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Streptomycin dependency in Escherichia coli K12 Whitlow, Karen J. 1975

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STREPTOMYCIN DEPENDENCY IN ESCHERICHIA COLI K12 by KAREN JEAN WHITLOW B.Sc, University of Simon Eraser, 19&9 M.Sc., University of Ottawa, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY the f a c u l t y of graduate studies Department of Biochemistry We accept t h i s thesis as conforming to the rje>quired s,tandard-THE UNIVERSITY OF BRITISH COLUMBIA July, 1975 (c) KAREN JEAN WHITLOW, 1975 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o lumbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f ^>J^>CJ^O^^JLr^i^ The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 - i -, ABSTRACT Twenty-three spontaneous streptomycin dependent (Sm^) E s c h e r i c h i a c o l i K 1 2 mutants were i s o l a t e d from a l o g phase c u l t u r e of w i l d - t y p e streptomycin s e n s i t i v e (Sm ) E s c h e r i c h i a /  c o l i K 1 2 . The metabolism of these mutants was c h a r a c t e r i z e d by a s t a t e of r e l a x e d c a t a b o l i t e r e p r e s s i o n d u r i n g growth on glucose. One o f the more r e a d i l y d e f i n e d c h a r a c t e r i s t i c s of these mutants i s the l o s s of the w i l d - t y p e phenotype o f s e n s i t i v i t y of growth to i n h i b i t i o n by L - v a l i n e . I n - s t u d i e s i n v o l v i n g both Sm and Sm3 E s c h e r i c h i a c o l i K 1 2 , i t was shown t h a t i n h i b i t i o n of growth by L - v a l i n e depended upon the l e v e l of a c t i v i t y of a r e g u l a t o r y enzyme i n the i s o l e u c i n e - l e u c i n e -v a l i n e ( i l v a ) b i o s y n t h e t i c pathway, acetohydroxy a c i d synthase (AHAS). Hence, under c o n d i t i o n s of c a t a b o l i t e d e r e p r e s s i o n , as observed i n the Sm d mutants or d u r i n g growth of the w i l d -type parent i n the presence of 5mM CAMP or on a c e t a t e , AHAS was derepressed and the s e n s i t i v i t y of growth to i n h i b i t i o n by v a l i n e was r e l i e v e d . That t h i s was a f u n c t i o n of the l e v e l of a c t i v i t y of t h i s enzyme was supported by the f i n d i n g that the s e n s i t i v i t y of AHAS to i n h i b i t i o n by L - v a l i n e was i d e n t i c a l i n both w i l d - t y p e and Sm d s t r a i n s . I n Sm d mutants a l l of the f i n a l products of the i l v a pathway - v a l i n e , i s o l e u c i n e , l e u c i n e and pantothenate were found to Hp®; i n -v o l v e d i n the r e g u l a t i o n of the s y n t h e s i s of AHAS. The c l o s e i n t e r r e l a t i o n s h i p observed i n t h i s work - i i -between dependency on streptomycin for growth and for the synthesis of catabolite sensitive enzymes, prompted a study of the influence of carbon source on a n t i b i o t i c requirement. Subsequently, i t was observed that Sm Escherichia c o l i K12 grew at l e a s t as well on an acetate medium devoid of a n t i -b i o t i c as on a glucose medium containing dihydrostreptomycin (DHSM). The same observation was made with Sm mutants of other strains of Escherichia c o l l . These observations are not e a s i l y explained on the basis of the current hypothesis of dependency based on a ribosomal s i t e of action for streptomycin (or DHSM). However, the experimental observations may possibly be explained on the basis of an eff e c t of the a n t i b i o t i c on the b a c t e r i a l membrane. Preliminary investigations showed that the character of the f a t t y acids i n the membrane of Escherichia c o l i K 1 2 varied both i n r e l a t i o n to carbon source and streptomycin phenotype. - i i i -TABLE OF CONTENTS Page No. INTRODUCTION 1 I. Proposed Sites of Streptomycin Action 1 I I . Proposed Mechanism of Streptomcyin Action 3 I I I . Genetics 13 IV. Streptomycin S e n s i t i v i t y 15 V. Streptomycin Resistance 17 VT. Streptomycin Dependency 18 VII. Physiology and Biochemistry of Smd Escherichia c o l i 19 VIII. Respiration i n Srad C e l l s 25 IX. Control of Acetohydroxy Acid Synthase i n Bacteria 27 X. Research Aims 33 METHODS AND MATERIALS 3k I. Organisms 3k i ) Nomenclature used i n describing the response of c e l l s to streptomycin 3^4-i i ) Maintenance of stock cultures 3k I i i ) I s o lation of Smd Escherichia c o l i K12 mutants 3k I I . Growth of Cultures and Preparation of C e l l Extracts 35 i ) Media 35 i i ) Estimation of growth 36 i i i ) Procedures f o r growing and harvesting cultures 36 a) general growth conditions » :.; 36 b) growth of a n t i b i o t i c - l i m i t e d S m a  Escherichia c o l i 36 c) growth of Smd E. c o l i K12 c e l l s used i n the investigation of multivalent end-product repression of AHAS 37 d) procedure f o r tes t i n g the growth of Smd E. c o l i cultures on paromomycin i n place of DHSM 37 - i v -Page No. e) growth of c e l l s for the assay of enzymes .. 37 f) adaptation of c e l l s to growth on acetate .. 38 g) harvesting cultures 38 iv) Preparation of c e l l extracts 38 I I I . Enzyme Assays 39 i ) Fumarase 39 i i ) Aconitase 39 i i i ) Glucose-6P Dehydrogenase 39 iv) Acetohydroxy Acid Synthase 1+0 v) Beta-galactosidase 1+0 IV. Quantitative and Qualitative Analysis 1+1 i) Protein 1+1 i i ) Estimation of amino acid excretion by Escherichia c o l i cultures 1+2 i i i ) Determination of P£03 (prototetrahydro-porphyrin IX) 1+3 iv) Extraction of Escherichia c o l i membrane l i p i d s and f a t t y acid analysis 1+3 PART A: I s o l a t i o n and Characterization of Sm Eacherichia c o l i K12,_ 1+5 RESULTS 1+5 I. I s o lation of Smd Escherichia c o l i K12 U5 I I . Derepression of Catabolite Sensitive Enzymes During the G-rowth of Smd Escherichia c o l i K12 on Glucose 1+5 I I I . Analysis of P503 Levels i n Sm Escherichia c o l i . 1+5 IV. Excretion of Amino Acids by Escherichia c o l i K12 Cultures 1+9 V. Growth of Smd Escherichia c o l i K12 i n the Presence of L-Valine 53 DISCUSSION 58 PART B: Regulation of Acetohvdroxv Acid Synthase i n Smd Escherichia c o i l K12 6l RESULTS ,61 I. E f f e c t of Carbon Source on the S e n s i t i v i t y of Growth to I n h i b i t i o n bv L-Valine i n Smd  Escherichia c o l i K12 .". 6l V-Page No. II. S e n s i t i v i t y of Growth to In h i b i t i o n by L-Valine i n Antibiotic-Limited Smd Escherichia c o l i K12 61 II I . A c t i v i t y of Acetohydroxy Acid Synthase i n Smd and Sms Escherichia c o l i K12 62 IV. End-Product In h i b i t i o n of Acetohydroxy Acid Synthase by-L-Valine i n Smd Escherichia c o l i K12 62 V. E f f e c t of DHSM Concentration on Enzyme Levels i n A n tibiotic-Limited Smd Escherichia c o l l K12. 66 VI. Multivalent End-Porduct Repression of Aceto-hydroxy Acid Synthase i n Sm Escherichia c o l i K12 '. 66 DISCUSSION 73 PART C: Control of Acetohydroxy Acid Synthase i n Escherichia c o l i K12 75 RESULTS 75 I. E f f e c t of Carbon Source on the In h i b i t i o n of Growth by L-Valine i n Escherichia c o l i K12 .... 75 I I . E f f e c t of CAMP on the S e n s i t i v i t y of Growth to Valine i n Escherichia c o l i K12 75 I I I . Oontrol of Acetohydroxy Acid Synthase by Catabolite Repression i n Escherichia collmK12 . 78 IV. Eff e c t of CAMP on the Excretion of the Branch Chain Amino Acids by Escherichia c o l i K12 78 DISCUSSION 82 PART D; Streptomycin Dependency i n Relation to Catabolite Repression In Smd Escherichia c o l i 85 RESULTS 85 I. Substitution of CAMP for DHSM i n the Growth Medium of Smd Escherichia c o l i K12 85 I I , E f f e c t of Carbon Source on the Requirement for DHSM i n Smd Escherichia c o l i K12 85 - v i -Page No. II I . 'Streptomycin Dependency' i n Sm^ Escherichia c o l i ' '. 88 IV. Phenotype of 'Smd' Escherichia c o l i K12 Grown Without DHSM 88 V. Examination of Catabolite Repression i n Sm3  Escherichia c o l i K12 Growing i n the Presence of Sublethal Concentrations of DHSM 91 VI. E f f e c t of Ethanol and DHSM on Ca.fcabolite Repression i n Smr Escherichia c o l i K12 Growing on Glucose 91 DISCUSSION 96 PART E: Effe c t of Acetate as a Carbon Source on 'Smd' and Sm3 Escherichia c o l i K12 100 RESULTS 100 I. Beta-galactosidase Induction . .100 I I . Growth on a Limiting Concentration of Acetate . 1 0 0 I I I . A n t i b i o t i c S e n s i t i v i t y 102 IV. Analysis of Patty Acids of the C e l l Membrane . . 1 0 5 DISCUSSION 109 GENERAL DISCUSSION 112 SUMMARY OF MAJOR FINDINGS 117 SUGGESTIONS FOR FUTURE WORK 118 BIBLIOGRAPHY , 119 - v i i -LIST OF TABLES No. Page No. I. Fumarase and G6PDH a c t i v i t i e s i n Smd and Sms  Escherichia c o l i K12 ij.6 II. Relationship between the a c t i v i t y of catabolite sensitive enzymes and P503 formation i n Sms  Escherichia c o l i 50 I I I . Coproporphyrinogenase and prot©porphyrinogen. . oxidase a c t i v i t i e s i n a n t i b i o t i c - l i m i t e d Sm Escherichia c o l i K12 52 j s IV. Excretion of amino acids by Sm and Sm Escherichia c o l i K12 after h» 6* and 8 hours growth on a glucose-salts medium Sh d 3 V. Amino acid excretion patterns i n Sm and Sm Escherichia c o l i K12 and Escherichia c o l i B a f t e r \\ hours growth on a glucose-salts medium. 