable amount of which was due to its containing a surprisingly large amount of radioactive material soluble in cold or hot tr ichloroacetic acid. The relative amount of radioactivity in the hot tr ichloroacetic acid soluble fractions doubled between 15 and 120 min, and accounted for 19% of the total of the ce l ls at the completion of the experiment. Achromobacter B81 also as similated a large percentage of the added radioactivity into the nucleic 59. acid fract ion, but in this instance the fraction appeared to be most im portant in the early stages of oxidative assimilation. In confirmation of the observations of Duncan and Campbell (55), the l ip id of P. aeruginosa appeared to be of significance in the early stages of oxidative assimilation. 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 experi ments was similar to that reported recently by Duncan and Campbell (55) and Cl i f ton (36), i .e. , the radioactivity appeared f i r s t in the cold t r i ch loro  acetic acid soluble components, from where it was distributed into the other fract ions. Al l four bacteria incorporated a large proportion of the assimilated carbon into nitrogenous cel l components, of which protein con tained most of the C'4 in each case. The concept (55) that oxidative as similation occurred by way of reincorporation of endogenously produced ammonia was found to be tenable. The two Achromobacter species ass imi l  ated a high proportion of the radioactivity into the nucleic acid fractions, at the expense of the l i p i d . This is part icular ly interesting in the case of A. viscosus. since this organism wi l l not grow in an inorganic salts 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 cel ls with no added vitamins or amino acids exhibited the highest amount of oxidative assimila 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 ratio (10.6) of endogenous oxygen uptake to ammonia production, which is a figure twice that normally obtained with obligate aerobes, and similar 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 tr ichloroacetic acid-soluble pool in the early stages of glucose oxidation, and the fraction insoluble in hot tr ichloroacetic 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 luids 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 cel ls of this organism, and 91% of the added c'4 remained in the supernatant f lu id at the end of the experiment. Moreover, more than 4 Mmoles of gluconic acid were present in the supernatant f lu id 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 specif ic 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 lu id during glucose oxidation Microorgan ism Compounds present at 15-45 min 60 min 120 min'' Acetobacter aceti Acetobacter xy1inum Azotobacter agi1 is Azotobacter vinelandii Glucose*** Gluconate*** Glucose++ Gluconate*** Glucose* Glucose* Glucose* Gluconate**** Glucose* Gluconate* Cellulose* Gluconate**** Gluconate* Cellulose** " Radioactive UV-absorbing material was present in a l l cases at 60 and 120 min. 62. A B 500J 0} •g 400. Q. g 300. 200. 100. I 0 0 F — _I00 30 60 90 M i n u t e s 30 6.0' 90 Minu tes T3 80 « T3 O 60 2 o 40 20 .2.0 1 o 3 "O O 1.5 O. X 0.5 FIG. 5 A. Oxygen uptake with 5 Hmoles of substrate and disappearance of glucose and Cl4 from supernatant f luids during experiments with washed- cel l suspensions of Acetobacter acet i . Oxygen uptake with glucose, 0 ; endogenously, 0 . Disappearance of glucose,Q,and CJ^, from super natant f lu ids . Endogenous oxygen uptake values were subtracted from the values reported for glucose oxidation. FIG. 5 B. Time course of NH3 production and incorporation into ce l ls of Acetobacter aceti during oxidation of 5 H m o ' e s of glucose-U-C^ by washed- cel l suspensions. NH3 production endogenously, 0 ; NH3 production in presence of glucose, 0 ; Cl4 incorporation into ce l l s , & . 63. could have been by pathways not involving gluconate, for whole cel ls and ce l l extracts, prepared with a 10 Kc Raytheon sonic osc i l l a to r , fa i led 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 equi l ib  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. Despite the very low endogenous oxygen uptake, ammonia production was relat ively high (Figure 5B). However, the presence of glucose did not result in ammonia reincorporation; in fact, ammonia production appeared to be s l i gh t  ly stimulated by glucose. Not surprisingly, only 2% of the radioactivity was incorporated into the ce 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 cel ls 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 cel l even when the substrate is being oxidized by way of the tr icarboxylic 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 ze inorganic nitrogen for the synthesis of organic compounds. In contrast to A,, acet ?, Acetobacter xyl inum oxidized glucose at a rapid, constant rate, until the break in the curve at 90 min, when 80% of the theoretical amount of oxygen required for complete oxidation of glucose was achieved (Figure 6A). Gluconate and °C-ketoglutarate were oxidized by induced enzymes, the Qn^'s for glucose, gluconate and °C-keto  glutarate oxidation being 78, Ih, and 39, respectively. The products detected in the supernatant f lu id during glucose oxidation were gluconic acid, which increased until 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. No keto acids were detected in the medium. The endogenous oxygen uptake followed a most unusual pattern, there was a rapid in i t i a l rate, but a l l oxidation soon stopped. The same result was obtained repeatedly. Despite the fact that A. xylinum required a complex nitrogen source for growth, endogenously produced ammonia appear ed to be reincorporated during the oxidation of glucose, without, however, the excretion of keto acids into the medium (Figure 6B). This is a similar situation 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 lu id (Table 5, Figure 7A). However, there was a second ary rate of oxidation which began after 67% of the oxygen for complete oxidation of glucose had been consumed, and continued l inearly 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 luids during experiments with washed- cel l suspensions of Acetobacter xylinum. Oxygen uptake with glucose, # ; endogenously, 0 . Disappearance of glucose, Q , and C ^ , • from super natant f l u ids . Endogenous oxygen uptake values were subtracted from the values reported for glucose oxidation. FIG. 6 B. Time course of NH3 production and C ' 4 incorporation into cel ls 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. M i n u t e s M i n u t e s FIG. 7 A. Oxygen uptake with 5 Hmoles of substrate and disappearance of glucose and from supernatant f luids during experiments with washed- cel 1 suspensions of Azotobacter v inelandi i . Oxygen uptake with glucose, 0 ; endogenously, 0 . Disappearance of glucose, Q , and C ^ , g§§ from supernatant f l u ids . 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 cel ls of Azotobacter vinelandii 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 rest, 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 respiration. Yet, there was no detectable loss of radioactivity from either the cel ls or from the supernatant f lu id after 45 min, although enough oxygen was consumed to oxidize completely 1 umole of glucose during this period. 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 °<-ketoglut- arate oxidation proceeded at a slow, steady rate. The Qo '^s for glucose, gluconate, and «C-ketoglutarate oxidation were 180, 20, and 23, respective ly . 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 wi l l in  crease the f ixation 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 rapidly, but without a secondary rate of oxidation (Figure 8A). Again there was very l i t t l e as similation of the glucose supplied, for at 120 min 83% of the theoretical amount of oxygen for complete oxidation had been consumed. Both gluconate and «^-ketoglutarate 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 in  corporated into ce l lu lar 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 luids during experiments with washed- cel l suspensions of Azotobacter ag i l i s . Oxygen uptake with glucose, © ; endogenously, 0,. Disappearance of glucose, • , and C14, HI from super natant f lu ids . 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 cel ls of Azotobacter ag i l i s during oxidation of 5 Hmoles of glucose-U-C^ by washed- cel l suspensions. NH3 production endogenously, 0 ; NH3 production in presence of glucose, 0 ; C^4 incorporation Into c e l l s , 69. i lated only 5% of the into ce l lu lar material, in spite of the excess of ammonia available to i t . 2. Incorporation of into cel ls during oxidative assimila- t ion The amount and patterns of the incorporation of from glucose are shown in Tables 6a, 6b, 7a and 7b. The amount of assimilated material, unlike that in most cases in the previous section, corresponded fa 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 radioactivity, while oxidizing glucose to 89% of the theoretical amount. None of these bacteria, however, excreted compounds into the medium which could be considered to act as "pacemakers'1 for oxidative assimilation, since the gluconate produced by A. acet i. and the cel lulose by A. xylinum were not metabolized further. Although ammonia was found to be the l imiting 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 ix nitrogen, and yet they exhibited a very low rate of glucose assimilation. The reason for this apparent anomaly may be that the energy requirements for nitrogen f ixat ion are great enough to limit oxidative assimilation severely. With these bacteria, therefore, the degree of assimilation may be a function of their eff iciency of f ix ing nitrogen, and it is true that A. vineland i i which is known to f ix nitrogen very e f f i c ien t l y , and produced large amounts of ammonia during glucose oxidation, assimilated much more radioactivity than did A. ag i l i s . 3. Incorporation of C ^ into the ce l l fractions soluble in  cold tr ichloroacetic acid The compounds extracted from the ce l ls by treatment with cold 70 Table 6a. from 5 H*™ into washed-cel1 suspensions of A. acet i Incorporation of c'^ fr   moles of glucose-U-c'^ Alcohol- Unfrac- Time Cold- soluble Hot Total in tionated (min) soluble* Lipid protein soluble* Residue fractions ce l ls Per cent of total C]l* added to vessel 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 cel 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 tr ichloroacetic acid soluble fractions. 71 Table 6b. Incorporation of c'^ from 5 Mmoles of glucose-U-C^ into washed-cel1 suspensions of A. xylinum Alcohol- Unfracr Time Cold- soluble Hot Total in tionated (min) soluble* Lipid protein soluble* Residue fractions ce l ls Per cent of total C I H added to vessel 15 1.5 0.58 0.20 0.90 5.1 8.28 8.10 30 2 0.87 0.30 1.29 8.3 12.7 12.5 120 2.3 1.7 0.64 1.88 22 28.5 29 Per cent of total C]k incorporated into cel 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 tr ichloroacetic acid soluble fract ions. 72. .Table 7a. Incorporation of c'^ from 5 Mmoles of glucose-U-C into washed-cel1 suspensions of A. vinelandi ? Alcohol- Unfrac- Time (min) Cold- soluble* Lip id soluble protein Hot soluble'* Res idue Total in fract ions t 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 fract ions 15 67 9.5 4.5 3.9 15 100 30 52 15.3 5.7 6.0 21 100 120 26.5 17.7 5.3 14.5 36 100 Cold and hot tr ichloroacetic acid soluble fractions. 73. Table 7b. from 5 Wm< into washed-cell suspensions of A. ag?1 is Incorporation of fro  5 Hmoles of glucose-U-c'^ Alcohol- Unfrac- Time Cold- soluble Hot Total in tionated (min) soluble* Lipid protein soluble* Residue fractions cel ls 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 1^ incorporated into cel l fractions 15 82 5.6 2.7 4.1 5.5 100 30 59.5 8.9 6.6 9.6 15.4 100 120 42 10.7 10.7 12.6 24 100 •A. " Cold and hot tr ichloroacetic acid soluble fractions. 74. 5% tr ichloroacetic acid accounted for essential ly a l l of the label of the cel ls of A. aceti (Table 6a), and a major amount of the radioactivity 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 demon strated in both Azotobacter species. With the exception of A. ag 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 acid. 4. Incorporation of into the ce l l fractions insoluble in  cold tr ichloroacetic acid In each of the bacteria except A. acet i . radioactivity was d i s  tributed among a l l the ce l l fract ions. 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 ce l ls which had been respiring for 120 min, revealed a correlation 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 acidic in nature, but paper chromatography in two solvent systems, as well as electrophoresis, showed no acidic compounds; the radioactive peaks cochromatogrammed with glucose only. The lack of acidic components in the residual fractions would indicate that poly- /3-hydroxybutyrate was not synthesized during the assimilation experiments, although Sobek and Cl i f ton (140) found low levels of this polymer in A. ag 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 residual* fraction Microorgan ism Per cent Cl4 incor porated "Glucose" (per cent dry wt)** Per cent counts total eluted H20 NH^ OH Azotobacter aqi1 is 23 0.4 2.5 97.5 A. vinelandii 3h 1.0 20 80 Acetobacter aceti 0.1 0.3 - - A. xylinum 80 3.8 80 20 * Insoluble in hot 5% trichloroacetic acid. As measured by the anthrone test. therefore, incorporated the major portion of the assimilated material into nitrogenous compounds, including proteins. 