55 VI. E f f e c t of metal ions on the amino acid excretion pattern i n Smd Escherichia c o l i K12 growing on a glucose-salts medium supplemented" with 1000 micrograms DHSM/ml 56 VII. E f f e c t of Fe.'(+++) on the doubling time of Smd  Escherichia c o l i K12 and the a c t i v i t y of AHAS during growth of the c e l l s on a glucose-salts medium"supplemented with 1000 micrograms DHSM/ ml 56 VIII. Relationship between AHAS a c t i v i t y and the i n h i b i t i o n of growth by valine i n Smd  Escherichia c o l i K12 65 IX. E f f e c t of DHSM concentration on the amounts of enzymes in a n t i b i o t i c - l i m i t e d Smd  Escherichia c o l i K12 70 X. Repression of AHAS by end-products i n Sm Escherichia c o l i K12 72 XI. E f f e c t of growth conditions on the l e v e l of AHAS i n Escherichia c o l i K12 79 XII. E f f e c t of CAMP on the excretion of the branch chain amino acids by Escherichia c o l i K12 81 XIII. E f f e c t of carbon source on the requirement of Smd Escherichia c o l i B for DHSM 87 - v i i i -No. Page No. XIV. Growth of Sm Escherichia c o l i i n r e l a t i o n to carbon source and drug supplement 90 XV. E f f e c t of carbon source and supplement on catabolitle repression i n 'Smal Escherichia c o l i K12 92 XVI. AHAS a c t i v i t y i n Escherichia c o l i K12 grown i n the presence of a sublethal concentration of DHSM 93 XVII. E f f e c t of growth conditions on the l e v e l of AHAS in Smr Escherichia c o l i K12 9U • • -~ d s XVlII^ArialysiS;of membrane f a t t y acids i n Sm , Sm and Smr Escherichia c o l i K12 grown on acetate and glucose 108 - i x -LIST OF FIGURES No. Page No. 1 . Physiological significance of P503 2 8 2 . The biosynthetic pathway to isoleucine, valine, leucine and pantothenate i n Escherichia c o l i ... 3 0 3 . Reduced-oxidised difference spectra of whole c e l l s of Sm3 and Smd Escherichia c o l i K 1 2 L+7 1+. E f f e c t of carbon source on the formation of P 5 0 3 i n Smd Escherichia c o l i B 1+8 5 . P5>03 formation i n Smd Escherichia c o l i K 1 2 grown with l i m i t i n g concentrations of DHSM 5 1 6 . S e n s i t i v i t y of the growth of Sm3 and Sra d  Escherichia c o l i K 1 2 to the presence of L-valine In the growth medium 5 7 7 . I n h i b i t i o n of growth by L-valine i n a n t i b i o t i c -l i m i t e d Smd Escherichia c o l i K 1 2 6 3 8 . E f f e c t of valine concentration on the i n h i b i t i o n of growth by valine i n a n t i b i o t i c - l i m i t e d Sm Escherichia c o l i K 1 2 61+ 9 . I n h i b i t i o n of acetohydroxy acid synthase by L>valine i n Smd and Sms Escherichia c o l i K 1 2 . . . . 6 7 10. Relationship between growth rate and DHSM con-centration i n Smd Escherichia c o l i K 1 2 6 8 1 1 . Determination of KDJJSJ^ for Smd Escherichia c o l i K 1 2 growing on glucose 6 9 1 2 . E f f e c t of carbon source on the i n h i b i t i o n of growth by L-valine In Escherichia c o l i K 1 2 7 6 1 3 . E f f e c t of CAMP on the i n h i b i t i o n of growth by L-valine i n Escherichia c o l i K 1 2 7 7 11+. I n h i b i t i o n of AHAS by L-valine i n Escherichia c o l i K 1 2 8 0 1 5 . Substitution of CAMP (5mM) for DHSM i n Smd  Escherichia c o l i K 1 2 8 6 1 6 . Non-dependency of fSm d | Escherichia c o l i K 1 2 growing on acetate 8 9 - X -No. Page No. 17. E f f e c t of growth conditions on the i n h i b i t i o n of growth by L-valine i n Smr Escherichia c o l i K12 : , 95 18. E f f e c t of carbon source on beta-galactosidase induction bv TMG i n Smd and Sm3 Escherichia c o l i K12 101 19. Growth of several Smu Escherichia c o l i strains and Sms Escherichia c o l i K12 on l i m i t i n g acetate supplemented with non-limiting lactose 103 20. Growth.of a Smd Escherichia c o l i K12 with successive gliquots of a l i m i t i n g concentration of acetate 101+ 21. E f f e c t of carbon source on the s e n s i t i v i t y of Sms Escherichia c o l i K12 to the addition of chloramphenicol or dihydrostreptomycin to the' growth medium 106 22. GLC of f a t t y acids i n the membrane l i p i d s of SrrF Escherichia c o l i K12 grown on glucose or acetate 107 - x i -ABBREVIATIONS ATP adenosine triphosphate DHSM dihydros trep tomyc in DNA deoxyribonucleic acid PAD+ f l a v i n e adenine dinucleotide i l v a isoleucine-leucine-valine IPTO isopropyl beta~D thio galactopyranoside NAD* nicotinamide adenine dinucleotide NADH reduced nicotinamide adenine dinucleotide NADP+ nicotinamide adenine dinucleotide phosphate NADPH reduced nicotinamide adenine dinucleotide phosphate Pm paromomycin RNA ribonucleic acid raRNA messenger ribonucleic acid rRNA ribosomal ribonucleic acid tRNA transfer ribonucleic acid Sm streptomycin Smd streptomycin dependent Smr streptomycin r e s i s t a n t Sm3 streptomycin sen s i t i v e Str A streptomycin gene TCA t r i c a r b o x y l i c acid TMG methyl beta-D thio galactopyranoside - x i i -ACKNOWLEDG-EMENTS I wish to thank Dr. W. J. Polglase for his encouragement and d i r e c t i o n throughout the course of this work and Dr. R. Poulson for many he l p f u l discussions. -1-INTRODUCTION Streptomycin i s one of a group of cl o s e l y related amino-glycoside a n t i b i o t i c s which includes streptomycin, dihydro-streptomycin, and bluenosamine. There are three g e n e t i c a l l y determined responses of bacteria towards the presence of streptomycin i n the growth medium: s e n s i t i v i t y , resistance, and dependence. Wild-type strains of bacteria are termed sens i t i v e to streptomycin since the presence of low concentrations o f the drug (10 ug/ml) i n the growth medium i s l e t h a l ; r e s i s t a n t strains grow either i n the presence or absence of the a n t i b i o t i c and can be described as having low l e v e l resistance (resistant to 25 yg streptomycin per ml or l e s s ) , or high l e v e l resistance (resistant to lev e l s of 1000 yg streptomycin/ml); dependent strains require the a n t i b i o t i c f o r growth. Streptomycin binds to DNA (Cohn et_ a l , 19&7) (and can in fact be used as a p r e c i p i t a t i n g agent for nucleic acids), and to the 30s component of the 70s ribosome of Escherichia c o l i (Kaji and Tanaka, 1968). It i s this l a t t e r property which has been expanded upon In attempts to explain the mechanism of action of streptomycin i n b a c t e r i a l c e l l s . I Proposed Sites of Streptomycin Action Spotts and Stanier (I96I) proposed the ribosome as the s i t e of action of streptomycin. This was based on the obser-vation that streptomycin dependent (Smd) mutants deprived of streptomycin stopped synthesizing protein but continued to synthesize DNA and RNA. Furthermore, the K i l l i n g action o f -2-streptomycin can be prevented or a l l e v i a t e d by the complete or p a r t i a l i n h i b i t i o n of protein synthesis. This would imply that i n order for the aminoglycoside to exert a bacter-i c i d a l e f f e c t , the normal ribosomal cycle i n protein synthe-s i s must be i n operation (Anand and Davis, I960; Blotz and Davis, 1962). Support i n favour of the ribosome as the s i t e of action i s based on several i n v i t r o studies. Reassociation of 30s and 50s ribosomal subunits from Sms (streptomycin sensitive) 1* cl and Sm (streptomycin resistant) or Sms and Sm strains of E s c h e r i c h i a - c o l i yielded ribosomes which were sens i t i v e , r e s i s t a n t or dependent on streptomycin for protein synthesis depending on the source of the 30s subunit (Cox et a l , 1961;; Cutler and Evans, 1967? Likover and Kurland, 1967a). Recon-s t i t u t i o n of the 30s subunit from the core p a r t i c l e s (16s RNA and 15 proteins) and the s p l i t proteins (6 proteins) pointed to the core p a r t i c l e as containing t h i s ribosomal factor (Staehelin and Meselson, I966; Traub e,t a l , 1966). Subse-quent studies were able to i d e n t i f y this factor as the S12 (P10) core protein (Ozaki et a l , I969). The Sms ribosomal binding s i t e for streptomycin was i d e n t i f i e d as the 30s subunit (Kaji and Tanaka, 1968). Moreover, the a f f i n i t y of the 30s subunit f o r streptomycin depended on the source of the 30s subunit. Thus, the 30s d s ribosomal subunit from Sm and wild-type Sm Escherichia c o l i bound streptomycin (Kaji and Tanaka, 1968j Zimmermann et a l , 1971) while that from a Smr mutant did not (Kaji and - 3 -Tanaka, 1968; Ozaki et a l , 1969). However, i n l i g h t of more recent observations, the b i o l o g i c a l s ignificance of streptomycin binding has been questioned. A search f o r a streptomycin binding s i t e on the cibosome revealed that streptomycin bound to the l6s RNA of the 30s subunit but not to the subunit proteins and, under these conditions, normal reconstitution of the 30s subunit was prevented (Biswas and Gorini, 1972). How.bver, exposure of the 30s subunit from Sm Escherichia c o l i to^streptomycin during reconstitution, induced a modified behavious that per-s i s t e d a f t e r streptomycin had been removed by d i a l y s i s ; the e f f e c t was reversed only by exposure of the modified ribosome to a high s a l t concentration:;(Biswas and Gorini, 1972). It was suggested, therefore, that streptomycin may not-actually bind to the ribosome but i t s presence modifies the assembly of the ribosome when a responsive S12 (P10) mutation i s present i n the genome (Garvin j3t a l . 1973). In Sm mutants, th i s modification would be required i n order f o r the ribosome to be functional. II Proposed Mechanism of Streptomycin Action In v i t r o assays of protein synthesis have shown that i n the presence of streptomycin, gross misreading of RNA codewords occurs with. Sm but not with Smr or Sm ribosomes (Gorini and Kataja, 196h,a; Davies et a l , 1965. ). This appeared to explain the observation that streptomycin causes pheno-typic suppression of certain auxotrophic mutations and amber and ochre mutations i n Sms and i n some classes of Smr strains of Escherichia c o l i . The a b i l i t y of streptomycin to cause suppression, however, i s very much reduced i n Smr strains (Gorini and Kataja, 1961+a, 1965; Orias and Gartner, I966). I n i t i a l l y , i t was suggested that the l e t h a l i t y of strep-tomycin i n Sms strains of bacteria was due to the promotion of f a u l t y protein synthesis (Gorini and Kataja, 1961+a), how-ever, certain b a c t e r i a l mutants of Escherichia c o l i have been shown to survive despite extensive misreading of mRNA (Gorini and Kataja, 1961+b). Furthermore, Likover and Kurland (1967b) observed that streptomycin had no misreading e f f e c t i n an incorporation system containing highly p u r i f i e d r i b o -somal and supernatant f r a c t i o n s . Translational errors could be introduced only by the addition of small amounts of de-natured DNA. Physiological concentrations of streptomycin i n h i b i t polypeptide synthesis and cause misreading In extracts con-taining Sm3 but not Smd or Smr ribosomes (Flaks e_t a l , 1962:). However, the importance of environment on the effects of streptomycin i n v i t r o must be emphasized. For example, i t has been observed that streptomycin i n h i b i t s polypeptide synthesis directed by various natural mRNAs at magnesium con-centrations optimal for peptide bond formation i n Sm3 extracts (Erdos and Ullmann, 1959; Schwartz, 1965; van Knippenberg et a l , 1965). Polypeptide synthesis directed by various natural mRNAs, on the other hand, can be stimulated by streptomycin i n the presence of supra-optimal magnesium concentrations (van Knippenberg et a l , 1965). Streptomycin has also been -5-shown to promote the a c t i v i t y of denatured DNA, rRNA, tRNA and other non-messenger-like polymers such as p o l y i n o s i n i c acid as templates for polypeptide synthesis (McCarthy et_ a l . 1966; Morgan et a l , 1967). Cells treated with streptomycin contain an abnormally high content of 70s ribosomes to which polypeptide chains are attached. This was interpreted as suggesting either (i) streptomycin interferes with termination of peptide form-ation or ( i i ) streptomycin interferes with i n i t i a t i o n of protein synthesis and freezes ribosomes as 70s particles,,, (Herzog, I96I4.). While Luzzatto ejb a l (1968) presented evi# dence i n support of a s p e c i f i c e f f e c t of streptomycin on the i n i t i a t i o n of protein synthesis,v;Scolnick et a l (1968) sup-ported the view that streptomycin i n h i b i t e d chain termination. Gorini et a l (1961+a, 1961+b) and Davies et a l (1965 ) using homopolynucleotide messengers, established that strep-tomycin could d i s t o r t the recognition region of the ribosome r e s u l t i n g i n the misreading of RNA codewords. However, systems employing homopolynucleotide messengers have been questioned^ In Sm c e l l s streptomycin causes complete In-h i b i t i o n of protein synthesis whereas In homopolynucleotide systems i n h i b i t i o n i s only p a r t i a l with much higher concen-trations of streptomycin, and the misreading i s so extensive that i t may even exceed the i n h i b i t i o n (Davies e_t a l , 196!j.:..; Davies et a l , 1965a). This had l e d to the b e l i e f that mis-reading and i n h i b i t i o n of protein synthesis caused by strep-tomycin are probably two d i s t i n c t expressions of drug action. -6-Modolell and Davis. (1969a) suggested that with natural mes-senger, streptomycin impaired the e f f e c t i v e binding of both amino acyl tRNA and peptidyl tRNA to the A s i t e (acceptor site) of sensitive ribosomes thus causing i n h i b i t i o n of chain elongation. This same d i s t o r t i o n was used as the basis for a unitary hypothesis explaining the several effects of strep-tomycin on the ribosome (Modolell and Davis, 1969b). For ex-ample the e f f e c t on chain termination reported by Scolnick et a l (I968), and on chain i n i t i a t i o n reported by Luzzatto et a l (I968). Presumably, i n ribosomes with r e s i s t a n t 3 0 s subunits, the d i s t o r t i o n by streptomycin i s much less severe due to the mutated S12 (P10) protein thus explaining the observed phenotyplc suppression i n the absence of protein i n h i b i t i o n (Sparling et a l , 1968). Lelongfet a l ( 1 9 7 1 ) reported that streptomycin caused the release of bound formyl methionyl tRNA fr o m a preformed i n i t i a t i o n complex but did not impair the f i r s t step i n the i n i t i a t i o n of polypeptide synthesis i e . the binding of formyl methionyl tRNA to the 3 0 s mRNA complex. This was interpreted as suggesting that the i n i t i a t o r tRNA is not i n i t i a l l y bound to the P s i t e (peptidyl s i t e ) but enters another s i t e , d i s t i n c t from the A s i t e and not affected by streptomycin. Once the i n i t i a t o r tRNA is engaged i n the P s i t e , however, strepto-mycin causes i t s release. Both Modolell and Davis ( 1 9 7 0 ) and Lelong et a l (1971) reported that the formation i n v i t r o of 3 0 s i n i t i a t i o n complex i s not impaired i n the presence of streptomycin, but formyl - 7 -methionyl tRNA i s released af t e r the joining of the 5 0 s sub-u n i t to the 30s. This was explained by the observations of Lelong e_t a l ( 1 9 7 2 ) who showed that p u r i f i e d i n i t i a t i o n factors F-^ , F^, and F^ protected ribosomes (30s and 70s) against the binding of streptomycin. It was suggested that the factors either bind or induce conformational changes i n the S12 (P10) region of the ribosome. The S12 (P10) protein appears to be essential for the occurrence of the i n i t i a t i o n steps on natural mRNA templates (Ozaki et a l , 1969). Thus i t was suggested that the streptomycin binding region of the 30s ribosomal subunit might also represent the point at which factors become normally attached or at l e a s t i s essential f o r these factors to promote i n i t i a t i o n on ribosomes. Several reports have appeared which demonstrate that the requirement for streptomycin for growth i n Smd strains i s suppressed by a mutation outside the Str A locus. The • g resultant Sm revertant .Tnutants display a wide variety of phenotypes not easily explained (Deusser et, a l , 1970). The suppressing mutation appears to be an altered 30s protein d i s t i n c t from the S12 (P10) protein and i d e n t i f i e d as SlO (Apiron e_t a l , 19&9J Deusser e_t a l , 1970; Kreider and Brownsteih, 1971). The SlO protein has been shown to bind d i r e c t l y to 1 6 s rRNA i n what i s considered to be the f i r s t step i n 3 0 s assembly (Mizushlma and Nomura, 1970). Elimination of this protein i n an in v i t r o r e c o n s t i t u t i o n mixture results i n a ribonucleoprotein p a r t i c l e with d r a s t i c a l l y reduced function -8-(Nomura et_ a l , 19&9). The S10 protein has also been impli<-cated as one of two 3 0 s proteins which can interact with free 2 3 s rRNA from the £ 0 s subunit (Schaup et a l , 1 9 7 1 ) . The exact mechanism by which a mutation i n this protein i s able to cause reversion from streptomycin dependence to i n -dependence, however, i s not known. Secondary mutations i n either Sj^'or Scj have also been . d , shown to suppress dependence of Sm mutants (Deusser et a l , 1 9 7 0 ; Birge and Kurland, 1 9 7 0 ; St'offler et a l , 1 9 7 1 ; Kr eider and Brownstein, 1 9 7 1 ; 1 9 7 2 ; Hasenback et a l , 1 9 7 3 ; Iton and Wittman, 1 9 7 3 ) . Gorini ( 1 9 & 9 ) reported that introduction of a ribosomal mutation termed ram I (P|j)& or Sj^) by transduction into several- Smd strains caused a reversion to streptomycin independence. The ram gene has been shown to define a 3 0 s ribosomal component which i s responsible f o r ribosomal am-b i g u i t y . The ram mutation was found to produce streptomycin independence i n Smd mutants which depended upon an increased l e v e l of ambiguity rather than upon streptomycin s p e c i f i c a l l y . In t h i s sense, the mutation did not a l t e r the phenotype of the c e l l but served as a genetic substitute f o r the drug. Gorini (19& 9 ) , i n f a c t , suggested that the s i t e of action of streptomycin