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 extend ed 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 ac id -c '4 was found in the metabolic pool, ammonia was probably assimilated via the ° (-ketoglutarate formed from glucose. The small amount of ce l lu lar material insoluble in cold t r i  chloroacetic acid confirmed the conclusion that A. acet? did not incorpor ate measurable amounts of ammonia into the ce l ls (Table 7a). One must 76, conclude, therefore, that A. acet? did not exhibit oxidative assimilation when glucose was the substrate, since the c'^ incorporated was almost entirely in the pool components, in the form of free glucose and gluconate. The ab i l i ty of A. xylinum resting ce l ls to synthesize cel lulose which accumulates extracel lular ly , complicated the study of oxidative as similation in this microorganism. Column chromatography of the hydro- lysed residual fraction revealed that 80% of the radioactivity was assoc iated with neutral compounds, which on paper chromatography and electro phoresis proved to be glucose and cel lobiose. Moreover, a large propor tion of the unhydrolysed material was insoluble in water, acid, or base, being soluble only in Schweitzer's reagent. These observations lead to the conclusion that cel lulose was the major component of the residual f ract ion. This does not mean that the cel lulose was formed intracel lu lar  ly, because extracellular f i b r i l s would also be in this fract ion. The release of radioactive amino acids by the hydrolysis of the residual fraction indicates that protein was synthesized by A. xylinum. There was also radioactivity incorporated into other nitrogen containing fractions, such as the alcohol soluble protein, and nucleic acids. It can be c a l  culated that about 11% of the added C 1^ was assimilated into ce l l material, and of this about half is nitrogenous, the result of the reincorporation of endogenously produced ammonia. C. Oxidative assimilation by starved cel ls of P. aeruginosa ATCC 1. Manometric observations Starvation of resting ce l ls of P. aeruginosa for 3 hr was found to decrease their rate of glucose diss imi lat ion, perhaps because it slowed 77. down the rate of pyruvate oxidation (Figure 9). Oxidation of ©(-keto glutarate was unaffected. This decrease in the rate of pyruvate oxidation occurred with ce l ls which had been starved with or without shaking, but shaking gave a more pronounced effect . 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 ce l ls on harvesting, although conditions were standardized as much as possible. There was no stimulation of the rate of oxidation of pyruvate when thiamine pyrophos phate (0.5 Hmole per Warburg flask) was added. Since glucose oxidation was more affected when cel ls were starved with shaking than without, shaken, starved ce l ls were used in the experiments with glucose-U-C^. | n the ex periment quoted below, the oxygen uptake at 120 min with starved cel ls 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 deter mined, it was found that shaking the ce l ls had greatly Increased endogen ous ammonia production (Table 9, Figure 10). Moreover, although both shaken and non shaken ce l ls reincorporated ammonia during glucose oxidation, the amount and the rate of uptake differed greatly. The curve of ammonia up take for the non shaken cel ls paralleled that for control c e l l s , except that less ammonia was assimilated, not a l l of that available being ut i l ized (Table 9, Figure 10). The ce l ls 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 ce l ls of Pseudomonas aeruginosa during glucose oxidation Time Non shaken cel ls NH3 present per vessel NH3 uptake (calculated) Shaken eel ls NH3 present per vessel NH3 uptake (calculated Endogenous Glucose Endogenous Glucose Min Mmoles umol es umoles umoles Mmoles Starvat ion per iod 0 180 0.010 0.080 0.010 0.550 Ass imi1 at ion period 0 5 15 30 120 0.200 0.225 0.310 0.450 1.100 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 0.200 0.120 0.140 0.155 0.470 0 0.355 0.350 0.345 0.210 Total endog NH3 prod'n 1.180 1.230 00 20 40 60 80 100 120 minutes FIG. 9. Oxygen uptake during oxidation of 5 Hmoles of . pyruvate or glucose by control and starved washed ce l l suspensions of Pseudomonas aeruginosa. . r 80. 20 4.0 60 80 100 120 minutes minutes FIG. 10. Production and uptake of NH3 by washed, starved cel ls 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  f luids 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 luids during glucose oxidation (Figure 11). In the experi ment where shaken, starved ce l ls oxidized glucose-cJ^, k umoles of pyruvate were present at 30 min, as compared to 2.6 umoles when freshly harvested ce l ls were used. In addition, chromatography and electrophoresis of the supernatant f lu ids , followed by scanning, revealed the presence of "cf-keto- glutarate, gluconate, and 2-ketogluconate. The sugar acids were most highly labelled at 5 min, whereas pyruvate and °C-ketoglutarate contained the high est amount of at 30 min. This is a pattern similar to that found with control c e l l s , although in this instance, the pyruvate concentration was highest at 15 min. There was very l i t t l e °C-ketoglutarate present in the Warburg supernatants; the ratio of optical density readings at:435mu and 390mjj was 2.15 in the keto acid assay throughout the experiment which is characterist ic of pyruvate. The rat io for ° f-ketog lu tara te was 1.05. How ever, the specif ic act iv i ty of the °C-ketoglutarate was high, since it could be detected on paper chromatograms by its radioactiv ity. When non shaken ce l ls 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 l Z f in the ce l ls Doudoroff and Stanier (53) reported that when starved ce l ls of P.. saccharoph?la were allowed to oxidize glucose, the oxidative assimila tion was more than doubled (to 50% of the added C 1^), over that of freshly 82. 20 4 0 60 80 100 120 minutes FIG. 11. Disappearance of C l i f and glucose from, and excretion of keto acid into supernatant f luids during oxidation of glucose- U-C14 by washed, starved cel ls of Pseudomonas aeruginosa. 83. harvested ce 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 results. Starved ce l ls 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 ibu  tion of the assimilated in starved cel ls was more similar to that in chloramphenicol treated, freshly harvested cel ls (55), than it was to the control ce l ls (Table 10), in that the cold tr ichloroacetic acid soluble pool components were highly label led, whereas label in the protein was low. However, although the radioactivity in the ce l ls in the presence of the ant ibiot ic remained mainly in the metabolic pool throughout the experiment, in starved ce l ls it was transferred slowly to protein and nucleic acids. 5. Analysis of the cold tr ichloroacetic acid soluble fractions When the cold tr ichloroacetic acid soluble pools from starved ce l ls were investigated by paper chromatography and electrophoresis, and the results compared to those obtained in experiments with freshly harvested and chloramphenicol treated ce l l s , it was found that a l l three types of ce l l s incorporated the c'^ mainly into the free amino acid pools. The amino acids were predominantly those which are neutral at pH 7.6 (alanine, serine, leucine, glycine, e tc . ) , as well as glutamic acid. The glutamate from the control and antibiotic treated ce l ls accounted for most of the radioactivity of the pools at 30 min, decreasing in label thereafter. There was no free glucose present. In the cold tr ichloroacetic acid soluble fractions from starved ce l l s , however, the main radioactive component during the entire experiment was glutamate, although it decreased in radioactivity 84. Table 10. Incorporation of C*4 from 5 Hmoles of glucose-U-C^ into washed, starved ce l l suspensions of Pseudomonas aeruginosa Time (min) Cold TCA** soluble Lipid Alcohol soluble protein Hot TCA** soluble Res idual fract ion Total in fract ions Per cent of total C , / + 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 ce l l fract ions* 5 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 (4) 9 (11) 29 (49) 100 * Figures in parentheses are values for control ce l ls (55)% *" Trichloroacetic acid. 85. 20 % C 1^ incorporated into cells "O •o •o o O 15 10 Control Chloromycetin 20 40 Starved 60 Minutes 80 100 120 F I G . 12. I n c o r p o r a t i o n o f Cl4 dur ing ox idat ion o f glucose-U-C'^ by washed, c o n t r o l , chloramphenicol (Chloromycetin) t r e a t e d , o r starved c e l l s o f Pseudomonas aeruginosa. 86. considerably during this time. There were also small amounts of radio- act ive °C _ketoglutarate, glucose, gluconic acid, 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 in BFW and EPB, and whose Rpicrate ' n 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 ce l ls might be due to a depletion of the cofactors or enzymes required for transamination. To test the cofactor theory, starved ce l ls were allowed to assimilate glucose-U-C^ in the presence of pyridoxal phosphate and pyridoxamine phosphate (8 Mg per f lask of each). A paral lel experiment was run in the absence of the cofactors. However, there Was no increase in the amount of incorporated into the ce l l s , nor any significant change in the pool when glucose was oxidized in the presence of these cofactors. Unlike P_. saccharophi la . which incorporates assimilated carbon into poly- -hydroxybutyrate, P_. aeruginosa does not form any primary product during the oxidative assimilation of glucose (55). This situation was emphasized by the results of these experiments with starved c e l l s . Starvation of P. saccharophila cel ls resulted in a doubling of assimilated material during glucose oxidation (53), but starvation of P. aeruginosa cel ls 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. One might postulate two reasons for the lack of protein synthesis in starved ce l l s . F i rs t , pyruvate was oxidized completely, but more slowly by these ce l l s , than by freshly harvested ones, which meant that not as much 0(-ketoglutarate was available for the incor poration of ammonia in the form of glutamate. In addition, highly labelled glutamic acid accumulated in the metabolic pool, 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 cel ls did not increase assimilation, a lack of transaminases could be responsible. The cel ls apparently have a control mechanism which prevented unlimited assimilation of carbon unless it could be incorporated into cel lu lar material. During glucose oxidation by P. aeruginosa, carbon was not as similated direct ly , but only after conversion to °C-ketoglutarate, which was, in turn, aminated to yield 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 act iv i ty , the incorporation of ammonia and carbon was slowed down, and glucose was oxidized more completely than in freshly harvested ce l l s . 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-ketoglutarate and glutamic acid dehydrogenase, recently there has been interest in a pos-88. s ib le additional route via pyruvate and alanine dehydrogenase (58,63,122,168). Both pyruvate and °C-ketoglutarate are present in the supernatant f luids dur ing the early stages of oxidative assimilation of glucose by P. aeruginosa, and disappear as the oxidation progresses, with concurrent uptake of ammonia. Since the tr icarboxyl ic acid cycle is functional in this microorganism, pyruvate can be converted to of-ketogtutarate (139). It has been shown that an NAOPH dependent glutamic acid dehydrogenase is present in ce l l extracts of J \ aeruginosa, but direct assays for 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 act iv i ty of extracts of this strain of P. aeruginosa is also low (160). Resting ce l l experiments were done with f_. aeruginosa and f_. fluorescens to try to differentiate between ammonia uptake direct ly through pyruvate, and that through the o(~-ketoglutarate derived from pyruvate. The aspartase route was not investigated. Two approaches were taken. F i r s t l y , 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 luores 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 lask, to resting ce 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 cel ls oxidizing °C-ketoglutarate resul t  ed in an apparent increase in oxygen consumption at the time the experiment was terminated (80 min) perhaps because the lag period was shorter (Table 11). However, the f inal oxygen uptake, at the time that the oxidation of the keto acid was complete was not increased. When 5 Hmoles of ammonia Table 11. Oxygen and ammonia uptake during the oxidation of 5 umoles of glucose, pyruvate, or °^-ketoglutarate in the presence and absence of 5 Hmoles of ammonia Oxygen uptake at NH3 uptake at Substrate 80 min 80 min (5 Hmoles NH3 added) Per cent theoretical Hmoles +NH3 Glucose 67 50 2.95 oC-Ketog1utarate 22* 28* 1.80 Pyruvate 80 59 1.00 Oxygen uptake had not ceased. were added, a relat ively 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 sat isf ied by the oxidation of the keto acids. In the experiments where the two keto acids were oxidized simul taneously, the second substrate was not added to the f lask unti 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 cel ls of P. aeruginosa, there is a lag period before oxygen uptake begins; however, pyruvate is oxidized im mediately. It is evident that the addition of pyruvate to cel ls oxidizing °C-ketoglutarate neither stimulated nor inhibited ammonia uptake (Table 12). Table 12. Oxygen and ammonia uptake during the oxidation of pyruvate and °<"-ketogl utarate Init ial Substrate Substrate added at 40 min Oxygen uptake % theoret ical at 100 min NH^  uptake Found Expected 60 min 100 min 100 min ^ K e t o - glutarate - % 28 umoles 1.5 1.8 umoles o("-Keto- glutarate pyruvate 34 3.0 2.9 (1.8+1.1) oC-Keto- glutarate <**-ketoglut. 37 3.0 3.3 (1.5+1.8) Pyruvate - 60 1.1 0.8 Pyruvate °C-ketoglut. 32 3.0 2.3 (1.5+0.8) Pyruvate pyruvate 65 1.9 1.9 (1.1+0.8) In fact , a l l combinations resulted in approximately the amount of am-9 U monfa uptake predicted, except in the situation where oC-ketoglutarate was added to ce l ls already oxidizing pyruvate, in which instance 30% more ammonia was incorporated than calculated. It is possible that pyruvate oxidation, l ike that of glucose, provided cofactors (such as reduced pyr id  ine nucleotides) or energy for the assimilation of ammonia by °C-keto glutarate. 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 radical ly altering in vivo cond it ions. One must conclude, there fore, 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 assimilation. Free pyruvate was not found in the metabolic pool of £. aeruginosa, and therefore, pyruvate did not appear to be assimilated without prior 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. However, the actual 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 comple t ion, how much only to oC-ketoglutarate, and how much of the of-keto glutarate was, in turn, oxidized. 