on the ribosome was the ram protein. It was proposed that streptomycin distorted the function of rara + and thereby introduced ambiguity into the c e l l s ie caused phenotypic suppression. Gorini ( 1 9 6 9 , 1 9 7 1 ) has also proposed that the ribosome plays an active r o l e i n c o n t r o l l i n g recognition e f f i c i e n c y - 9 -of tRNA. It was suggested that the ribosome has a tRNA recognition s i t e which would provide an additional l e v e l of s p e c i f i c i t y for the mRNA-tRNA inte r a c t i o n . Theoretically, this recognition s i t e would be distorted by the binding of streptomycin to the 30s subunit of the ribosome such that the degree of ribosomal r e s t r i c t i o n would be decreased and, therefore, the f i d e l i t y of the ribosomal machinery affected, cl Hence, i n Sm strains the r e s t r i c t i o n would be too great and would prevent normal t r a n s l a t i o n unless streptomycin was s present, while i n Sm strains streptomycin would cause too great a relaxation of r e s t r i c t i o n and thereby prevent protein synthesis and r e s u l t i n c e l l death. Although the e f f e c t of streptomycin action has been explained on the basis of a ribosomal s i t e of action, despite many-,attempts, the exact mechanism of the i n t e r a c t i o n of streptomycin with the ribosome i s unknown. This issue i s complicated by the fact that not a l l the observed effects of streptomycin can be s a t i s f a c t o r i l y explained by a^-Kibosomal s i t e of action. For example: amino acid substitution induced by streptomycin i n whole c e l l s has not been demonstrated; streptomycin-treated c e l l s can be protected from the k i l l i n g action of streptomycin by a s h i f t from aerobic to anaerobic conditions (Geiger e_t a l , 191+6; Williamson and White, 1956); streptomycin exerts a p r e f e r e n t i a l effect on the synthesis of c e r t a i n enzymes (Fitzgerald et a l , 191+8; Roote and Polglase, 1955; Goodman and Spotts, 1967). Aside from the ribosomal theory of streptomycin action, -10-e a r l i e r theories of streptomycin action emphasized an effect on the c e l l membrane. On treatment with streptomycin, Escherichia c o l i was shown to lose nucleotides (Roth^ejb a3L, I960; Rosano e_t a l , I960; Anand and Davis, i960), amino acids (Anand and Davis, i960), and potassium (Dubin and Davis, 1961) into the culture medium. Moreover, addition of streptomycin to Escherichia  c o l i s t r a i n ML-35 which i s const i t u t i v e for beta-galactosi-dase but lacks a beta-galactosidase transport system, caused the c e l l s ' t o become less cryptic (Dubin and Davis* 19&1)• As suggested by the observed etotagonism of streptomycin action by chloramphenicol, growth i s required i n Sm3 c e l l s for the b a c t e r i c i d a l e f f e c t of streptomycin (Plotz and Davis, 1962). These findings supported the suggestion that an early e f f e c t of streptomycin i s to injure the c e l l membrane of growing bacteria. Thus, the observation that there i s a biphasic uptake of streptomycin i n Sm3 c e l l s l e . an i n i t i a l rapid adsorption on the c e l l membrane followed by i n t r a c e l l u l a r accumulation, was interpreted as uptake re s u l t i n g from damage to the c e l l membrane (Anand and Davis, I960; Plotz et a l , 1961). Sm mutants were found to exhibit the same i n i t i a l uptake o f streptomycin but f a i l e d to show a secondary uptake. This was interpreted as suggesting that resistance depended on an a l t e r a t i o n i n the membrane forming apparatus:? such that streptomycin could not damage the c e l l u l a r membrane (Anand et a l , i960). Analysis of the phospholipid composition of 1 1 -Smr mutants of Escherichia c o l i B and S h i g e l l a sonnei revealed that the amount of a phospholipid generally i d e n t i f i e d as a bis-phosphatidic acid was very much reduced;-(Yamagami e_t a l , 1 9 7 0 ) . Furthermore, Carlson arid Bockrath ( 1 9 7 0 ) found that streptomycin accumulation varied depending^ on whether the b a c t e r i a l c e l l s grew i n a glucose defined medium or i n nutrient broth. Other groups have suggested that strepto-mycin accumulation i n b a c t e r i a l c e l l s i s mediated by a perm-ease (Hurwitz and Rosano, 19&5; Andry and Bockrath, 197U). Growth conditions can influence streptomycin resistance. r For example, the number of spontaneous Sm mutants was shown to increase s i g n i f i c a n t l y i n Escherichia c o l i and V i b r i o  comma c e l l s grown under anaerobic conditions, plus or minus streptomycin, compared to aerobic conditions (Farkas-Himsley, 1 9 6 l ) . Furthermore, continued subculture of Salmonellae i n a chemically defined medium containing succinate as a carbon source led to an increase i n streptomycin resistance; sub-sequent subculture i n nutrient broth returned s e n s i t i v i t y (Voets, 1 9 6 9 ) . The e f f e c t of the growth medium on the s e n s i t i v i t y of a micro-organism to an a n t i b i o t i c has been well documented (Trainer, I96I4.). For example, increase i n the l i p i d content of the gram p o s i t i v e micro-organisms B a c i l l u s s u b t i l i s . Staphylococcus aureus and Streptococcus f a e c a l i s by growth on g l y c e r o l (termed 'fattened c e l l s ' ) , caused an increased resistance to p e n i c i l l i n s (Hugo and Franklin, 1 9 6 8 ) and to phenols (Hugo and Stretton, I 9 6 6 ) . Furthermore, i f the l i p i d • 1 2 -content was decreased, resistance decreased proportionally. The addition of streptomycin to Sm3 S e r r a t i a marcescens has been shown to cause s i g n i f i c a n t alterations i n l i p i d composition. Addition of 5 - 1 0 ug streptomycin/ml culture medium, caused the almost complete elimination of c y c l i c ' depsipeptides (li|. membered c y c l i c rings containing serine e s t e r i f i e d to C^Q and OH-fatty acids) while the t o t a l l i p i d phosphorous was increased more than two f o l d and was due to an increase i n phosphatidyl ethanolamine (Bermingham et a l , I 9 7 6 ) . As a r e s u l t , i t was suggested that low con-centrations of streptomycin may cause large alterations i n b a c t e r i a l l i p i d composition leading to changes i n the membrane and r e s u l t i n g i n an increased permeability to ions. Other findings also demonstrate that streptomycin may have an effect on l i p i d s . For example, the l i p i d content was doubled i n a Smr human s t r a i n of Mycobacterium tuberculosis (H^yR ) com-pared to a Sms s t r a i n (Chandraseckhar et a l , 1958) and, i n s a Sm avian s t r a i n , the streptomycin treated bacteria had a higher phospholipid and g l y c o l i p i d content than the control (Motomiya, I 9 6 0 ; Yamaguchi et a l , i960). Studies with Sm mutants have also indicated that streptomycin may af f e c t the b a c t e r i a l membrane. Laurence and Scruggs (I966) hypothesized that the synthesis of an altered enzyme (due to the presence of streptomycin) might r e s u l t i n a change i n the s t r u c t u r a l components of San and Sm c e l l s . An unusual compound was detected in the c e l l wall of Sm , Staphlococcus aureus ( 2 0 9 0 ) , B a c i l l u s cereus -13-and B a c i l l u s megaterium. It was characterized as having per mg - 1.1+5 juraole leucine equivalents, O.lj.2 jjmole reducing equivalents and 0.29 ymole glucosamine equivalents. Landman and Burchard (19&2) proposed that Sm^ c e l l s re-quired streptomycin for c e l l wall formation possibly by combining with the c e l l membrane and maintaining various functions. They observed that Sm Salmonellae paratyphi B c e l l s deprived of streptomycin formed long filaments. More-over, i f Sm c e l l s depleted of streptomycin were grown on soft agar, L-forms appeared analogous to the e f f e c t of pen-s i c i l l i n i n Sm c e l l s . The L-forms required streptomycin for growth but at a much reduced concentration i e . 150-300 yg streptomycin/ml compared to the parent which required 2000 ug/ml. Thus, although the more recent investigations of strep-tomycin action are concerned with a ribosomal s i t e of action for the a n t i b i o t i c , there i s a considerable body of informa-t i o n which supports, i n addition, an ef f e c t of streptomycin at the l e v e l of the membrane. I l l Genetics The three responses of bacteria towards streptomycin -s e n s i t i v i t y , resistance, and dependence, although phenotypi-c a l l y d i s t i n c t , are genotypically relat e d . Genetic analysis of these responses has been carried out i n d e t a i l ; i t appears that s e n s i t i v i t y , dependence, and single-step resistance are point mutations determined by multiple a l l e l e s of a single genetic locus (Hashimoto, I960) mapping near malt A (Newcombe -14-and Nyholm, 1950). This locus i s referred to as the Str A locus and has been shown to code f o r the S12 (P10) ribosomal protein (Traub and Nomura, I968) which contains approximately 200 residues (Craven et a l , 19&9). T ° date, no Smr mutants resis t a n t to d high l e v e l of streptomycin or Sm mutants of Escherichia c o l l are known which are due to alterations i n proteins other than S12. Isono (197ir)» however, has isolat e d mutants of B a c i l l u s  stearothemophilus p a r t i a l l y sensitive to or dependent on streptomycin which have an altered S5 ribosomal protein. Genetic analysis of Smr mutants has distinguished four classes. Compared to the wild-type (Str A +) a l l e l e , these four classes were ordered with respect to increasing incom-petence f o r phenotypic suppression (Breckenridge and Gorini, 1970), The res i s t a n t mutations were found to map at two sit e s i n the Str A cistro n , one group of mutants mapped at one locus while the remaining three groups mapped at a second. Low l e v e l resistance i e 25 u g streptomycin/ml or less i s defined outside the Str A locus by an episomal transfer?