2. Assimilation of ammonia in the presence of inhibitors Arsenite, at the concentration of 10~3M, was found to inhibit , by 80-90%, the uptake of both ammonia and oxygen by resting ce l ls of f_. aerug inosa in the presence of °C-ketoglutarate or pyruvate (Table 13 ) . Endog enous oxidation was also inhibited, although to a lesser extent. Experi ments with ce l l extracts showed that the glutamic acid dehydrogenase of P. aeruginosa, and the alanine dehydrogenase of B_. cereus (122) are unaffected Table 13. Inhibition of oxygen and ammonia uptake by 10"3M arsenite during the oxidation of pyruvate and °C-ketoglutarate by washed cel l suspensions of f_. aeruginosa Initial Substrate Substrate added at hO min Inhibition by arsenite Oxygen uptake NH3 uptake «»C-Ketogl utarate tm % 93 % 85 <K -Ket og1ut a rat e pyruvate 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 ox idation. In this connection, Fairhurst et al_. (58) reported that the 93. formation of alanine by B. cereus resting ce l ls 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 oxida tion which provides both the reduced cofactors, and the energy necessary, ammonia assimilation does not occur. The second inhibitor used was fluoroacetate, at the concentra tions of 10 and 25 Hmoles per f lask, in hopes of blocking glucose oxidar tion at c i t ra te . However, glucose dissimilation was affected very l i t t l e by the inhibitor, and the uptake of ammonia proceeded as usual. It was then learned that neither acetate nor fluoroacetate are converted to c i t ra te by acetate grown ce l ls of P_. aeruginosa, although pyruvate is (139). This observation would explain these findings with glucose grown £. aeruginosa ce l l s . It is interesting, therefore, that ammonia was as similated during acetate oxidation by P. aeruginosa, and that the endog enous oxygen uptake was inhibited 35%. The use of fluoroacetate was abandoned with this strain of P. aeruginosa, and experiments were done with a second s t ra in , P. aeruginosa 120 Na, but this too appeared to be unaffected by the inhibitor, since oxygen uptake with glucose was in  creased 25%, and endogenous oxygen consumption was decreased 36%. B. Experiments with P. fluorescens A 3.12 Resting cel ls of P. fluorescens were found to have an unchanged rate of glucose oxidation in the presence of 10 and 25 umoles of f luoro acetate, but the total oxygen uptake was reduced 25%. There was a s l ight in i t i a l lag in oxygen uptake, and the curve broke at a different point. 94. In addition, the endogenous oxygen consumption was found to be decreased 54%. When 50 umoles of the inhibitor were used per f lask, 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 inal oxygen uptake with pyruvate was decreased from 67% of theoretical to 45%, but there was no such decrease with acetate. Oxidation of the keto acid appeared to have ceased at the end of the experiment. A manometric experiment was then done, in which ammonia assimila tion was followed during pyruvate oxidation in the presence of 50 umoles of fluoroacetate per f lask. The results are shown in Figure 13 and Table 14. In the control experiment, ammonia uptake followed oxygen uptake, and when oxidation ceased, ammonia was evolved. In the presence of f luoro acetate, both ammonia and oxygen consumption remained linear unti l the ex periment was terminated at 80 min; however, levels were much lower than in the control . 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 luores 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 acid. In contrast, ammonia was certainly 95. minutes F I G . 13. Uptake of oxygen and NH3 by washed cel ls 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 cel ls of Pseudomonas fluorescens Time Control Plus Fluoroacetate Inhibit ion NH^  uptake 0 2 uptake NH^  uptake 0 2 uptake 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 control , since oxygen uptake had not stopped at this time. assimilated via °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 e q far in favour of amino acid synthesis. Supporting data for the primary role of ©C-ketoglutarate in ammonia assimilation by P. aeruginosa came from the starved cel l ex periments, where a large amount of labelled glutamate accumulated in the metabolic pool under circumstances where protein synthesis was not occur r ing. However, there was l i t t l e radioactive alanine in the pool. One would expect, since pyruvate was present in large quantities in the medium, that, i f ammonia were incorporated to any extent via alanine dehydrogenase, then alanine would contain the highest amount of C 1 ^ and that glutamic 97. acid would be of lower specif ic act iv i ty , because of the lack of ©("-keto glutarate for its synthesis. In addition, investigation of the metabolic pools of freshly harvested cel ls of JP. aeruginosa after glucose assimila t ion , showed glutamic acid to be the most highly labelled component. Therefore, the data from these experiments were consistent with the oxida tion of pyruvate to cf-ketoglutarate, amination to form glutamate, follow ed by transamination to yield alanine and other amino acids. Although the experiments with P. aeruginosa and P. f1uorescens in the presence of inhibitors gave negative results, they did serve to emphasize the interrelationships between cel 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 cel ls has been indicated in a number of experiments (19,34,66,100,110,175), and the cofactor re 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 wi l l have to take another direct ion. Alternative approaches would be either the study of ce l l extracts, or short term experiments, similar to those performed with algae by Calvin's group, to determine whether alanine is formed direct ly through pyruvate, or only by transamination with glutamate. III. Oxidative Assimilation into the Cytological Fractions of Normal. Chloramphenicol Treated, or Starved Cells of Pseudomonas aerug inosa ATCC 9027 A. Chemical composition of cytological fractions 98. Recent work in this laboratory on cytological fractions of f_. aeruginosa ce l ls has been done mainly with lysozyme-versene disrupted cel ls (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 rag ments (75). Accordingly, an alternate method of breaking cel ls was sought. The French pressure c e l l , used at 15,000 to 17,000 lbs pressure, was found to give an eff ic ient breakage of small volumes of £_. aeruginosa ce l l suspensions. Since it was desired to obtain representative "membrane", ribosomal, and cytoplasmic fractions from these cel ls in as pure a state as possible, several c r i t e r i a were set up to determine the purity of the fractions, as follows (30): "membranes"- contain most of the glucose oxidizing act iv i ty , 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 act iv i ty , most of the RNA, have an RNA:protein ratio approaching unity, and contain l i t t l e DNA; cytoplasm- contains no glucose oxidizing act iv i ty , l i t t l e RNA, and almost a l l of the DNA. 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 re 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 (30) for P. aeruginosa, and also resemble those of JE. col ? fractions (149). If magnesium was omitted from the buffer in which the ce l ls were broken, most of the RNA appeared in the cytoplasm. There is probably contamination of at least the "membrane" fraction with cel l wall material, and some particles of "membrane" appear with the ribosomes. B. Incorporation of glucose-P-C^ into cytological fractions Three experiments were performed: 99. Table 15. Distribution of glucose oxidizing act iv i ty , protein and nucleic acids in various cytological fractions Fract ion Glucose oxid izing act iv ity Protein RNA DNA RNA/ Prot units* %total mg %tota1 mg %total mg %total Cell free extract 675 100 64 100 18 100 3 100 0.30 "Membranes" IX 381 62 9.8 15.7 1.2 6.7 0.03 1 0.12 "Membranes" 2X 80 20 2.7 4.3 0.3 1.7 0 0 0.11 "Membranes" IX wash 33 6 4.4 7.0 1.1 6.2 0.10 3.4 0.25 "Membranes" 2X wash 0 0 1.3 2.1 0.7 3.9 0 0 0.54 40T xg pel let 86 14 3.8 6.1 2.1 1.2 0 0 0.55 Ri bosomes 20 5 12.5 20 11.7 66 0 0 0.94 Cytoplasm 0 0 28 45 0.7 3.9 2.8 96 0.03 Total 600 62.5 17.8 2.93 Recovery (%) 89 97.5 99 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 wi l l be referred to as the "con t r o l " experiment, and the ce l ls used wil l be referred to as "freshly har vested" or "normal". (b) Cells which had oxidized glucose-C^ in the presence of 200 Hg of chloramphenicol per ml were disrupted, the constituents physic a l ly fractionated, and then each of these fractions chemically fractionated. (c) Cells which had been starved for 3 hr under aseptic conditions, and then allowed to oxidize glucose-c'^ were disrupted, the constituents physically fractionated, and each of these fractions chemic a l ly fractionated. When the relative incorporation of radioactivity into these physical fractions was determined, it was found that the "membranes" IX, ribosomes, and cytoplasm accounted for most of the labell ing of the cel l extracts. Table 16 gives the incorporation of C^** into each of the f rac  tions from the three types of experiments, expressed as a percentage of the radioactivity in the ce l l extracts. 1. "Membrane" fractions The percentage of radioactivity which was incorporated into the "membrane" fractions was similar in the three types of ce l l s , and remained quite constant during the experiments, but the actual number of counts in  creased with time in each case. The control "membranes" contained a s l ight ly lower percentage of the C ^ Q f the extracts than did those from chloramphen icol treated or starved ce l l s . On chemical fractionation of the "membranes", the distr ibution 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 cel ls of Pseudomonas aeruginosa (Results expressed as per cent of the c'4 of the ce l l extract) Time "Membranes" Ribosomes Cytoplasm (min) A* B* C* A B C 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 ce l ls (3.53xlo6 C p m added). B- Chloramphenicol treated ce l ls (3.70xlo6 cpm added). C- Starved ce l ls (3.94x106 cpm added). was in the residual f ract ion, whereas that in chloramphenicol treated or starved ce l l "membranes" during the early stages of glucose assimilation was found in the l ip id (Figure 14). However, as the oxidation of glucose progressed, there appeared to be a marked transfer of radioactivity from the l ip id to the protein residue in the starved cel l "membranes", and a s l ight shi f t from the l ip id label to the residue label in the antibiotic treated ce l l s . The l ip id from the control cel l "membranes" contained l i t t l e radioactiv ity, although l ip id was found experimentally to make up 21% of the dry weight of this f ract ion. There seemed to be less inhibition by chloramphenicol of C14 in- corporation into the residual fractions, presumably protein, of the "mem branes" than into the cytoplasm. At 120 min, based on the percentage of C 102. Table 17. Incorporation of C l Z f into chemical fractions of "membranes" during oxidation of glucose-U-C^ by control, chloramphenicol treated, or starved resting ce l ls of Pseudomonas aeruginosa Alcohol- Time soluble Hot (min) protein Lipid soluble* Residue Control Per cent of Cl4 in cel l extract 5 1.4 1.5 1.2 9.8 30 1.9 1.0 2.2 9.0 120 1.3 0.5 1.5 9.9 Per cent of C 1 i f in "membranes" 5 11.3 10.6 8.4 70 30 13.6 7.1 15.4 64 120 10.0 4.1 10.7 76 Chloramphen icol treated Per cent of C 1 / f in ce l l extract 5 1.3 6.4 1.7 6.3 30 1.1 7.7 2.2 5.0 120 0.9 6.3 2.3 6.5 Per cent of Cl^ in "membranes" 5 8.5 41 11.0 41 :3o 7.0 48 13.5 31 120 5.4 39 14.5 41 Starved Per cent of C'4 in eel 1 ext ract 5 0.2 12.5 1.0 3.4 30 0.2 11.5 1.6 4.7 120 0.2 8.6 1.6 7.8 Per cent of C'4 in "membranes" 5 1.0 73 6.0 20 30 1.0 64 9.0 26 120 1.0 48 9.0 43 * Hot tr ichloroacetic acid soluble f ract ion. 103. o I 0) c o £ £ o C h e m i c a l f r a c t i o n a t i o n of membranes Residue _ > ® 20 40 . 60 80 Minutes 100 120 FIG. 14. Incorporation of into the protein residue and l ip id of the "membrane" fractions during oxidation of glucose-U-Cl4 by wash ed cel ls of Pseudomonas aeruginosa. Control ce l ls 8 — — © , chlor amphenicol treated cel IsB — — — El , starved ce l ls A •—•— »A. 104. in each f ract ion, the inhibition was 48% and 86%, for "membranes" and cytoplasm, respectively. Accordingly, the 30 and 120 min "membrane1 residue samples were hydrolysed, and analysed by column chromatography. 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 ce l l s , but only about 10-15% in the other two. Table 18. Column chromatography of the residual fractions from "membranes" of control , chloramphenicol treated, or starved ce l ls Fract ion Dowex-50 H20 (neutral and ac idic) Dowex-1 , H 2 ° , (neutral) Dowex-1 HCl (ac id ic) Dowex-50 NH^ OH (amino acids) cpm* % cpm % cpm % cpm % Control-30 0.99 16 0.48 8 0.51 8 4.68 84 Chioram.-30 1.29 55 0.79 33 0.51 22 1.12 45 Starved-30 0.88 25 0.46 13 0.42 12 2.64 75 Control-120 1.46 11 - - - - 11.30 89 Chloram.-120 1.08 31 - - - - 2.46 69 Starved-120 1.35 15 0.63 7 0.72 8 7.58 85 cpm x 10"3 The non amino acid radioactivity was found, on further fract iona tion by column chromatography on Dowex-1 resin, to consist of about equal quantities of neutral and acidic compounds, but there was so l i t t l e radio act iv i ty in these materials that their identities could not be established. 105. The neutral compounds may represent cel l wall carbohydrates, or sugars from the "membranes" themselves. Studies have shown that, in some bacteria, hexose (glucose or mannose) makes up to 20% of the dry weight of the mem branes, probably in the form of a glycol ipid (70). The acidic compound found in these "membranes" could come from this glycol ipid also, or from the rhamnolipid which has been found to be formed by P_. aeruginosa cel ls (80,81,93). Chloramphenicol treatment of the ce l ls caused l i t t l e or no inhibition of labell ing of these non amino acid components, but did result in a drastic reduction (75%) of incorporation of c'** into the "membrane" protein, after correction for non proteinaceous radioact iv i ty. In freshly harvested ce l l "membrane" fract ions, the act iv i ty of the protein was high, while in the starved ce l l "membranes" during the early stages of assimila t ion, protein was of low specif ic act iv i ty , although this increased rapid 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 posit ive compounds. No diaminopimelic acid could be demonstrated. Table 19 gives the specif ic act iv i ty of the nucleic acids at 5 min and 120 min in each of the "membrane", ribosomal, and cytoplasmic fract ions. Although the RNA of the "membranes" made up only 6-7% of the total RNA of the cel l extract, it was of high specif ic act iv i ty 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 radioactivity into the ribosomes, which con tained 20% of the protein, and 66% of the total RNA of the ce l l extracts, was low throughout each experiment, indicating a slow turnover of the 106. Table 19. Specific act iv i ty of nucleic acid from cytological fractions of control , chloramphenicol treated, or starved cel ls of Pseudomonas aeruginosa Fract ion mg RNA i h fraction cpm in RNA Specific act iv i ty cpm/mg RNA 5 min 120 min 5 min 120 min 5 min 120 min "Membranes" Control 0.058 0.072 760 3,600 13,100 50,000 Chloramphenicol 0.060 0.076 1,120 3,060 18,700 40,300 Starved 0.062 0.062 710 3,080 11,400 49,900 R i bosomes 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 Cytoplasm 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 protein and nucleic acid (Table 16). In the control ce l l s , the percentage of added c'^ incorporated into the ribosomes decreased with time, although the actual amount of radioactivity remained fa i r l y constant (Figure 15). This decrease in apparent label l ing was shown, by a chemical fractionation, C 1 4 incorporation into ribosomes 6^ 5. 4 . S 3- 2. l_ \ Starved Chloromycetin Control 2 0 4 0 6 0 80 Minutes 100 120 FIG. 15. Incorporation of Cl4 into the ribosomal fractions during oxidation of glucose-U-C.14 by washed, control , chloramphenicol (Chloromycetin) treated, or starved ce l ls of Pseudomonas aerugin osa. 108. to be the result of a low Incorporation of c'^ into the nucleic acid of the hot tr ichloroacetic acid extract, while the amount of c'^ in the prot ein residue increased during the experiment, and made up most of the radio act iv i ty of the fraction (Figure 16, and Table 20). On the other hand, with chloramphenicol treated or starved ce l l s , there was an increase both in absolute and in relat ive incorporation of label into the ribosomes over that of the control . The ribosomal RNA of the former ce l ls was much more highly labelled than that of the control ce l l s , whereas protein contained l i t t l e C ^ . A calculation of the specif ic act iv i ty of the ribosomal RNA showed that it was low in a l l three cases (Table 19). However, in ant i  biot ic treated ce l ls early in the experiment, and in starved cel l ribosomes in the later stages, the specif ic act iv i t ies were much higher than that of the control . 3. Cytoplasmic fractions In the ce l ls 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 relat ive amount of radioactivity incorporated into this fraction did not change s ignif icant ly during the course of each ex periment, but as with the "membranes" and ribosomes, the absolute number of counts increased. Changing the conditions of the experiments proved to cause only a sl ight variation in the relative amount of cytoplasmic cl4 (Table 16). The signif icant change occurred in the distr ibution of radio act iv i ty 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 tr ichloroacetic acid pool components, or the l ip id and alcohol soluble protein. This ce l lu lar compon-1 109. Chemical fractionation of ribosomes lOO Protein • I I I 20 40 60 80 100 120 Minutes FIG. 16. Incorporation of C'4 into the RNA and protein resi- . due of the ribosomes during oxidation of glucose-U-C'4 by washed ce l ls of Pseudomonas aeruginosa. Control ce l ls 0 — — 0 , chloramphenicol treated ce l ls • • , starved ce l ls & • 110 Table 20. Incorporation of into the protein residue and the RNA of the ribosomal fractions during oxidation of glucose-U-C^ by control, chloramphenicol treated, or starved resting ce l ls of Pseudomonas aeruginosa Time (min) Hot soluble* Residue Hot soluble* Residue Control Per cent of C ^ in cel l extract Per cent of c'^ in ribosomes 5 1.0 2.4 - 30 70 30 0.41 2.2 . 16 84 120 0.29 1.9 13 87 Chloramphenicol treated Per cent of O1* in ce l l extract Per cent of c'^ j n ribosomes 5 2.5 3.3 43 57 30 2.4 2.0 54 46 120 3.2 1.7 65 35 Starved Per cent of C ^ in ce l l extract Per cent of c'^ i n ribosomes 5 1.6 1.5 52 48 30 2.0 1.7 55 45 120 3.2 2.0 61 39 " Hot tr ichloroacetic acid soluble fract ion. I l l o I £ a) CL O >» U Chemical fractionation of cytoplasm 100 80. 1 6Qj 20. &— A A  Pool Protein A » 7 # ^ I __Pool Protein •vJ.lTrj 3 1 — » » • „ U Protein y 2 0 4 0 .60 80 Minutes 100 120 FIG. 17. Incorporation of C*4 into the cold tr ichloroacetic , . acid soluble pools and residual fractions of the cyto plasm during oxidation of glucose-U-Cl^ by washed cel ls of Pseudomonas aeruginosa. Control ce l ls 6 chlor amphenicol treated cel ls B <- S , starved cel ls A • — • — • A . Table 21 Incorporation of c'4 into chemical fractions of the cytoplasm dur g1ucose-U-cl4 oxidation by control , chloramphenicol treated, or starved resting cel ls of Pseudomonas aeruginosa Alcohol Time Cold soluble Hot (min) soluble* protein Lipid soluble* Res idue Control Per cent of c l^ in eel 1 extract 5 38 2.6 1.3 0.8 22 30 28 2.9 0.6 1.3 35 120 17 2.9 0.4 1.3 51 Per cent of c l^ in cytoplasm 5 49 4.1 2.1 1.1 29 30 36 3.8 0.7 1.6 44 120 22 2.8 0.5 1.6 65 Chloramphenicol treated Per cent of Cl4 in ce l l extract 5 66 0.9 1.4 1.6 2.6 30 61 1.5 1.2 1.7 8.0 120 60 2.0 1.1 2.2 9.3 Per cent of c l^ in cytoplasm 5 92 1.2 2.0 2.2 3.6 30 85 2.0 1.6 2.2 10.4 120 81 2.6 1.4 3.0 12.4 Starved Per cent of C'4 in ce l l extract 5 64.5 0.20 0.3 3.0 30 68.0 0. 15 0.5 4.5 120 51.5 0.20 1.6 12.9 Per cent of c'4 in cytoplasm 5 95 0.30 0.4 4.4 30 93 0.20 0.65 6.2 120 78 0. 35 2.4 19.5 * Cold and hot tr ichloroacetic acid soluble fract ions. 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 prior starva tion of the ce l l s . However, in starved ce l l s , the rate of labell ing of the protein continued at a rapid rate until the end of the experiment. In both the chloramphenicol treated and starved ce l l s , the t r i  chloroacetic acid soluble pool made up a very much larger proportion of the radioactivity of the cytoplasm than it did in the control ce l ls (Fig ure 17 and Table 21). The pool from starved ce l ls at 5 min contained 95% of the cytoplasmic label , and this decreased only 15% during the course of the experiment, as the label in the protein residue rose. The incorpora tion c'4 into l ip id and alcohol soluble proteins was extremely low. With chloramphenicol, 92% of the radioactivity was in the metabolic pool at 5 min, decreasing to 81% at 2 hr. The residual protein fraction increased s l ight ly in radioactivity during this period, although there was 86% less c'4 incorporated than in the control . Ether extraction of the cold t r i  chloroacetic acid soluble pools, followed by chromatography and electro phoresis of the aqueous solutions and scanning to detect the radioactive components, showed that, as with the metabolic pools from whole ce l l s , the c'4 was mainly in the form of amino acids in a l l three experiments. The distr ibution of radioactivity was similar to that found in pools of the different types of whole ce l l s , in that glutamate was highly label led, and accumulated in the starved cel l fract ions. Although the actual amount of c'4 incorporated into the hot t r i  chloroacetic acid soluble fraction of the cytoplasm was low, a calculation of the specif ic act iv i ty of the nucleic acid in this fraction showed that 114 It was highly labelled (Table 19). The nucleic acid extracted with hot tr ichloroacetic acid proved to be almost a l l RNA, presumably soluble RNA, which has a higher rate of turnover than does ribosomal RNA. The cyto plasmic DNA, which was degraded by the DNase treatment of the extracts to reduce their v iscosi ty , was a part of the cold tr ichloroacetic acid pool. The in i t i a l specif ic act iv i ty of the RNA formed in the presence of chlor amphenicol was higher than that in the other two experiments. C. Experiments with the cytoplasmic proteins 1. Amount of radioactivity contained in the "pH 5 enzyme" To obtain some estimate of the incorporated into the synth et ic enzymes of the cytoplasm, the following experiment was performed. Each of the cytoplasmic fractions from the three types of ce l ls was adjust ed to pH 5.4 with acetic acid, allowed to stand in the cold 20 min, and centrifuged. Counts were made on aliquots of each fraction before this treatment, and on the supernatant solution after ac idi f icat ion and centri- fugation. Since the enzymes responsible for the activation of amino acids, and their transfer to soluble RNA and ribosomes, are precipitated by ac idi f icat ion 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 prior starvation of f_. aeruginosa c e l l s , there was a definite 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 c e l l s . This would help to explain why, in the starved ce l l s , protein syn thesis was greatly reduced. Chloramphenicol caused a complete inhibition 115. Table 22. Percentage of Cl4 removed by acidi f icat ion 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 radioactivity into these fract ions, except at 2 hr, when a sl ight labell ing was found, and shows that protein synthesis was occurring. 2. Effect of starvation on the act iv i ty of the aminoacyl-s-RNA  synthetases Additional information on the state of the amino acid activating enzymes during starvation of P. aeruginosa ce l l s , and during glucose assim i lat ion by these ce l l s , was gained by assaying the formation of aminoacyl- s-RNA by these synthetases. Preliminary experiments indicated that only the cytoplasmic enzymes were capable of activating the amino acids. An experi ment was therefore carried out in which the cel ls were starved, allowed to oxidize glucose, then disrupted and physically fractionated. Samples were taken at intervals during the starvation and assimilation periods. The amount of enzyme which was limiting for the formation of aminoacyl-s-RNA in the zero time sample was determined, and then the relative act iv i t ies of the enzymes of the other time intervals were assayed in the same manner. 116. Protein determinations were done on each fract ion, and the specif ic act iv i ty (cpm per ug protein) was calculated. 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 ce l ls of Pseudomonas aeruginosa Fract ion Protein incorporated Specific Activity Mg/0.003 ml 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 Period of ass imi1 at ion 15 min 6.5 3320 510 30 min 8.0 4130 515 120 min 8.4 4480 535 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. This would explain the low incorporation of radioactivity from glucose into protein by starved ce l ls during the early stages of assimilation. As glucose oxida 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 account ed for only a small proportion of the cytoplasm of the starved ce l l s , at least during the early stages of the experiment, compared to that for the control ce l ls (Table 22). One would think from the reactivation curve in Figure 18, that the proteins of the pH 5 enzyme would be preferential ly synthesized during assimilation, but this did not appear to be the case. The cytological distr ibution of the carbon assimilated from glucose was perhaps not surprising, since it was known from a previous chemical fractionation of whole cel ls (55) that the incorporation of label occurred mainly into proteins, and from preliminary experiments that cyto plasmic proteins constituted by far the largest chemical fraction of the c e l l . Therefore, the finding that 60-65% of the of the cel l was in the soluble proteins of the cytoplasm in the control experiment was con sistent with these observations (Table 21). However, the high specif ic act iv i ty of the cytoplasmic proteins, which are mainly enzymes, was some what unexpected, since the cel ls 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, a l  though the synthesis of soluble protein was inhibited 86%, glucose oxida tion proceeded at a rapid rate. Since nearly 20% of the total radioactivity of the control cytoplasm was found in the pH 5 enzyme fract ion, some of the newly synthesized soluble protein was concerned in protein synthesis. The lower incorporation of into this fraction in starved ce l ls corresponded to the decrease of protein synthesis in these c e l l s . During endogenous respiration, the act iv i ty 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 their endogenous breakdown (75). An effect of chloramphenicol on the metabolic pool components similar to that found in these experiments with P_. aeruginosa, has been reported in Vibrio cholera (78). In this case also, the amount of amino acids in the pool was higher, but its qualitative 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 coefficient 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 cel l extracts, the chloramphenicol RNA appeared in the particulate fraction with the ribosomes. There are two main schools of thought as to just what this high molecular weight chloramphen icol RNA represents. Gros et a l . (76) showed that in !E. col i. chloramphen icol RNA possessed properties similar to those of a rapidly labelled RNA, termed "messenger RNA", and that in common with messenger RNA, it st imul ated amino acid incorporation into protein. There is normally a rapid break down of messenger RNA, but Gros and his group believe that this does not occur unless protein elongation is completed, and that chloramphenicol stabi l izes this RNA by stopping protein synthesis. The antibiotic 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 fraction of RNA enters the ribosomes intact, as does chloramphenicol RNA, providing that the organisms, after removal from the ant ib iot ic , are placed in a medium which permits protein synthesis. The high molecular weight RNA, then, is unstable until it is combined with protein. Thus, there are two proposed templates for protein synthesis: the ribosomal RNA, or a transient, DNA-like messenger RNA. Despite the prol i ferat ion 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 ce l ls of Shigella flexner? in the presence of chloramphenicol is a functional s-RNA, with the expected base rat ios. The s-RNA of this organism was found to be increased by 160% after h hr of incubation in a complete medium with the ant ib iot ic . Extracts of S_. flexneri were prepared by grinding, however, which Aronson and Spiegelman (10) found resulted in the solubi l iz ing 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 ce l ls in the presence of chloramphenicol gave r ise to an increase in the radioactivity 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 fraction from high speed centrifugation, and result in a high in i t i a l incor-121. poration of C l i f into the ribosomal RNA. Normal ribosomal RNA has a slow turnover (47), as shown by the very low specif ic act iv i ty of this fraction in the control c e l l s , whereas chloramphenicol RNA is constantly turning over (88). Cytoplasmic RNA, although only a minor part of the total RNA, was also of high specif ic act iv i ty in the chloramphenicol treated c e l l s , during the f i r s t stages of glucose oxidation. 