-able factor which codes f o r an enzyme capable of detoxifying streptomycin (Rosenkranz, 19&k', Gund ersen, 1963)* Enzymes have been described which destroy the b i o l o g i c a l a c t i v i t y of streptomycin through adenylation (Harwood and Smith, 1969) or through phosphorylation (Ozanne et a l , I969). Both g alterations prevent;streptomycin :from.~binding to Sm ribosomes (Benveniste and Davies, 1973). Since i t has also been postu-lated that a permease f a c i l i t a t e s the entry of streptomycin -15-into b a c t e r i a l c e l l s (Hurwitz and Rosano, 1965; Andry and Bockrath, 19710, another type or resistance could obviously aris e by mutation of t h i s enzyme protein to a nonfunctional protein. Most Smd mutants are more correc t l y referred to as 'drug dependent' based on the findings of Gorini et a l (1967) that these mutants would grow on 3^*ethanol or 200 P g par-omomycin (Pm)/ml i n place of streptomycin. A l l of these drugs were shown to be capable of causing misreading i n v i t r o and phenotypic suppression In vivo and thus, i t was postulated tha^t they f u l f i l l e d the same function i n the Sm^ c e l l . d It i s also possible to d i s t i n g u i s h four classes of Sm mutants (Momose and Gori n i , 1971). They d i f f e r widely;!,in (i) t h e i r dependence on Sm, Pm, and ethanol, ( i i ) th e i r re-sistance to three drugs: Smf Pm, and Kanamycin, ( i i i ) t h e i r r e v e r s i b i l i t y to drug-independence through secondary mutations external to the Str A locus, and (iv) the l e v e l of r e s t r i c t t i o n imposed on phenotypic suppression. Genetic analysis has revealed three classes of Sm mutants: Drug , Sm Et , d and Sm (Momose and Gorini, 1971). These r e s u l t s , as well as the results with Smr mutants i e . the lack of phenotypic homogeneity, contradict the commonly held assumption that the Str A gene product i s s p e c i f i c a l l y the target of streptomycin action (Momose and Gorin i , 1971). IV Streptomycin S e n s i t i v i t y Streptomycin s e n s i t i v i t y had i n i t i a l l y , as previously -16-mentioned, been attributed to gross misreading of RNA code-words i n the presence of streptomycin (Davies et; a l , 1965a). It i s now thought, however, that the s e n s i t i v i t y r e s u l t s from the i n h i b i t i o n of protein synthesis (Modolell and Davis, 1969a). The question of the dominance of Sm over Sm strains (Lighthown, 1957) has been explained by the proposal that streptomycin interferes with chain termination or i n i t i a t i o n i e . only one SmS ribosome on each polysome would then be necessary to i n h i b i t the t r a n s l a t i o n process i n the presence of streptomycin. Although streptomycin i s l e t h a l to Sm c e l l s , low l e v e l s (-3~h ug/*nl culture medium) have been shown to cause pheno-typic suppression of c e r t a i n mutations. For example, Gorini and Kataja (1965) have described a Sms amino acid auxotroph/. of Escherichia c o l i which l o s t ^ i t s auxotrophy i n the presence of sublethal concentrations of streptomycin. It was postulat that streptomycin activated a suppression mechanism which functioned to repair phenotypically, mutations i n a wide vari e t y of c i s t r o n s . Streptomycin has also been shown to cause suppression of amber and ochre r l l mutants of phage T1+ i n a Sm Su s t r a i n of Escherichia c o l i (Orias and Gartner, 1966). Kirschmann and Davis (1969) observed that phenotypic suppression i n a n t i b i o t i c sensitive Escherichia c o l i was not a property peculiar to streptomycin; sublethal concen-trations of chloramphenicol, t e t r a c y c l i n , erythromycin, and - 1 7 -spectinoraycin (reversible i n h i b i t o r s of the ribosome) were shown to cause suppression. It was suggested, therefore, that phenotypic suppression by borderline concentrations of ribosomal i n h i b i t o r s need not necessarily depend on an a l t e r -ation of the recognition region of the ribosome i e . the streptomycin sensitive s i t e . Rather, i t might be explained by a p a r t i a l i n h i b i t i o n of the ribosome r e s u l t i n g i n a changed environment i n a manner that would influence the frequency of misreading. V Streptomycin Resistance High l e v e l Smr mutants display a variety of phenotypes. They d i f f e r widely i n t h e i r a b i l i t y to cause phenotypic sup-pression (Breckenridge and Gorini, 1970) which i s generally i n f e r i o r to the a b i l i t y of streptomycin to cause suppression in the Sms parent (Gorini and Kataja, 1965). The re s i s t a n t mutants are characterized most c l e a r l y by the properties of the r e s i s t a n t ribosome. These ribosomes do not bind streptomycin i n contrast to the ribosomes of s the wild-type Sms parent ( K a j i and Tanaka, 1 9 6 8 ) . Sm and P Sm ribosomes can also be distingiushed on the basis of ' several physical properties (Leon and Brock, 1 9 6 7 ; Wolfe and Hayne, 1 9 6 8 ) . Cross resistance between a n t i b i o t i c s has not been dem-onstrated i n Smr c e l l s . Thus, while Smd strains can grow on paromomycin or neamine, Smr strains are sensitive to these a n t i b i o t i c s (Gorini et_ a l , 1 9 6 7 ; Szybalski and Cocito-Vandermeulen, 1 9 5 8 ) . -18-VI Streptomycin Dependency Sm c e l l s have been considered to constitute a class of resi s t a n t s . However, they are d i s t i n c t i n that cross de-pendency i n Sm c e l l s is well documented (Gorini et a l , 1967). For example, Gado and Horvath (1963), using Escherichia c o l i , reported that several organic solvents could be used as sub-cl s t i t u t e s f o r streptomycin i n Sm c e l l s i e . methanol, i s o -propanol, ethanol and acetone. Streptomycin i s reportedly required i n Smd c e l l s to maintain normal protein synthesis. In v i t r o studies with ribosomes from starved Sm c e l l s revealed that low conceh-. cl trations of streptomycin (10""5M) could maintain Sm ribosomes as 70s p a r t i c l e s i n 10~3M magnesium r e s u l t i n g i n i n v i t r o protein synthesizing a c t i v i t y with poly U or bacteriophage MS2 RNA (Davies, 196b,). Under assay conditions i n which the divalent cation content was comprised of both magnesium and calcium, Likover and Kurland (1967a) demonstrated that optimal cl polypeptide synthesis i n v i t r o on Sm ribosomes was substan-t i a l l y dependent on low concentrations of streptomycin; this requirement was shown to be related to the 30s subunit of ci. Sm strains and could not be s a t i s f i e d by a related amino-glycoside, such as neomycin. However, i n Smd c e l l s , special conditions such as the use of starved Smd c e l l s (Davies, 196ij.) or incubation mixtures containing calcium (Likover and Kurland, 1967a) must be employed i n order to demonstrate a requirement for streptomycin for i n v i t r o polypeptide synthesis. - 1 9 -VII Physiology and Biochemistry of SmQ Escherichia c o l i Soon afte r the discovery of streptomycin (Schatz _et a l , I9I4.U), the f i r s t Sm organism was iso l a t e d ( M i l l e r and Bohnhoff, 191+7). Subsequently, many attempts have been made to define the nature of streptomycin dependency. As a means of more c l e a r l y understanding this dependency, the physiology and biochemistry of Sm organisms i s reviewed with p a r t i c u l a r ci attention to Sm Escherichia c o l i . The concentration of streptomycin required to permit a maximal growth rate varies amongst dependent st r a i n s . How-ever, the concentration i s generally i n the order of several hundred ug/ml growth medium. At concentrations below t h i s c r i t i c a l l e v e l , the growth rate of the Sm culture is a d i r e c t function of the concentration of streptomycin In the medium (Paine and Finland, 1914-8; Schaeffer, 1 9 5 0 ; Spotts, 1 9 6 2 ; Coukell and Polglase, 1 9 6 9 a ) . If streptomycin i s re-moved from the medium, c e l l d i v i s i o n ceases, growth becomes arithmetic^,.,v nuclear d i v i s i o n continues and the organisms form longffilaments (Delaport, 191+9; Demerec e_t a l , 191+9; Schaeffer, 1 9 5 0 ) . I n i t i a l attempts to show the i n t r a c e l l u l a r accumulation of streptomycin f a i l e d due to the fact that growth of Sm organisms does