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 ce 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 endogen ous respiration, than as a manifestation of the decrease in protein syn thesis (29,75). If the latter situation were the case, one would expect that the in i t i a l label, l ike that in the antibiotic treated ce l l s , should be high. Cl i f ton (36), and Duncan and Campbell (55) have suggested that assimilation may serve to replenish the endogenous reserves of micro organisms, 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 lipoprotein, which contains most of the ce l l l i p i d , was the in i t i a l s i te of incorporation of lys?ne-C l Z f in B_. megaterium. 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 alcohol, the amino acid was found to be associated with the l ip id portion. In this connection, Silberman and Gaby (137) have shown that the l ip id of P. aeruginosa con tains bound amino acids, but the effect of chloramphenicol on the uptake 122. of amino acids by this microorganism was not investigated. In contrast to the results of Hunter et_ aj_. (28,89,90), in which chloramphenicol was shown to inhibit the incorporation of c'^ amino acids into B_. megater ium membrane l ipoprotein, there was a marked increase in the incorporation of from glucose into the alcohol-ether soluble fraction of the ''membranes" of P. aeruginosa in the presence of the antibiotic (Table 17). Duncan and Campbell (55) have also reported an increased labell ing of the total l ip id of the ce l l when protein synthesis was inhibited in this microorganism. This increase may be the result of more substrate being available for l ip id synthesis when protein synthesis is not occurring, or it may be due to the formation, by a chloramphenicol insensitive route, of lipo-amino acid com plexes in P. aerug inosa. but not in B_. megater ium. These lipid-amino acid complexes would then accumulate during the inhibition of protein synthesis. Since glucose C ^ was used in the experiments with P. aeruginosa, it is not known whether the radioactivity was in the l ip id per se. or whether the c'^ was in the form of amino acids attached to the l i p i d . Chloramphen icol 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 prior to the production of aminoacyl s-RNA, one could visual ize how the antibiotic would have no effect on their 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 in i t i a l increase in label of the l ip id component of the "membranes" was found in starved cel ls (Table 17). As the experi ment progressed, the rate of C 1 ^ incorporation into l ip id decreased, as protein synthesis increased (Figure 14). This phenomenon may be explained 123. by either of the two explanations previously considered. Unlike chlor amphenicol treated c e l l s , however, the relative label in the total l ipids of the starved cel ls was not higher than that of the control (see Section I, C 4). Many reports have appeared concerning the presence of small amounts of RNA in the membrane fraction of microorganisms (1,24,30,43,89, 142), and the high specif ic act iv i ty of this RNA after exposure of E. col? ce l ls to P3 2 or has also been demonstrated (43,142,158). It has been suggested that this RNA is not merely an art i fact arising as a result of the fractionation procedure, but that it may be an integral part of r ibo 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" fraction could be separated into two components by centrifugation at 25,000xg, which yielded a pellet ("membranes") and the supernatant from which ribosomes could be sedimented at 100,000xg. McQuillen et a]_. (112) have shown that some ribosomes of JE. co 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 ce l l wall synthesis, but instead later obtained evidence that it might consist of messenger RNA, since, after infection of the organism with T 2 bacteriophage, "membrane" RNA had base ratios similar 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 specif ic act iv i ty , and therefore appears to be different than the ribosomal RNA, whose label : is low (Tables 17, 19 and 20). The high incorporation of C 1 ^ into the "membrane" RNA of 124. control cel ls could be explained by its possible function as a messenger suggested by Suit (150), which would result in a rapid turnover, and an increased label . The same interpretation of results could be used for the starved ce l l s , with the additional argument that this RNA may also be ut i l ized as an endogenous reserve, and thus be resynthesized during glucose oxidation. The in i t i a l high speci f ic act iv i ty of the membrane RNA in an t i  biot ic treated ce l ls would be a manifestation of the formation of chlor  amphenicol RNA, some of which would be attached to the "membrane" ribosomes. IV. Species Specif ic i ty 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 their aminoacyl-s-RNA synthet ase act iv i ty 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 replace the s-RNA. Chloramphenicol (200 Mg per ml) produced no inhibition 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 ce 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 raising 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 ce l l fractions Fraction s-RNA Incorporation of C'4 into s-RNA cpm Cytoplasm ( 0.025 ml ) - 700 Cytoplasm ( 0.050 ml ) - 910 Cytoplasm ( 0.025 ml ) + 24,210 Cytoplasm ( 0.050 ml ) + 21,220 "Membranes" ( 0.050 ml ) - 770 "Membranes" ( 0.050 ml ) + 1,150 Cytoplasm ( "Membranes" ( P.025 0.025 ml ) ml ) - 800 Cytop 1 asm ( "Membranes" ( 0.025 0.025 ml ) ml ) + 26,130 Cytoplasm ( R i bosomes ( 0.050 0.050 ml ) ml ) - 900 Cytoplasm ( Ribosomes ( 0.050 0.025 ml ) ml ) + 21,440 Cytoplasm ( Chloramphen ico 0.050 ( 80 ml ) H9 ) + 19,740 126. would cause a di lut ion of the c'^ amino acids added. Hunter et, aK (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 fract ion, whereas, i f the ionic strength were low, these enzymes appeared in the cytoplasm. In an effort to determine whether the use of a high ionic strength suspending f lu id 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 cu l t to obtain an extract of low viscosity by the use of DNase treatment. However, satisfactory extracts were f ina l l y prepared, and sep aration of the fractions by centrifugation proceeded normally. These f rac  tions were tested for their ab i l i ty 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 was cor respondingly lower (Table 25). It 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 l iver that heated cyto plasm could be used as a source of s-RNA. Since this would simplify the experiments considerably, this 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 Cytoplasm "Membranes" 1 + 9060 fraction used in the experiments reported in Table 24. The control ex periment in this case contained 0.5 mg s-RNA (Table 26). The data in Table 26 show that the heated cytoplasm did not cause a stimulation of counts incorporated into the alcohol precipitate. 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 precipitate, as indic ated by the controls in which no enzyme was added. The heated cytoplasmic fraction 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 similar situation with yeast s-RNA and E. co 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 s-RNA Heated pH 5 fraction of cytoplasm Incorporat ion of Cl4 ml cpm Cytoplasm - - 400 Cytoplasm 0.25 7250 Cytop 1 asm + - 5270 Cytoplasm - 0,10 1460 None - 0.10 1380 Cytop1 asm - 0.25 2660 None - 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. co l i B, and bakers' yeast were selected for study. It was thought that, i f any species spec i f ic i ty 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 possible, since sometimes an enzyme wi 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 fract ion. 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 homol ogous enzyme system, and Table 29 gives the per cent of radioactivity 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, since, with the exception of Achromo- bacter B81, there was an almost constant amount of c'^ incorporated into each s-RNA (40,000 cpm). 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 eff iciency than the homologous s-RNA. None of the bacterial s-RNA's reacted well with the yeast enzyme, and only the enzyme from E. col i in  corporated much into the yeast s-RNA. The E. col? synthetases incor porated 68% of the counts of the homologous bacter ia l , or 74% of the homologous yeast, system into the yeast s-RNA. A higher amount of incorporation of amino acids into a heterol  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 their own. Doctor and Mudd (51), studying interspecif ic reactions between yeast, E. c o l i . and rat l iver systems, reported a similar phenomenon, es pecial ly with the rat l iver enzymes. They suggested several reasons for these anomalous f indings, but none was established as being responsible Table 27 Incorporation of C H amino acids into s-RNA's of various microorganisms by homologous and heterologous enzymes Enzyme P. aeruq. 9027 P. aeruq. 120 Na P. f l uo r .A 3.12 Achr.'B8l E. col i Yeast cpm cpm cpm cpm cpm 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 luor . A 3.12 51,230 35,980 38,250 48,250 55,130 17,030 Achr. B81 17,370 13,830 13,750 20,980 24,330 6,320 E. co l i 32,930 28,180 26,030 33,400 39,930 10,850 Yeast 15,130 8,000 13,380 13,700 26,980 36,150 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^^ 5-RNA P. aerug. 9027 P. aeruq. 120 Na P. f luor . A 3.12 Achr. B81 . E. co l i Yeast P. aeruq. 9027 100 60 82 110 110 24 P. aeruq. 120 Na 132 100 96 179 144 38 P. f luor . A 3.12 125 83 100 230 138 47 Achr. B81 42 31 37 100 61 18 E. col i 79 65 70 160 100 30 Yeast 37 19 36 66 68 100 Table 29 Percentage of C 1 4 amino acids incorporated into s-RNA's based on the amount found in the system homologous for the s-RNA Enzymex^ ^x"s-RNA P. aerug. 9027 P. aeruq. 120 Na P. f luor . A 3.12 Achr. B81 E. col i Yeast P. aeruq. 9027 100 63 75 54 106 21 P. aeruq. 120 Na 125 100 87 87 132 33 P. f luor . A 3.12 134 94 100 125 144 44 Achr. B81 83 64 66 100 116 30 E. co l i 83 71 65 81 100 27 Yeast 43 22 37 38 74 100 133. for their observations. In the case of Achromobacter B81 in these experi ments, it 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 heterol  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 different amounts of s-RNA's in different species, or, where multicomponent s-RNA's exist , 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 acid, or group of amino acids, incorporated into the s-RNA's of the six microorganisms by the homologous enzyme systems. The incorporation of some of the amino acids was found to be very low, especially that of prol ine, 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 Chlorella hydrolysate (Merck, Sharpe and Dohme Ltd.) is given in Tab1e 31. The pattern of incorporation of the amino acids by the bacteria does ref lect , to some degree, their ava i lab i l i t y , and also the composition of the c e l l s . The leucine group was incorporated to the largest extent in a l l of the homologous reactions, accounting for from 31% (E. col i ) to 52% (Achromobacter) of the total radioactivity in the s-RNA's. 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.—• •- p. aeruq. P.. aerug. P. f luor . Achromo. E. col ? Yeast Amino acid 9027 120 Na A 3.12 B81 B Basic group* % 19*4 % 10.6 % 18.5 % 14.0 % 22.3 % 25.2 Asp. group* 9.5 9.9 9.0 18.4 11.9 6.9 . 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 Val . 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 - lys, h is , arg. Aspartic acid group - asp, gly, ser. Glutamic acid group - glu, thr . Valine group - va l , met. Leucine group - leu, i leu, phe. Table 31. A comparison of the amino acid composition Of Chlorel la hydrolysate* and of that of several bacteria (144,146) Amino acid Bacteria Chlorel la hydrolysate Lys ine 6.4 Hist idine 2.0 Arginine 5.9 Aspartic acid 10.2 Ser ine 4.6 Glyc ine 9.2 Glutamic acid 11.2 Threon ine 5.5 Alanine 10.4 Proline 4.2 Tyros ine 2.7 Val ine 7.2 Meth ion ine 3.0 Leucine 8.7 Isoleuc ine 5.4 Phenylalan ine 3.5 % amino acid 13.7 24.0 16.7 10.2 17.6 % amino ac id % C 14 7.4) 2.5 8.9 ) 18.8 18.5 8.8 ) 2.9 \ 4.4 1 16.1 12.9 10.4 I 2.9 J 13.3 13.0 7.4 5.8 7.4 5.8 5.9 6.1 5.9 * 14.2 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 valine is of higher specif ic act iv i ty than the other amino acids. 136. of most of the bacteria. This was somewhat surprising, for the amounts of these amino acids in the bacteria as shown by analysis (Table 31) (144,146), and their specif ic act iv i t ies in the Chlorel la 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 tyros ine was also higher than one would have expected from the data in Table 31, whereas that of alanine was lower. The only enzyme systems which catalysed the formation of prolyl-s-RNA were those of E. col?, £. fluorescens. and yeast. The lack of incorporation of glutamate, glycine, and proline has been reported previously. Glutamyl-s-RNA synthetase was found by Alford et a h (4) to be easily inactivated by mild procedures during its isolat ion, and Zubay (183) was unable to detect any incorporation of glutam ate 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 this 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 iver can be preserved in an active form only at the temperature of l iquid 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, isoleucine, phenylalanine, val ine, methionine, and tyrosine) is common, and could be the result either of the presence of multicomponent s-RNA's for these amino acids, or the inadvert ent selection of conditions favouring their reaction. It is known that in £. c o l i . there is more than one s-RNA for leucine, isoleucine, valine and methionine, but this is also true for serine, 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 radioactivity 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 proline (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 combina tions (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 act iv i t ies which were tested for , although in some cases, the incorporation was very low. Even with the two Pseudomonas strains, 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. There were,'nevertheless, some interesting findings especially in the cases where the heterologous reac tion was greater than the homologous one. In. some of these instances, the increase in incorporation was due to a corresponding increase in uptake Table 32a Incorporation of C 1 4 amino acids by P_. aeruginosa ATCC 9027 enzyme into heterologous s-RNA's, and by heterologous enzymes into P. aeruginosa ATCC 9027 s-RNA Micro organism P. aeruginosa ATCC 9027 P. aeruginosa 120 Na P. fluorescens A 3.12 Achromobacter B81 E. co l i B Yeast Amino acid: s-RNA s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. cpm x 10~3 cpm x 10-3 cpm x 10-3 cpm x 10-3 cpm x 10-3 cpm x 10-3 Bas ic* 8.0 4.0 3.3 4.3 6.9 1.7 5.3 3.6 6,0 0.8 2.0 Asp.* 3.9 3.8 3.4 6.5 1.9 3.0 2.4 4.9 4.7 1.7 1.3 Glu.* 3.8 2.0 1.1 2.3 1.2 1.7 1.3 1.6 2.5 0.9 0.4 Ala. 2.4 1.2 0 1.4 0 0 1.9 0.3 0 0 0 Pro. 0.5 0.5 0 1.1 0 0 0 1.0 3.8 0 0.5 Tyr. 2.3 5.5 3.2 3.5 3.9 1.5 1.0 2.9 3.1 0 0.9 Va l . * 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 Tota l : k].0 54.5 26.0 51.2 30.7 17.4 23.2 32.9 43.8 15.1 8.7 * Basic group - his, arg, lys. *Valine group - va l , met. Aspartic group - asp, gly, ser. Glutamic group - glu, thr. Leucine group - leu, i leu, phi . 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 Micro- organ ism: P. aeruginosa ATCC 9027 P. aeruginosa 120 Na P. fluorescens A 3.12 Achromobacter B81 E. co l i B Yeast Amino acid: 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 12.9 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 53.5 35 35.8 35 41.3 37.9 26.4 27.2 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 Micro- organ ism: P. aeruginosa 120 Na P. aeruginosa ATCC 9027 P. fluorescens A 3.12 Achromobacter B81 E. co l i B Yeast Amino acid: s-RNA s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. cpm x 10~3 cpm x 10-3 cpm x 10-3 cpm x 10-3 cpm x 10-3 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 Tota l : 43.2 26.1 54.5 36.0 36.7 13.8 37.5 28.2 57.5 8.0 13.6 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 Micro- 1 organism: P. aeruginosa 120 Na P. aeruginosa ATCC 9027 P. fluorescens A 3.12 Achromobacter B81 E. col i B Yeast Amino acid: s-RNA s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. Bas ic 10.4 12.8 7.4 8.5 11.5 8.4 25.7 16.4 14.7 5.5 21.4 Asp. 9.9 12.9 7.0 16 6.4 16.6 7.5 13.6 11.1 20.4 12.7 Glu. 2.0 4.3 3.6 0 1.6 5.5 3.4 0 2.6 0 6.1 Ala. 0 0 2.2 0 1.9 4.5 6.2 0 0 0 1.6 Pro. 0 0 1.0 0 0 0 4.7 4.1 2.6 0 4.7 Tyr. 14.4 12.2 10.1 13 10.7 18.3 9.1 11.3 10.8 0 6.0 Val . 21.1 19 17.9 14.9 21.1 18.7 13.3 20.4 13.9 74 9.9 Leu. 42.1 38.8 48.7 47.6 46.7 27.5 29.9 34.2 44.5 0 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 Micro organism: P. fluorescens A 3.12 P. aeruginosa ATCC 9027 P. aeruqinosa 120 Na Achromobacter B81 E. col i B. Yeast Amino acid: s-RNA s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. cpm x 10~3 cpm x 10-3 cpm x 10-3 cpm x 10-3 cpm x 10-3 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 Tota l : 38.2 30.7 51.2 36.7 36.0 13.7 48.2 26.0 55.1 13.4 17.0 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 Micro- 1 organ ism: P. fluorescens A 3.12 P. aeruginosa ATCC 9027 P. aeruqinosa 120 Na Achromobacter B81 E. col i B Yeast Amino acid: s-RNA s-RNA Enz. s.RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. Bas ic 18.5 22.5 8.5 11.5 8.5 9.7 16.4 15.3 13.6 6.6 20.3 Asp. 9.0 6.3 12.6 6.4 16 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 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 S - R N A Micro organism: Achromobacter B81 P. aeruginosa ATCC 9027 P. aeruginosa 120 Na P. fluorescens A 3.12 E. col i B Yeast Amino Acid: s-RNA s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. cpm x 10"3 cpm x 10"3 cpm x 10-3 cpm x 10-3 cpm x 10-3 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 Leu. 11 8.1 6.3 11.2 4.1 21.5 7.3 10.4 11.4 2.1 2.0 Tota l : 21.0 23.2 17.4 37.5 13.8 48.5 13.7 33.4 24.3 13.7 6.3 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 Micro- organ ism: Achromobacter B81 P. aeruqinosa ATCC 9027 P. aeruqinosa 120 Na P. fluorescens A 3.12 E. col i B Yeast Amino acid: s-RNA s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. Bas ic 14 22.9 9.6 25.7 8.4 16.4 9.9 19.1 14.7 22.2 16.2 Asp. 18.4 10.3 17.3 7.5 16.6 11.6 11.9 10.3 12.9 5.7 18.1 Glu. 10.6 5.6 9.8 3.4 5.5 4.2 4.3 3.2 5.2 4.5 4.9 Ala. 1.6 8.1 0 6.2 4.5 4.0 0 3.7 1.1 1.1 0 Pro. 0 0 0 4.7 0 2.0 0 5.8 4.6 10.3 3.3 Tyr. 0 4.5 8.4 9.1 18.3 5.5 5.5 8.9 4.1 2.7 9.1 Val . 19.2 13.8 19.1 13.3 18.7 11.9 18.7 17.8 14.2 39.2 17.6 Leu. 52.4 35 35.8 29.9 27.5 44.5 57.9 3K2 46.7 15.1 30.8 Table 36a. Incorporation of C ' 4 amino acids by E. col? enzymes into heterologous s-RNA's, and by heterologous enzymes into E_. col ? s-RNA Micro organism: E. col i B P. aeruginosa ATC 9027 P. aeruginosa 120 Na P. fluorescens A 3.12 Achromobacter B81 Yeast Amino Acid: s-RNA s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. cpm x 10~3 cpm x 10-3 cpm x 10-3 cpm x 10-3 cpm x 10-3 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 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 7.0 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 Tota l : 39.9 43.8 32.9 57.5 28.2 55.1 26.0 24.3 33.4 27.0 10.8 Table 36b Percent of each amino acid incorporated by JE_. col ? enzymes into heterologous s-RNA's, and by heterologous enzymes into E. col i s-RNA Micro organism: E. coli B P. aeruqinosa ATCC 9027 P. aeruqinosa 120 Na P. fluorescens A 3.12 Achromobacter B81 Yeast Amino acid: 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 4.1 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 Table 37a Incorporation of C H amino acids by yeast enzymes into heterologous s-RNA's, and by heterologous enzymes into yeast s-RNA Micro organism: Yeast P. aeruginosa ATCC 9027 P. aeruginosa 120 Na P. fluorescens A 3.12 Achromobacter B81 E. col i B Amino acid: s-RNA s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. cpm x 10"3 cpm x 10-3 cpm x 10-3 cpm x 10-3 cpm x 10-3 cpm x 10-3 Bas ic 9.1 2.0 0.8 2.9 0.4 3.5 0.9 1.0 3.0 1.7 6.0 Asp. 2.5 1.3 1.7 1.7 1.6 2.3 1.2 1.2 0.8 1.7 1.2 Glu. 1.3 0.4 0.9 0.8 0 0 0 0.3 0.6 0.4 1.5 Ala. 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 Total : 36.1 8.7 15.1 13.6 8.0 17.0 13.4 6.3 13.7 10.8 27.0 Table 37b Percent of each amino acid incorporated by yeast enzymes into heterologous s-RNA's, and by heterologous enzymes into yeast S-RNA Micro- organ ism: Yeast P. aeruqinosa ATCC 9027 P. aeruqinosa 120 Na P. fluorescens A 3.12 Achromobacter B81 E. col i B Amino acid: s-RNA s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. s-RNA Enz. Bas ic 25.2 23.6 5.1 21.4 5.5 20.3 6.9 16.2 22.2 17.4 24.2 Asp. 6.9 15.2 11.4 12.7 20.4 13.2 8.9 18.1 5.7 18.4 Glu. 3.5 11.3 6.1 6.1 0 0 0 4.9 4.1 5.6 Ala. 1.1 0 0 1.6 0 0 2.6 0 1.1 0 6.2 Pro. 6.4 5.4 0 4.7 0 9.1 0 3.3 10.4 2.3 8.0 Tyr. 12.7 10.4 0 6.0 0 5 4.2 9.1 2.7 12.3 0 Val . 20.3 14 51 9.9 74 13.8 55 17.6 39.2 18.7 34.6 Leu. 24.7 27.2 26.4 39.6 0 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 luores cens leucyl-, isoleucyl-, and phenylalanyl- s-RNA's as that of its own. In the case of the second £. aeruginosa s t ra in , valine - methionine incor poration was also doubled, but neither of the other two pseudomonads re acted as well with the basic amino acids as did P. aeruginosa ATCC 9027. The increase in heterologous reaction between E. co l i enzyme, and P. aerug inosa 120 Na s-RNA, could also be attributed mainly to the leucine group, and partly to these amino acids in the s-RNA's of the other strains of £. aeruginosa and of £. fluorescens. The incorporation of prol ine, for which E. co l i has an active enzyme, was nearly tr ipled 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 especially 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. co l i enzyme has also been reported by Clark and Ezyaguirre (31), and Benzer and Weisblum (14). However, the results with valine in this heterologous system are in contrast to those of Loft f ie ld and Eigner (105), who, using an assay procedure similar to that employed here, and valine as the sole amino acid present, found that there was only 18% as much incor poration into yeast s-RNA by the E. co l i enzyme as into the homologous E. co l i s-RNA. The yeast s-RNA and the E. co l i s-RNA were shown to be in com-151. petit ion for the valine. The presence in the;.Chlorel la hydrolysate of methionine, which is not separated paper chromatographically from valine, would explain the differences between these results and those of Loft f ie ld and Eigner (105). The yeast enzymes proved to be much more versati le 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 fa i r l y well with al l of the bacterial s-RNA's, and there was generally a fu l l complement of amino acids incorporated, although the incorporation was low in each case. Despite the high cross reaction exhibited between the E. co l i enzyme, and the yeast s-RNA, the reverse cross was no better than that with the other bacteria. The poor incorporation of radioactivity by the yeast enzyme into bacterial s-RNA's may be a problem of slow reac tion 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 spec i f ic i ty 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 related. In the months since this investigation was in i t iated, there have been several extensive experiments reported in which similar results were obtained to those presented here. The latest demonstration of the lack of spec i f i c i ty between s-RNA's and aminoacyl-s-RNA synthetases has been in the work of Yamane and Sueoka (176), who showed that the enz ymes of many microorganism wi l l interact to a greater or lesser extent 152. with E. co l i s-RNA. The one case in which there does appear to be specif  ic i ty for some amino acids is that of yeast, since many reports have in  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 wel l . This situation has been veri f ied in these experiments, although the yeast enz yme appeared to show a greater interaction with the bacterial s-RNA than vice versa (Table 37). 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 studies, since hybrid formation appears to occur only be tween closely related bacteria, there being, for example, very low hybrid ization between E_. col i and Pseudomonas. The lack of spec i f i c i ty 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 in oxidative assimilation by the aerobic bacteria studied. Thus, in both strains of Pseudomonas aeruginosa, and in Pseudomonas fluorescens. Achromobacter B81, Achromobacter viscosus. Azotobacter ag i l i s . Azotobacter vinelandii and Acetobacter xylinum, the major part of the radioactivity incorporated into the ce l ls during gl ucose-U-C^ oxidation was found in the proteinaceous fract ions. When more detailed studies were done with P. aeruginosa, under circumstances in which protein synthesis was greatly reduced, oxidative as similation was also markedly decreased. The addition of chloramphenicol to cel ls oxidizing glucose-C^ resulted in a reduction of about 25% in the total amount of oxidative assimilation, and a large part of the assimilated material was found in the metabolic pool as free amino acids. Starving the ce l ls prior to glucose oxidation, i .e. , aerating the ce l ls in a non nutrient medium, proved to decrease oxidative assimilation to a greater extent (40%) than did the presence of the ant ib iot ic . There are several possible ex planations for this phenomenon which are, in each instance, based on the use of protein and nucleic acid as major endogenous reserves by P. aerug inosa (29,75). One result of the starvation period was a greatly diminished act iv i ty of the aminoacyl s-RNA synthetases, which are necessary for protein synthesis. A second was the accumulation of radioactive glutamate in the metabolic pool, perhaps due to a lack of transaminases. A third result was the decrease in the rate of pyruvate oxidation. This led to a slower forma tion of ©f-ketoglutarate, 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 sacr i f i ce of v i ta l ce l lu lar constituents, which were resynthesized as soon as a substrate became ava i l  able. The studies of oxidative assimilation in P_. aeruginosa were ex tended to an investigation of the cytological sites 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 cel l extracts. This indicated that these enzymes were turning over rapidly during glucose oxidation. Experiments with chloramphenicol revealed, that although this 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 diss imi lat ion, either by freshly harvested or by starved ce l l s , into the fraction of the cytoplasmic pro tein which includes aminoacyl s-RNA synthetases. Further experiments with starved cel ls showed that the act iv i ty of these enzymes was decreased dur ing the starvation period, but Was rapidly restored during oxidative as- s 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 spec i f i c i ty between the enzymes and s-RNA's of other microorgan isms. Three of the bacteria which had been found to have a pattern of oxidative assimilation similar to that of P.. aeruginosa were selected - a second strain of JP. aeruginosa, as well as P. fluorescens. and Achromobacter 681. For comparative purposes, a more distantly 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 its poor cross reaction with IP. aeruginosa. It was found that there was l i t t l e spec i f ic i ty 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. co l i enzyme system. In the past few months, the same general conclusion as to the lack of species spec i f i c i ty 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 if starved cel ls were used, the incorporated into the RNA was greatly increased, supporting the contention of Duncan and Campbell (55) and of Cl i f ton (36) that oxidative assimilation takes place to replace endogenous reserves. Chloramphenicol also increased the incor poration of Cl^ into the ribosomal RNA, in agreement with previous reports on its action (9,66,67). The nitrogen for synthesis of these components in JP. aeruginosa was found by Duncan and Campbell (55) to be derived from the ammonia pro 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 paralleled by the uptake of endogenously produced ammonia. Like the strain 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 excre tion of pacemaker compounds, whose presence in the surrounding medium dur ing glucose dissimilation increased the extent of oxidative assimilation. 