not appreciably diminish the content of strep-tomycin i n the medium (Ruben and St^inglass, 1 9 5 1 ) . However, Engelberg and Artman (I96I, x) were able to demonstrate the Xlx cl uptake of ^"C-labelled streptomycin by a Sm mutant of Escherichia c o l i . Subsequently, Spotts ( 1 9 6 2 ) estimated that - 2 0 -d a Sm organism grown with optimal concentrations of strep-tomycin i r r e v e r s i b l y bound approximately 2 5 0 , 0 0 0 molecules of streptomycin. F i t z g e r a l d et a l ( 1 9 U & )» i n studies using Sm mycobacteria demonstrated that streptomycin i n h i b i t e d adaptive enzyme formation. Polglase confirmed these findings i n studies with Sm3 and Smd Escherichia c o l i (Roote and Polglase, 1 9 5 5 ; Polglase, 1 9 5 6 ; Polglase ejb a l , 1 9 5 6 ; Peretz and Polglase, 1 9 5 7 ) . It was suggested that the process i n h i b i t e d i n Sm organisms and requiring streptomycin i n Sm^ organisms was the synthesis of inducible enzymes (Peretz and Polglase, 1 9 5 7 ) . Incubation of Sm^ organisms i n culture medium lacking strep-tomycin does not a f f e c t DNA synthesis or function; however, the t o t a l RNA content of the c e l l s i s greatly increased and the synthesis of some, but not a l l , enzymes Is arrested or decreased (Spotts and Stanier, 1 9 6 1 ) . This led Spotts and Stanier to propose that the s i t e of streptomycin action i n the c e l l s was at the l e v e l of the ribosome. However, since the d i f f e r e n t i a l effects on enzyme synthesis observed during growth of the c e l l s i n the absence of streptomycin was assumed to be random, i t i s d i f f i c u l t to explain why the synthesis of only s p e c i f i c enzymes i s affected. The hypothesis of Spotts and Stanier (1961) implied that r d Sm and Sm mutants should employ the same metabolic path-s ways as Sm organisms when grown under optimal conditions. This was questioned by Bragg and Polglase ( 1 9 6 2 ) when i t was observed that Sm organisms excreted quantities of valine 2 1 -into the medium far i n excess of that observed with the Sms parent or a Srar/mutant (Bragg and Polglase, 1 9 6 2 ; Tirunarayanan et a l , 1 9 6 2 ) . The excretion of valine by Sm Escherichia c o l i was estimated to account for 10% of the glucose carbon (Bragg and Polglase, 1 9 6 2 ) . Moreover, the addition of streptomycin to Sm3 Escherichia c o l i growing on glucose was shown to markedly af f e c t the excretion of extra-c e l l u l a r metabolites i e . i n place of pyruvate, valine was excreted. Smr Escherichia c o l i produced l a c t i c acid from glucose when grown i n a n t i b i o t i c supplemented medium but f a i l e d to do so i n the absence of the a n t i b i o t i c . cl The formation of valine by Sm mutants was explained as representing a neutral end product of an aerobic pathway of glucose metabolism; during anaerobic growth or growth i n the absence of streptomycin, these c e l l s produced l a c t i c acid and alanine. Hence, i t was suggested that valine and l a c t i c acid were alternative secondary products of glucose metabolism (Bragg and Polglase, 1961+). Subsequent studies revealed that the l e v e l of aceto-hydroxy acid synthase EC I ) . . 1 . 3 . 1 8 (AHAS), the enzyme control-l i n g the f i r s t step i n valine biosynthesis, was enhanced i n d s a n t i b i o t i c supplemented Sm Escherichia c o l i compared to Sm or a n t i b i o t i c limited Smd c e l l s . The feedback control of valine biosynthesis through end-product i n h i b i t i o n of AHAS by valine (Umbarger and Brown, 1 9 5 8 ) was normal and, there-fore, the enhanced l e v e l of enzyme was explained as a dere-pression of enzyme synthesis i n the presence of the a n t i -2 2 -b i o t i c (Bragg and Polglase, 1965; Coukell and Polglase, 1965) . A s i m i l a r finding was reported by Polglase (1965;:^) i n a Smd Escherichia c o l i K12. Escherichia c o l i K12 i s d i s t i n c t from other strains of Escherichia c o l i i n that i t s growth i s inh i b i t e d by the presence of L-valine (10 M) i n the growth medium (Bonner, I9I4.6). This was explained by Temple et a l (1965) by the increased s e n s i t i v i t y of AHAS i n K12 to i n h i b i -t i o n by L-valine. Since AHAS also catalyzes the second step i n isoleucine biosynthesis, i n h i b i t i o n of this a c t i v i t y leads to a s i t u a t i o n of 'isoleucine starvation* within the c e l l s r e s u l t i n g i n in h i b i t e d growth. Polglase (1965 ), however, found that the growth of a Sm Escherichia c o l i K12 was valine i n s e n s i t i v e despite a normal s e n s i t i v i t y of AHAS to valine. The observed i n s e n s i t i v i t y was explained as a con-sequence of a derepression of AHAS synthesis. Moreover, i t was found that when suboptimal l e v e l s of the a n t i b i o t i c were employed, this derepression was a function of the streptomycin concentration i n the growth medium. Polglase (1966a) also established that, l i k e the Sm3 parent, AHAS was controlled by multivalent end-product re-pression i n several strains of Sm Escherichia c o l i . Max-imum repression was observed only with growth of the organims i n the presence of L-valine, L-leucine, L-isoleucine and pan-tothenate. In some cases a repression was observed with L-valine alone but this effect varied with the s t r a i n . Extention of these studies to wild-type Sms Escherichia  c o l i B revealed that catabolite repression was also a mechan-23-ism by which AHAS was controlled i n this s t r a i n (Coukell and Polglase, 1969b). Hence, th i s work had uncovered a new form of AHAS reg-u l a t i o n . In l i g h t of t h i s , a more detailed analysis of the control of AHAS a c t i v i t y i s warranted and w i l l be dealt with i n a separate section. Analysis of a culture f i l t r a t e of a Sm Escherichia  c o l i B/r showed that i n spite of an enhanced AHAS a c t i v i t y and hence acetohydroxybutyrate formation, isoleucine was not excreted by these c e l l s (Coukell and Polglase, 1966). It was suggested, therefore, that i n these mutants another control s i t e repressed isoleucine formation. Further analysis of enzymes i n the isoleucine pathway i n Escherichia c o l i K12 revealed that the f i r s t enzyme i n the pathway of isoleucine biosynthesis, threonine dehydratase (EC I4..2.1.16) also known as threonine deaminase, was derepressed i n Smd mutants; the properties of the enzyme were not s i g n i f i c a n t l y d i f f e r e n t from those of the enzvme from the wild- type Sm° parent (Desai and Polglase, 1966a, 1967a, 1967b). Desai-? and Polglase (1966b) extended t h e i r studies to Escherichia  c o l i K12 and found a low l e v e l of threonine dehydratase a c t i v i t y ; i t was suggested that this contributed to the valine s e n s i t i v i t y of t h i s s t r a i n . The synthesis of catabolic enzymes i n bacteria has been shown to be repressed when the c e l l s are grown on an energy r i c h carbon source such as glucose. Magasanik (1961) coined the phrase 'catabolite repression' i n reference to this form -21+-of control. Coukell and Polglase (1969b) studied several d catabolite s e n s i t i v e enzymes i n Sm mutants of Escherichia  c o l i B and Escherichia c o l l E i e . fumarase, c i t r a t e synthase, aconitase, i s o c i t r a t e dehydrogenase and beta galactosidase. They established that beta galactosidase synthesis could be induced by the gratuitous inducer IPTG during growth of the c e l l s on glucose and further, that a l l the catabolite sen-s i t i v e enzymes studied were derepressed i n Smd c e l l s i r r e -spective of carbon source; the l e v e l of the glucose insen-s i t i v e enzymes glucokinase and glucose 6-phosphate dehydrog-enase (G6PDH) were comparable to the wild-type Sms parent. Coukell and Polglase (1969b) also reported that the c e l l y i e l d , defined as the milligram dry weight of c e l l s produced per micro mole of carbon source consumed.,/ was re-d duced by one-third i n Sm c e l l s during aerobic growth of the c e l l s on glucose compared to the Sm3 parent. This suggested that the decreased e f f i c i e n c y of aerobic glucose u t i l i z a t i o n i n Smd c e l l s was responsible for the observed relaxation of catabolite repression. While aerobic u t i l i z a t i o n of glucose appeared to be impaired, the anaerobic u t i l i z a t i o n of glucose was found to be s i m i l a r i n Sm^ and Sm3 c e l l s (Kamitakahara and Polglase, 1970). Several glucose sensitive enzymes were shown to be d s p e c i f i c a l l y repressed i n Sm c e l l s grown under conditions of a n t i b i o t i c l i m i t a t i o n Ie. less than 100u g streptomycin/ ml of growth medium (Coukell and Polglase, 1969a). Strep-tomycin supplementation of these a n t i b i o t i c starved c e l l s re--25-sulted i n a rapid derepression of catabolite sensitive enzymes. Moreover, i t was found that the half-maximal growth rate of Sm mutants on l i m i t i n g concentrations of dlhydro-streptoraycin C^DJJSM^ * a r e d u c e d analogue of streptomycin, varied with the nature of the carbon source. The