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 s t i l l 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 into the sur rounding medium. These organisms, by virtue of their ab i l i ty to f ix 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 results, it would seem that an investigation of oxida t ive 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 assimilation. This oxidation would provide cofactors, such as reduced pyrimidine nucleotides, as well as energy for transport of the aminated compound across ce l l barr iers. 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 paral lel uptake of ammonia and disappearance of ©C-ketoglutar ate 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 cel 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 ipids were generally highly labelled in most of the bacteria. From a cytological investigation of JP, aeruginosa, most of the l ip id was found to be in the "membrane" f ract ion, its label being increased by treatment of the cel ls with chloramphenicol, or by prior starvation. The reason for this increase was not established, but it may be the result of the channeling of more substrate into l ip id when protein synthesis is decreased, the latter process usually taking precedence. This increase, although substantial, especially in the case of the antibiotic treated ce 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. Polynucleotide phosphorylase in isolated bacterial membranes. J . B io 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 Bacter ium c o l i . Z. Physiol. Chem. Hoppe-Seyler's 255: 14r26. 3. Adler, E., G. Gunther, and J .E . Everett. 1938. Uber den enzymatis chen abbau und aufbau der glutaminsaure in hefe. Z. Physiol. Chem. Hoppe-Seyler's 255: 27-35. 4. Alford, M.A., M. Brotman, M.A. Chudy, and M.J. Fraser. 1963. The activation of glutamic acid and glutamine in mammalian t issue. Can. J . Biochem. Physiol. 4l_: 1135-1145. 5. Al len, E.H., E. Glassmann, and R.S. Schweet. I960. Incorporation of amino acids into ribonucleic acid. I. The role of activating enzymes. J . B io l . Chem. 2J5_: 1061 -1068. 6. A l l i son , R.M., and R.H. Burris. 1957. Kinetics of f ixation of n i t  rogen by Azotobacter v inelandi i . J . B io l . Chem. 224: 351-364. 7. Altenbern, R.A., and R.D. Housewright. 1951. Alanine synthesis and carbohydrate oxidation by smooth Brucella abortus. J . Bact e r i d . 62: 97-105. 8. Apgar, J . , R.W. Hoiley, and S.H. Mer r i l l . I960. Countercurrent d i s  tribution of yeast 'soluble ' 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. 10. Aronson, A. I . , and S. Spiegelman. 1961. On the nature of the r ibo nucleic acid synthesized in the presence of chloramphenicol. Biochim. Biophys. Acta 5_3_: 84-95. 11. Barker, H.A. 1936. The oxidative metabolism of the colorless, alga, Prototheca zopfi i. J . Cel lular Comp. Physiol. 8: 231-250. 12. Bautz, E.K.F., and B.D. Hal l . 1962. The isolation of T-4 specif ic RNA on a DNA-cellulose column. Proc. Natl . Acad. Sc i . U.S. 48: 400-408. 13. Be l l , D., R.V. Tomlinson, and G.M. Tener. 1964. Chemical studies on mixed soluble ribonucleic acids from yeast. Biochemistry 3_: in press. / 159. 14. Benzer, J . , and B. Weisblum. 1961. On the species spec i f ic i ty of acceptor RNA and attachment enzymes. Proc. Natl. Acad. Sc i . U.S. kj_: 1149-1154. 15. Berezorskaya, N.N. 1962. Some properties of a purif ied enzyme system catalysing the direct amination of pyruvic acid. Akual'ne. Vopr. Sovrem. Biokhim. 2: 130-137. 16. Berg, P. 1961. Specif ic i ty of protein synthesis, p. 293-324. In J.M. Luck, F.W. Al len, and G. McKinney (eds.), Annual review of biochemistry, Vol . 30. Annual Reviews, Inc., Palo Alto, Cal i f- orn ia. 17. Berg, P., F.H. Bergmann, E.J. Ofengand, and M. Dieckmann. 1961. The enzymic synthesis of amino acyl derivatives of ribonucleic acid. The mechanism of leucyl-, valy l-, isoleucyl-, and methi- onyl ribonucleic acid formation. J . B io l . Chem. 236: 1726-1734. 18. Bernheim, F. 1963. Effect of some surface active drugs on assim i lat ion of ammonium ions by a strain 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. Bio phys. 21: 451-457. 20. Bezborodov, A.M. 1963. The presence of alanine dehydrogenase and fumarase in ce l l free extracts of Streptomyces species. Mikro- biologiya 3J2: 20-26. 21. Blnnie, B., E.A. Dawes, and W.H. Holmes. 1959. Metabolism of Sarc- ina lutea. IV. Patterns of oxidative assimilation. Biochim. Biophys. Acta 40: 237-251. 22. Bolton, E.T., and B.J. McCarthy. 1962. A general method for the isolation of RNA complementary to DNA. Proc. Natl . Acad. Sc i . U.S. 48: 1390-1397. 23. Braunstein, A.E. 1957. Les roies principales de 11 assimilation et dissimilation de l 'azote chez les animaux, p. 339-389. _[n F.F. Nord (ed.), Advances in enzymology, Vol . 19. Interscience Publ ishers, L td . , New York. 24. Brooks, P., A.R. Crathorn, and G.D. Hunter. 1959. Site of synthes is of the peptide component of the cel l wall of Baci1lus mega- terium. 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 . Bu l l . soc. chem. b i o l . 43: 593-599. 26. Burk, R.R., and J.A. Pateman. 1962. Glutamic acid and alanine de hydrogenases determined by one gene in Neurospora crassa. Nature 160. 196: 450-451. 27. Burris, R.H. 1942. Distribution of isotopic nitrogen in Azoto bacter v inelandi i . J . B io l . Chem. 143: 509-517. 28. Butler, J .A.V. , G.N. Godson, and G.D. Hunter. 1961. Observations on the s i te and mechanism of protein biosynthesis in Baci11 us  megaterium, p. 349"362. Jhn R.J. Harris (ed.), Protein bio synthesis. Academic Press, New York. 29. Campbell, J . J .R . , .A.F . Gronlund, and M.G. Duncan. 1963. Endogen ous metabolism of Pseudomonas. Ann. N.Y. Acad. Sc i . 102; 669- 677. 30. Campbell, J.J.R., L.A. Hogg, and G.A. Strasdine. 1962. Enzyme distr ibution in Pseudomonas aeruginosa. J . Bacteriol . 83: 1155-1160. 31. Clark, J .M. , and J.P. Eyzaguirre. 1962. Tyrosine activation and transfer to soluble ribonucleic acid. Purif ication and study of the enzyme of hog pancreas. J . B io l . Chem. 237: 3698-3702. 32. C l i f ton , C.E. 1937. On the poss ib i l i ty of preventing assimilation in respiring ce l l s . Enzymologia 4: 246-253. 33. C l i f ton , C.E. 1946. Microbial assimilations, p. 269-308. Jn, F.F. Nord (ed.), Advances in enzymology, Vol . 6. Interscience Publ ishers, Inc., New York. 34. C l i f ton , 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 ton , C.E. 1962. Oxidative assimilation and distr ibution of glucose in Bacillus cereus. J . Bacteriol . 83: 66-69. 36. C l i f ton , C.E. 1963. Endogenous metabolism and oxidative assimila tion of typical bacterial species. Symposium on endogenous metabolism with special reference to bacteria. Ann. N.Y. Acad. Sc i . 102: 655-668. 37. C l i f ton , C.E. , and W.A. Logan. 1939. On the relation between as similation and respiration in suspensions and in cultures of Escherichia col ?. J . Bacteriol . 3_7_: 523~540. 38. C l i f ton , C.E. , and J.M. Sobek. 1961. Endogenous respiration of Bacillus cereus. J . Bacteriol . 82: 252-256. 39. Cohen, P.P., and G.W. Brown. I960. Ammonia metabolism and urea b io synthesis, p. 161-245. Jn. M. Florkin and H.S. Mason (eds.), Comparative biochemistry, Vol . II. Academic Press, Inc., New York. 161. 40. Conway, E.J. 1950. MicrodIffus ion analysis and volumetric error. 3rd ed. Crosby, Lockwood and Sons Ltd. , London. 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 iab 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. Volkin. 1959. Nucleic acid metabolism and ribonucleic acid heterogeneity in Escherichia c o l i . J , Bacteriol. 2§.: 41-48. 44. Crick, F.H.C. 1957. Discussion - Deoxyribosenucleic acid and pro tein synthesis, p. 26. _hn The structure of nucleic acids and their role in protein synthesis. Cambridge University Press, Cambridge. 45. Crouch, D. 1963. Oxidative metabolism of Hydrogenomonas f a c i l i s . Doctoral dissertation. Stanford University. Stanford, Ca l i f  ornia. 46. Dagley, S., and A.R. Johnson. 1953. The relation between l ip id and polysaccharide content of Bacterium c o l i . Biochim. Biophys. Acta JJ_: 158-159. 47. Dagley, S., A. White, D.G. Wild, and J . Sykes. 1962. Synthesis of proteins in ribosomes by bacteria. Nature 194; 25-27. 48. De Ley, J . , and J . Schel l . 1959. Oxidation of several substrates by Acetobacter acet i. J . Bacteriol . 27_: 445-451. 49. DeTurk, W.E. 1957. Effect of streptomycin on ammonia assimilation by P. aeruq inosa. Abstracts Fed. Proc. j_6: 292. 50. Doctor, B.P., and C M . Connelly. 1961. Separation of yeast amino acid-acceptor ribonucleic acids by counter current distr ibution in modified Kirby's system. Biochem. Biophys. Res. Comm. 6: 201-204. 51. Doctor, B.P., and J .A. Mudd. 1963. Species spec i f ic i ty of amino acid acceptor ribonucleic acid and aminoacyl soluble ribonucleic acid synthetases. J . B io l . Chem. 238: 3677-3681. 52. Doudoroff, M. 1940. The oxidative assimilation of sugars and re lated substances by Pseudomonas saccharophi la . Enzymologia 9_: 59-72. 53. Doudoroff, M., and R.Y. Stanier. 1959. Role of poly-yff-hydroxy- butyric acid in the assimilation of organic carbon by bacteria. Nature 183: 1440-1442. 162. 54. Duncan, M. 1962. Oxidative assimilation of glucose by Pseudo monas aeruginosa. M.Sc. Thesis, The University of Br i t ish Columbia. Vancouver, B.C. 55. Duncan, M., and J.J.R. Campbell. 1962. Oxidative assimilation of glucose by Pseudomonas aeruginosa. J . Bacteriol . 84: 784-792. 56. El Hawari, M.F.S., and R.H.S. Thompson. 1953. Separation and est imation of blood keto acids by paper chromatography. Biochem. J . 5J.: 341-347. 57. E l l i o t , W.H., and E.F. Gale. 1958. Glutamine-synthesizing system of Staphylococcus aureus: its inhibition by crystal violet 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. Microbiol. Jj>,: 106-120.. 59. Fincham, J.R.S. 1951. The occurrence of glutamic dehydrogenase in Neurospora and its apparent absence in certain mutant strains. J . Gen. Microbiol. £: 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 bacter ia . Nature 182: 800-801. 61. Fraser, M.J. 1962. A sensitive method for measurement of aminoacyl- ribonucleic acid synthetase ac t i v i t i es . Can. J . Biochem. Physiol. 40: 653-666. 62. Fraser, J . J . , and D.B. Klass. 1963. Partial puri f icat ion and prop erties of prolyl-RNA synthetase of rat l i ver . Can. J . Biochem. Physiol. 4]_: 2123-2139. 63. Freeze, E., and J . Oosterwyck. 1963. The induction of alanine de hydrogenase. Biochemistry 2_: 1212-1216. 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, Vol . III. Academic Press, Inc., New York. 65. Gaffron, H. 1935. Uber den stoffwechsel der Purpurbakterein. Bio chem. Z. 27_5_: 301-319. 66. Gale, E.F. 1947. The assimilation of amino acids by bacteria. I. The passage of certain amino acids across the ce l l wall and their concentration in the internal environment of Streptococcus faec  al i s . J . Gen. Microbiol. J_: 53"76. 67. Gale, E.F. 1953. Assimilation of amino acids by Gram positive bact er ia , and some actions of antibiotics thereon, p. 287-371. Jn. M.L. Anson, K. Bailey, and J.Y. Edsall (eds.), Advances in pro tein chemistry, Vol . VIII. Academic Press, New York. 163. 68. Giacomoni, D., and S. Spiegelman. 1962. Origin and biologic in  dividual i ty of the genetic dictionary. Science 138: 1328- 1331. 69. Giesberger, G. 1936. Beitrage zur Kenntis der Gattung Sp?r?11 urn Ehbg. Dissertation, University of Utrecht. Quoted from / C l i f ton , C.E. 1946. Microbial assimilations, p. 269-308. _ln_ F.F. Nord (ed.), Advances in enzymology, Vol . 6. Interscience Publishers, L td . , New York. 70. Gilby, A.R., A.V. Few, and K. McQuillen. 1958. The chemical com position of the protoplast membrane of Micrococcus lysodecktic- us. Biochim. Biophys. Acta 29_: 21-29. 71. Goldman, D.S. 1959. Enzyme systems in mycobacteria. VII. Puri f  ication, properties and mechanism of action of the alanine de hydrogenase. Biochim. Biophys. Acta 3.4: 527-539. 72. Goodman, H.M., and A. Rich. 1962. Formation of a DNA-soluble RNA hybrid and its relation to the or ig in , evolution and degeneracy of soluble RNA. Proc. Natl. Acad. Sc 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 io l . Chem. 2j$6: 54-60. 74. Gronlund, A .F . , and J.J.R. Campbell. 1961. Nitrogenous compounds as substrates for endogenous respiration in microorganisms. J . Bacteriol . . 8l_: 721-724. 75. Gronlund, A .F . , and J.J.R. Campbell. 1963. Nitrogenous substrates of endogenous respiration in Pseudomonas aeruginosa. J . Bact e r i o l . 86: 58-66. 76. Gros, F., S. Naono, C. Woese, C. Willson, and G. At ta ld i . 1963. Studies on the general properties of Escherichia co l i messenger ribonucleic acid, 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 as paragine. J . B io l . Chem. 187: 111-125. 78. Haldar, D., H. Bal, and A.N. Chatterjee. 1962. Action of some ant i  biotics in Vibrio cholera. Ann. Biochem. Exptl. Med. 22: 191- 196. 79. Hartmann, G., and V. Coy. 1961. Fraktionierung der aminosaure spe- zifischen lossichen ribonukleinsaure. Biochim. Biophys. Acta 47_: 612-613. 80. Hauser, G., and M.L. Karnovsky. 1954. Studies on the production of 164. glycolipide by Pseudomonas aeruginosa. J . Bacteriol . 68: 645- 654. 81. Hauser, G., and M.L. Karnovsky. 1958. Studies on the biosynthesis of L-rhamnose. J . B io l . Chem. 233: 287-291. 82. Hestrin, S. 1948. The reaction of acetyl chloride and other carboxy l i c acid derivatives with hydroxylamine and its analytical appl ication. J . B io 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 pro tein synthesis. J . B io l . Chem. 231: 241-257. 84. Hoi ley, R.W., J . Apgar, B.P. Doctor, J . Farrow, M.A. Marini, and S.H. Me r r i l l . 1961. A simplif ied procedure for the preparation of tyrosine and valine acceptor fractions of yeast "soluble r ibo nucleic ac id" . J . B io l . Chem. 236: 200-202. 85. Holme, T . , and H. Palmstierna. 1955. On the glycogen of Escherich ia col? B; its synthesis and breakdown and its specif ic labe 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 tab i l i ty of a bacterial ribonucleic acid. J . B io 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 fraction of Bacillus megaterium. Biochem. J . Z l : 369-376. 90. Hunter, G.D., and G.N. Godson. 1962. The f inal stages of protein synthesis and the role of l ipids in the process. J . Gen. Micro b i o l . .29.: 65-75. 91. Ichihara, H., H. Kanagawa, and M. Uschida. 1955. Studies on aspart- ase. J . Biochem. (Tokyo) 42: 439-447. 92. Jackson, W.T., and M.J. Johnson. 1961. Pathway of sucrose oxidation in Torulopsis ut M Is. J . Bacteriol . 81.: 182-188. 93. Jarvis, F.G., and Johnson, M.J. 1949. A glycolipide produced by Pseudomonas aeruginosa. J . Am. Chem. Soc. 21 : 4124-4126. 165 94. Kel ler, E.B., and R.S. Anthony. 1963. The two leucine acceptor RNA's from E. c o l i . Abstracts Fed. Proc. 22: 231. 95. Kretovich, V.L. 1958. The biosynthesis of dicarboxylic amino acids and enzymic transformation of amides in plants, p. 319~340. .In. F.F. Nord (ed.), Advances in enzymology, Vol . 20. Interscience Publishers Inc., New York. 96. Kretovich, V .L . , and M. Kasperek. 1961. Amino acid synthesis from pyruvate in different plants. 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 io l . Chem. Jj6_7_: 77-100. 99. Lacks, S., and F. Gros. I960. A metabolic study of the RNA-amino acid complexes in Escherichia col?. J . Mol. B io 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. 1946. Fermentation of acide /ff-hydroxybutyrique. Helv, Chem. Acta 2j9_: 1303-1310. 103. Levine, S., H.J.R. Stevenson, E.C. Tabor, R.H. Bordner, and L.A. Chambers. 1953. Glycogen of enteric bacteria. J . Bacteriol . 66: 664-670. 104. Lipmann, F. 1958. Introduction, Symposium on amino acid activation. Proc. Natl. Acad. Sc i . U.S. 44: 67~73. 105. Lof t f ie ld , R.B., and E.A. Eigner. 1963. Species spec i f i c i ty of transfer RNA. Acta Chem. Scand. T7_: S117-S122. 106. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. 1951. Protein measurement with the Folin-phenol reagent. J . B io l . Chem. 19J.: 265-275. 107. McCarthy, B.J., and E.T. Bolton. 1963. An approach to the measure ment of genetic relatedness among organisms. Proc. Natl. Acad. Sc i . U.S. 5.0: 156-164. 108. Macrae, R.M., and J .F . Wilkinson. 1958. Poly-/?-hydroxybutyrate metabolism in washed suspensions of Bacillus cereus and Bac? 1 lus 166. megaterium. J . Gen. Microbiol. J9_: 210-222. 109. McCormick, N.G., and H.O. Halvorson. 1964. Purif ication and prop erties of L- alanine dehydrogenase from vegetative cel ls of Bacillus cereus. J . Bacteriol . 8_7_: 68-74. 110. McLean, D.C., and K.C. Fisher. 1947. The relationship between oxy gen consumption and the ut i l iza t ion of ammonia for growth in Serratia marcescens. J . Bacteriol. 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 ac id: the influence of metall ic ions on carbohydrate and fat storage. Biochem. J . 3_2_: 1571-1582. 112. McQuillen, K., R.B. Roberts, and R.J. Britten. 1959. Synthesis of nascent protein by ribosomes in Escherichia c o l i . Proc. Natl . Acad. Sc i . U.S. 45_: 1437-1444. 113. Marmur, J . , S. Falkow, and M. Mandel. 1963. New approaches to bact er ial taxonomy, p. 329-377. Jn. C.E. Cl i f ton (ed.), Annual re view of microbiology. Annual Reviews, Inc., Palo Alto, Cal i fornia. 114. Matthaie, J .H . , O.W. Jones, R.G. Martin, and M.W. Nirenberg. 1962. Characteristics and composition of RNA coding units. Proc. Natl . Acad. Sc i . U.S. 48: 666-676. 115. Midwinter, G.G., and R.D. Batt. I960. Endogenous respiration and oxidative assim?lation in Nocardia cora l l lna . J . Bacteriol . 79: 9-17. 116. Mortenson, L.E. 1962. Inorganic nitrogen assimilation and ammonia incorporation. Jn. I.C. Gunsalus and R.Y. Stanier (eds.), The bacteria, Vol . III. Academic Press, Inc., New York. 117. Nirenberg, M.W., and Matthaie, J .H. 1961. The dependence of ce l l - free protein synthesis in JE. col? upon naturally occurring or synthetic polyribonucleotides. Proc. Natl. Acad. Sc 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 part icles within chloromycetin-inhibited Escherichia col?. J . Mol. B io l . U 204-217. 120. Norris, F.C., and J.J.R. Campbell. 1949. The intermediate metabol ism of Pseudomonas aeruginosa. III. The applicat ion of paper chromatography to the identif ication of gluconic and 2-keto- gluconic acids, intermediates in glucose oxidation. Can. J . Res. C. 27_: 253-261. 167. 121. Norris, F.C., J.J.R. Campbell, and P.W. Ney. 1949. The intermed iate metabolism of Pseudomonas aeruginosa. I. The status of endogenous respirat ion. Can. J . Res. p_. 27_: 157-164. 122. O'Connor, R.J., and H.O. Halvorson. I960. Intermediate metabolism of aerobic spores. V. The purif icat ion and properties of ].- alanine dehydrogenase. Arch. Biochem. Biophys. 9J.: 290-299. 123. O'Connor, R.J., and H. Halvorson. 1961. The substrate spec i f ic i ty of L-alanine dehydrogenase. Biochim. Biophys. Acta 48: 47-55. 124. Olson, J .A . , and C.B. Anfinson. 1953. Kinetic and equilibrium studies on crysta l l ine {.-glutamic acid dehydrogenase. J . B io l . Chem. 202: 841-856. 125. Pickett, M.J., and C.E. C l i f ton . 1943. The effect of selective poisons on the ut i l i za t ion of glucose and intermediate compounds by microorganisms. J . Cel lular Comp. Physiol. 22: 147-165. 126. Pollak, J .K. , and D. Fairbairn. 1955. The metabolism of Ascaris lumbricoides ovaries. II. Amino acid metabolism. Can. J . Bio chem. Physiol. 3J.: 307-316. 127. Quastel, J .H . , and B. Woolf. 1926. The equilibrium between 1- aspartic acid, fumaric acid, and ammonia in presence of resting bacteria. 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. in 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 spec i f ic i ty in activation and transfer of leucine from carrier ribonucleic acid to r ibo somes. J . B io 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 analysis, p. 680-684. In S.P. Colowick, and N.0. Kaplan (eds.), Methods in enzymology, Vol . III. Academic Press, Inc., New York. 132. Schweet, R.S., F.C. Bovard, E. Al len, and E. Glassman. 1958. The incorporation of amino acids into ribonucleic acid. Proc. Natl. Acad. Sc i . U.S. 44: 173-177. 133. Sguros, P.L., and S.E. Hartsel l . 1952. Aerobic glucose d iss imi la  tion by Achromobactier species. I. Fate of the carbon substrate. J . Bacteriol . 64: 811-819. 168. 134. Siegel, B.V., and C.E. C l i f ton . 1950. Energy relationships in carbohydrate assimilation by Escherichia c o l i . J . Bacteriol . 60: 573-583. 135. Siegel, B.V., and C.E. C l i f ton . 1950. Energetics and assimilation in the combustion of carbon compounds by Escherichia c o l i . J . Bacteriol . 60: 583-593. 136. Sierra, G., and N.E. Gibbons. 1962. Production of poly-/?-hydroxy- butyrate granules in Micrococcus ha1oden?tr?f ?cans. Can. J . Micro. 8: 249-253. 137. Silberman, R., and W.L. Gaby. 1961. The uptake of amino acids by l ipids of Pseudomonas aeruginosa. J . Lipid Res. 2: 172-176. 138. Smith, I. i960. Chromatographic and electrophoretic techniques, Vol . I, 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 Br i t ish Columbia. Vancouver, B.C. 140. Sobek. J .M. , and C.E. C l i f ton . 1962. Oxidative assimilation and C '4 distr ibution in Azotobacter ag i l i s . Proc. Soc. Exptl. B io l . Med. 109: 408-411. 141. Speyer, J . F . , A. Lengyel, C. Bas i l io , and S. Ochoa. 1962. Synthetic polynucleotides and the amino acid code. IV. Proc. Natl. Acad. Sc i . U.S. 48: 441-448. . 142. Spiegelman, S. 1959. Protein and nucleic acid synthesis in subcel lular fractions of bacterial c e l l s , p. 81-103. Jn, Recent prog ress in microbiology, Symposium Vl l th International Congress for Microbiology, Stockholm. Charles C. Thomas Publishing Co., Springfield, I l l ino is . 143. Steward, F.C., and J .F . Thompson. 1954. Proteins and protein metab olism in plants, p. 513~594. _ln_ H. Neurath, and K. Bailey (eds.), The proteins, Vol . 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 bact er ial genera with reference to aquatic productivity. J . Fish. Res. Bd. Canada 20: 729~734. 145. Struck, J . Jr., and I.W. Sizer. 1959. The substrate spec i f ic i ty of glutamic dehydrogenase. Arch. Biochem. Biophys. 86: 260-266. 146. Sueoka, N. 1961. Correlation between base composition of deoxyribo nucleic acid and amino acid composition of protein. Proc. Natl . Acad. Sc i . U.S. 47_: 1141-1149. 147. Sueoka, N., and T. Yamane. 1962. Fractionation of aminoacyl-169. acceptor RNA on a methylated albumin column. Proc. Natl . Acad. Sc i . U.S. 48: 1454-1461. 148. Sueoka, N., and T. Yamane. 1963. Fractionation of aminoacyl-ac ceptor 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 fraction of Es cherichia col? and its relation to cell-wall synthesis. J . Bact e r i o l . 84: 1061-1070. 150. Suit, J .C. 1963. Location of deoxyribonucleic acid l ike ribonucleic acid in a membrane fraction of Escherichia c o l i . Biochim. Bio phys. Acta 72: 488-490. 151. T iss ieres, A. 1959. Some properties of soluble ribonucleic acid from Escherichia c o l i . J . Mol. B io 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 . Bacteriol . 86: 434-444. 153. Trevelyn, W.E., and J.S. Harrison. 1952. Studies on yeast metabol ism. I. Fractionation and microdetermination of cel l carbohyd rates. Biochem. J . 5jO: 298-303. 154. van Nie l , C.B. 1936. Arch. Microbiol. J: 323-327. Quoted from Doudoroff, M., and R.Y. Stanier. 1959. Role of poly-j0-hydr- oxybutyrate in the assimilation of organic carbon by bacteria. Nature J83_: 1440-1442. 155. van N ie l , C.B., and E.H. Anderson. 1941. On the occurrence of f e r  mentative assimilation. J . Cel lular Comp. Physiol. JJ7: 49"56. 156. Virtanen, A. I., T..Z. Csaky, and N. Rautamem. 1949. On the forma tion of amino acids and proteins in Torula ut ?1 is on nitrate nutr i t ion. Biochim. Biophys. Acta 3_: 208-214. 157. Virtanen, A. I . , and J . Tarnanen. 1932. Die enzymatische spaltung und synthese der asparaginsaure. Biochem. Z. 250: 193-211. 158. Volkin, E., and L. Astrachan. 1956. Intracellular distr ibution of labeled ribonucleic acid after phage infection of Escherichia  c o l i . Virology 2: 433-437. 159. von Ehrenstein, G., and F. Lipmann. 1961. Experiments on hemoglobin synthesis. Proc. Natl. Acad. Sc i . U.S. 47_: 941-950. 160. von Tigerstrom, M.D. 1963. Unpublished results. 161. Wall, J .S . , A.C. Wagenknecht, J.W. Newton, and R.H. Burris. 1952. Comparison of the metabolism of ammonia and molecular nitrogen 170. in photosynthesizing bacteria. J . Bacteriol. 6j_: 563"573. 162. Warner, A.C.I . 1956. The actual nitrogen sources for growth of heterotrophic bacteria in non-limiting media. Biochem. J . 64: 1-6. 163. Warren, R.A.J., A.F. E l l s , and J.J.R. Campbell. 1959. Endogenous respiration of Pseudomonas aeruginosa. J . Bacteriol . 29_: 875" 879. 164. Webster, G.C. 1961. Isolation of an alanine-activating enzyme from pig l i ver . Biochem. Biophys.. Acta 49_: 141-152. 165. Webster, G . C , and J .E . Varner, 1955. Aspartate metabol ism and asparagine synthesis in plant systems. J . B io l . Chem. 215; 91- 99. 166. Weisblum, G., S. Benzer, and R.W. Holley. 1962. A physical basis for degeneracy in the amino acid code. Proc. Natl. Acad. Sc i . U.S. 48: 1449-1453. 167. Wiame, J . F . , and M. Doudoroff. 1951. Oxidative assimilation by Pseudomonas saccharophila with C^4 labelled substrates. J . Bact e r i o l . 62: 187-193. 168. Wiame, J .M., and A. Pierard. 1955. Occurrence of an L_(-)-alanine dehydrogenase in Bacillus subtil is . Nature 176: T073-1075. 169. Williams, A .E . , and R.H. Burris. 1952. Nitrogen f ixation by blue green algae and their nitrogenous composition. Am. J . Botany 2£: 340-342. 170. Wilkinson, J .F . 1958. The extracellular polysaccharides of bacteria. Bacteriol . Rev. 22: 46-73. 171. Wilkinson, J .F . 1959. The problem of energy storage compounds in bacteria. Exptl . Cell Res., Suppl. 7_: 111-130. 172. Wilner, B., and C.E. C l i f ton . 1954. Oxidative assimilation by Bac i l  lus subti l i s . J . Bacteriol . 6_7_: 571-575. 173. Winzler, R.J. 1940. The oxidation and assimilation of acetate by baker's yeast. J . Cel lular Comp. Physiol. 15_: 343-354. 174. Winzler, R.J., and J.P. Baumberger. 1938. The degradation of energy yielding compounds in the metabolism of yeast c e l l s . J . Cel lular Comp. Physiol. . 1 2 : 183-211. 175. Winzler, R.G., D. Bink, and V. du Vigneaud. 1944. Biotin in fermen tat ion, respiration, growth and nitrogen assimilation by yeast. Arch. Biochem. Biophys. 5j, 25-40. 176. Yamane, T . , and N. Sueoka. 1963. Conservation of spec i f ic i ty between 171. amino acid acceptor RNA and aminoacyl-s-RNA synthetases. Proc. Natl . Acad. Sc i . U.S. %0: 1093-1100. 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 lexner i . J . Gen. Microbiol. 2J: 521-527. 178. Yee, R.B., and H.M. Gezon. 1963. Ribonucleic acid of chloramphen- icol-treated Shigel la f lexneri . J . Gen. Microbiol. 32.: 299" 306. 179. Zachau, H.G., M. Tada, V/.B. Lawson, and M. Schweiger. 1961. Frak- tionierung der loslichen ribonucleinsaure. Biochim. Biophys. Acta 5_3_: 221-223. 180. Zel i tch, I., E.D. Rosenblum, R.H. Burris, and P.W. Wilson. 1951. Comparison of the metabolism of ammonia and molecular nitrogen in Clostridium. J . Bacteriol . 62: 747~752. 181. Zel i tch, I., P.W. Wilson, and R.H. Burris. 1952. The amino acid composition and distr ibution of 5 in soybean root nodules sup pl ied N'5 enriched N2. Plant Physiol. ZJ: 1-8. 182. Z i l l i g , W., D. Schachtschnabel, and W.Z. Krone, i960. Untersuchun- gen zur biosynthese der protein. IV. Zusammensetzung, Funktion und Spezifitat der loslichen ribonucleinsaure aus Escherichia  col i. Z. Physiol. Chem. 3J8: 100-114. 183. Zubay, G. 1962. A theory on the mechanism of messenger-RNA synthes i s . Proc. Natl. Acad. Sc i . U.S. 48: 456-460. 

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