highest KDHSM was found with the richest energy y i e l d i n g carbon sources i e gluconate glucose glycer o l l a c t a t e . An anomo-lous but s i g n i f i c a n t finding was that Smd Escherichia c o l i B exhibited normal catabolite repression when grown with glucon-ate as the carbon source. The statement was made at this time that 'catabolite repression may play an important role i n establishing the quantitative requirement for the a n t i b i o t i c f o r growth of Sm Escherichia c o l l ' (Coukell and Polglase, 19&9a). Moreover, i n keeping with the hypothesis that streptomycin affects protein synthesis, i t was suggested that ' in vivo the primary e f f e c t of the a n t i b i o t i c on protein synthesis i s augmented by the secondary e f f e c t on catabolite repression.' VIII Respiration i n Smd C e l l s Evidence has accumulated over the past few years which has been interpreted as suggesting that streptomycin may (3. play a role i n the electron transport system i n Sm Escherichia  c o l i . Umbreit (19^9) proposed that streptomycin i n h i b i t e d pyruvate and other keto acids from entering the terminal r e s p i r a t i o n system by i n h i b i t i n g the condensation of pyruvate to oxaloacetate. It was suggested that Smd and Smr c e l l s did -26-not possess the a b i l i t y to effect this condensation and were, therefore, not sensitive to streptomycin. However, t h i s point could not be proven conclusively (Smith et a l , 19U9). Later work reinforced the hypothesis that pyruvate metabolism was the s i t e of streptomycin action (Umbreit, 1 9 5 3 ; Barkulis, 1 9 5 3 ; Rosanoff and Sevag, 1 9 5 3 ) * Schaeffer ( 1 9 5 2 ) , using Sm B a c i l l u s cereus, reported that streptomycin was necessary f o r the synthesis of cyto-chromes and some porphyrins. For example, i t was observed that coproporphyrin was excreted into the medium of Sm c e l l s grown anaerobically i n the presence or absence of streptomycin. Engelberg and Artman (1961 ) confirmed these results and -showed that c e l l s grown with suboptimal concentrations of streptomycin had a lower l e v e l of oxidase enzymes. In a series of a r t i c l e s , Bragg and Polglase supplied further evidence i n support of terminal r e s p i r a t i o n as the s i t e of streptomycin action. They observed that streptomycin and i n h i b i t o r s of terminal r e s p i r a t i o n such as cyanide, azide, and amobarbital, had a si m i l a r e f f e c t on the metabolism of Sms c e l l s (Bragg and Polglase, 1 9 6 2 ) . Furthermore, strepto-mycin was shown to Inhibit i n v i t r o a pathway of electron transport i e . the succinate-triphenyltetrazolium chloride (TTC) reductase system, i n extracts of c e l l s grown i n the absence of the a n t i b i o t i c - Sms and Smr but not i n those grown i n i t s presence - Smr and Smd^(Bragg and Polglase,1963. ). Later, Kamitakahara and Polglase ( 1 9 7 0 ) reported that, s d i n contrast to the Sm parent, Sm Escherichia c o l i B were - 2 7 -d e f i c i e n t i n a pigment absorbing at 5 0 3 nanometers i n a d i f -ference spectrum of whole c e l l s . On the basis of th e i r i n -vestigation, they speculated that this pigment was involved i n an oxidative energy-yielding pathway i n Escherichia c o l i which used NADPH as an i n i t i a l substrate. Hence, i t was sug-gested that the deficiency i n thi s pigment and the impaired aerobic energy metabolism i n Sm strains of Escherichia c o l i were related phenomena. Poulson and Polglase ( 1 9 7 3 ) , have since i d e n t i f i e d the 5 0 3 pigment as prototetrahydroporphyrin IX, a compoundcapable of being oxidised to protoporphyrin IX, a precursor i n the biosynthesis of cytochromes. Pig. 1. To date, most of the research e f f o r t directed into un-raveling; the s i t e of streptomycin action has been centered on the use of Smr and Sms organisms. However, the study of the nature of dependency i n Sm c e l l s should ultimately define the l e t h a l action of thi s and perhaps related aminoglycoside a n t i b i o t i c s i n Sm3 c e l l s . The importance of the study of Smd mutants was remarked upon by Spotts ( 1 9 & 2 ) who wrote the ^ •analysis of the r e l a t i v e l y neglected, bizarre and c l i n i c a l l y unimportant phenomenon of dependence has an equal i n t r i n i s i c p r o b a b i l i t y of furnishing clues to the mechanism of action (of streptomycin) i n the b a c t e r i a l c e l l . ' IX Control.:'of Acetohydroxy Acid Synthase i n Bacteria Acetohydroxy acid synthase (AHAS) i s an enzyme shared by the common pathways of L-isoleucine and L-valine biosynthe-s i s . With pyruvate a s t he substrate, I t catalyzes the form-ation of acetolactate; with alpha-Ketotoutyrate as the substrate, - 2 8 -Fig. 1 Physiological Significance of P503 glycine succinate aminolevulinic acid porphob11inogen uroporphyrinogen I I I auto-oxidation prototetra-hydroporphyrin IX (P503) coproporphyrinogen III Coproporphyrinogenas e .protoporphyrinogen IX "Protoporphyrinogen oxidase protoporphyrin IX Fe cytochrome Based on the findings of Poulson and Polglase (1973) -29-i t catalyzes the formation of alpha-aceto-alpha-hydroxy-butyrate (Strassman et a l , 1 9 5 3 , 1 9 5 5 ; Strassman e_t a l , 1 9 5 ^ , 1 9 5 6 ) . The two pathways also have the subsequent enzymes i n common - reductoisomerase ( i i ) , dihydroxy dehydrase ( i i i ) , and transaminase B ( i v ) . F i g . 2 . The regulatory enzyme i n valine biosynthesis.^ AHAS, i s under feedback control by the end-product L-valine (Umbarger and Brown, 1 9 5 8 ) . S i m i l a r l y , L-isoleucine biosynthesis i s controlled by threonine deaminase which i s under feedback control by the end-product L-isoleucine (Umbarger, 1 9 5 6 ) . The formation of four of the f i v e i l e - v a l enzymes i i control-led by multivalentrepression (Freundlich et a l , 1 9 6 2 ) while the remaining enzyme, reductoisomerase, i s induced by the product of AHAS (Arfin et a l , 1 9 6 9 ) . The f i v e enzymes of the i l v a pathway map i n the same region of the b a c t e r i a l chromosome and constitute the ' i l v a ' operon (Ramakrishnan and Adelberg, 1 9 6 5 a ) . There i s evidence for the existence of two forms of AHAS.-The a c t i v i t y required f o r valine formation i s in h i b i t e d by L-valine (Umbarger and Brown, 1 9 5 8 ) , while the second AHAS a c t i v i t y i s in s e n s i t i v e to feedback i n h i b i t i o n . The presence of the second a c t i v i t y has been established i n Salmonella  typhimurium and Escherichia c o l i (Blatt et a l , 1 9 7 2 ; O'Neill and Freundlich, 1972) and i t appears to be involved i n is o -leucine biosynthesis. The two forms of AHAS i e . the valine sensitive and the valine i n s e n s i t i v e forms have been designated AHAS I and t h r e o n i n e i cc- keto — butyrate pyruvate pyruvate a-aceto -a -hydroxy-butyrate — a - ace to -l a c t a t e . I Acetohydroxy A c i d Synthase I I Reducto isomerase I I I Dihydroxy Dehydrase IV Transaminase B 11 a , £- d ihydroxy -0- methy I -v a l e r a t e — a - k e t o -methy l --> va lera te L - i s o l e u c i n e I I I IV c t , £ - d i h y d r o x y -^ i s o v a l e r a t e L-va l ine L—leuc ine (X-keto -pantoate pantoate I pantothenate i O I Fig. 2. The biosynthetic pathway to isoleucine, valine, leucine and pantothenate in Escherichia coli. -31-AHAS II respectively (Blatt et a l , 1972; O'Neill and Freundlich, 1972). AHAS II synthesis i s reportedly under the control of multivalent repression by L-valine, L-leucine, and L-isoleucine whereas the synthesis of AHAS I i s repressed only by L-valine and L-leucine. Previous reports have estab-l i s h e d that pantothenate, a fourth product of the i l v a path-way, also contributes to the repression of AHAS; however, the importance of i t s r o l e i n the regulation of this enzyme i s not known (Freundlich and Umbarger, 1963). The finding that the growth of Escherichia c o l i K12 i s inh i b i t e d by L-valine, i s explained by the absence i n this s t r a i n of the feedback i n s e n s i t i v e form of AHAS - AHAS I I . O'Neill and Freundlich (1973) have shown that these two forms of AHAS are coded fo r by d i f f e r e n t genes i n the i l v a operon. It has been suggested that the immature form of threonine deaminase bound to val-tRNA, leu-tRNA or ile-tRNA represses the i l v a pathway (Calhoun and H a t f i e l d , 1973). This i s sup-ported by the finding that c e l l s grown i n the presence of the three branch chain amino acids and under conditions which re-s t r i c t the formation of t h i s enzyme, have a derepressed AHAS and dihydroxy acid reductase. K l i n e et a l (1971+), however, established that although threonine deaminase may be involved i n a repression mechanism when i t i s present, i t i s hot essential for the multivalent regulation of the i l v a gene cl u s t e r . The presence of a single AHAS a c t i v i t y i n Escherichia  c o l i K12 strains has been questioned by O'Neill and Freundlich -32-on the basis of a number of observations. For example, Ramakrishnan and Adelberg (1965b) isolated an i l v ADE operon 0 C mutant of Escherichia c o l i K12 which displayed growth i n -_2 s e n s i t i v i t y to high concentrations of L-valine (10 M). More-over, starvation of an ile-auxotroph of Escherichia c o l i K12 for isoleucine allowed the formation of residual amounts of an AHAS which was not in h i b i t e d by L-valine (O'Neill and Freundlich, 1972). The maximum i n h i b i t i o n of AHAS by L-valine reported i n Es c h e r i c h i a ' c o l i K12 i s of the order of Q0% (O'Neill and Freundlich, 1973); however, the degree of i n h i b i t i o n i s de-pendent upon the concentration of pyruvate i n the assay mix-ture (Umbarger and Brown, 1958). I n h i b i t i o n of AHAS by valine i n strains such as Escherichia c o l i B which exhibit valine-i n s e n s i t i v e growth, i s approximately 60$. -33-Research Aims Studies were undertaken to characterize Smd Escherichia  c o l i K12. Previously, studies with Sm Escherichia c o l i had focused on a single Sm mutant of the various strains of Escherichia c o l l . However, i n order t o establish the exis-tence of a general phenotype, i t was considered necessary to i s o l a t e several Smd mutants of the same s t r a i n , i n this case Escherichia c o l l K12. As a basis f o r this study, the findings of previous studies i n this lab were extended i e . i t had previously been observed that Smd Escherichia c o l l mutants exhibited: (i ) derepressed catabolite sensitive enzymes ( i i ) valine excretion during growth on glucose ("iii) lack of In h i b i t i o n of growth by L-valine i n Sm Escherichia c o l l K12 (iv) low l e v e l s of a pigment, absorbing at 5>03 nanometers, which has been i d e n t i f i e d as prototetrahydroporphyrin IX (v) v a r i a t i o n i n the DHSM requirement depedding on the carbon source Having thus obtained a general understanding of the phenotype of Sm Escherichia c o l i K12, i t was hoped that this would serve 'as a basis for experiments which would lead to a better understanding of the nature of streptomycin dependency. -3U-MBTHODS AND MATERIALS I Organisms (i) Nomenclature used i n describing the response of c e l l s to  streptomycin a) Wild-type Escherichia c o l i c e l l s are sensitive to streptomycin concentrations greater than 10 Ug/ml growth medium. These are described by the notation Sms. b) Streptomycin-resistant Escherichia c o l i grow either in the presence or the absence of streptomycin and are de-r scribed by the notation Sm . c) Streptomycin-dependent Escherichia c o l i require streptomycin for growth and are described by the notation Smd. ( i i ) Maintenance of stock cultures Cultures were maintained on agar plates containing a basal s a l t s medium and 0,k% glucose and in 20 ml test tubes c i containing 5 ml of the same glucose-salts medium. Sm mutants were grown in the presence of 1000 ug dihydrostreptomycin/ml of growth medium. Transfers were made at approximately two week intervals and the dependency of Smd mutants on dihydro-streptomycin was checked with each transfer by testing growth i n the presence and absence of the a n t i b i o t i c . Cultures were grown without shaking at 3 7 ' C and stored under refrigerated conditions. ( i i i ) I s o l a t i o n of Sm Escherichia c o l i K12 mutants Spontaneous Sm mutants were i s o l a t e d from a wild-type Escherichia c o l i K12 culture as follows: 100 ml of an - 3 5 -Escherichia c o l i K12 culture were harvested a s e p t i c a l l y during log phase growth on a glucose-salts medium. The c e l l p e l l e t was resuspended i n 2ml of s t e r i l i z e d isotonic saline and 0.2ml of this suspension was spread, using a glass spreader, over the surface of a heart infusion broth agar plate contain-ing 1000 yg dihydrostreptomycin (DHSM)/ml. After 21+ hours incubation at 37'C, mutants either resistant to or dependent on DHSM could be detected. The ind i v i d u a l colonies were picked o f f the plates and tested for dependency on DHSM by demonstrating a requirement for the a n t i b i o t i c during growth on glucose. Dependency was tested several times to insure that the Sm mutants were stable. II Growth of Cultures and Preparation of C e l l Extracts (i) Media The Davis-Mingioli ( 1 9 5 0 ) medium minus c i t r a t e was used throughout these experiments. I t contained: K^HPO^ ( 0 . 7 $ ) , K E 2 V 0 k ( G » 3 $ ) , (NH^) 2S0^ (0.1%), MgS0^-7H20 (0.02$). The f i n a l pH was adjusted to 7.0. Due to i t s greater s t a b i l i t y , the reduced analogue-of streptomycin l e . dihydrostreptomycin (DHSM) was used through-out these studies for the growth of Smd mutants. For the optimum growth of these mutants, DHSM was f i l t e r s t e r i l i z e d and added to the medium to give a f i n a l concentration of 1000yg/ml. Carbon sources were autoclaved separately and added to the medium as required. In certain experiments, the medium was supplemented with -36-a metal ion solut i o n . The stock solution contained per 100 ml: The metal ion solution was used at a concentration of 1 m l / l i t r e of medium. ( i i ) Estimation of growth The growth of a culture was monitored by measuring t u r b i d i t y at 1+20 nanometers using a Beckman Model B spectro-photometer. ( i i i ) Procedures f o r growing, and harvesting cultures (a) general growth conditions Cultures were grown at 37'C i n a New Brunswick reciprocal shaking water bath set at approximately 200 rpm. Where more vigorous aeration was required, culture flasks were f i t t e d with rubber stoppers containing two lengths, of glass tubing and a i r saturated with water was bubbled through the flask. I t was found that evaporation could be minimized i f the a i r was bubbled through water before entering and after leaving the f l a s k . (b) growth of a n t i b i o t i c - l i m i t e d Sm^ Escherichia c o l i Smd c e l l s were grown without shaking on 0,il+$ glucose-salts medium ,'Containing 30 y g DHSM/ml at 37'C. Growth was turbid af t e r an incubation period of 21+-1+8 hours. How-ever, several transfers were made before the c e l l s were con-FeSQh.7H20 MnCl2.1+H20 CuSOh.5lH20 CoCl2*6H20 C i t r a t e Zn(acetate)p« 2H?0 500 mg 20 mg 25 mg 200 mg 15 mg 5 mg 10 rag 5 mg -37-sidered to be a n t i b i o t i c - l i m i t e d . Before use, the c e l l s were grown overnight at 37'C with shaking. (c) growth of Smd E. c o l i K12 c e l l s used i n the i n v e s t i -gation of multivalent end-product repression of AHAS Smd E. c o l l K12 cultures were grown at 37'C with shaking i n an 0,1$ glucose s a l t s medium containing 1 0 0 0 yg DHSM/ml, 10 " 3 M concentration of the L-amino acids leucine, isoleucine, and valine, and 10" alcium pantothenate. The following combination of end-products were used: i ) v a l i i i ) pan + i l e l i e pan + leu leu pan + val pan iv) val + leu + i l e i i ) val + i l e val + leu v) val + leu 4 i l e + pan i l e + leu Cultures were grown from an ODj^o o f 0 ml to 0.8 and harvested. (d) procedure for testing the growth of Smd E. c o l i cultures on paromomycin i n place of DHSM Since the simultaneous presence of DHSM and paromomycin i s reportedly l e t h a l to the Smd c e l l , cultures were pregrown on 0.3$ ethanol before they were tested for growth on paromomycin ( 2 0 0 ug/ml growth medium) (Gorini et a l , 1 9 6 7 ) . (e) growth of c e l l s for the assay of enzymes Log phase cultures were used f o r the preparation of c e l l extracts to be used i n enzyme assays. Cultures were started from a parent culture grown overnight under the appropriate growth conditions. Experimental cultures were inoculated to an i n i t i a l OD^o of 0*1 and harvested at an 0 D ^ 2 0 between 0.8-1,0... C e l l s were usually used immediately - 3 8 -for the assay of enzymes. However, for the assay of AHAS, the p e l l e t was often stored at O'C f o r up to 72 hours. I t has been established previously, that under these conditions, AHAS i s stable f o r approximately 6 days (Desai and Polglase, 1 9 6 5 ) . (f) adaptation of c e l l s to growth on acetate Ce l l s were adapted to growth on acetate by inoculating 15 ml of O.U-0.8$ acetate-salts medium contained i n 125 ml flasks shaking vigorously at 37'C. Turbid growth usually required no longer than 1+8 hours. However, several transfers were made before the c e l l s were considered to be adapted. (g) harvesting cultures Cultures were harvested by centrifuging at 9,000 rpm fo r 10 min i n a Sorval centrifuge. The c e l l p e l l e t was washed once with 0.1M potassium phosphate buffer pH 7.0 or isotonic s a l i n e . C e l l s to be used f o r the preparation of cell;-.extracts used i n the assay of enzymes, were suspended i n the appro-pr i a t e buffer. (iv) Preparation of c e l l extracts C e l l extracts were prepared by sonication using either a Bronson 20-K.C. sohicator for 30 seconds f o r preparation of 3.5-5.0 ml of extract or a Bronwill 20-K.C. sonicator for 1+ min for preparation of 12 ml or more of c e l l extract. A l l operations were carried out at l+'C. In general the c e l l extract was prepared using a concentration of 1 gram of c e l l s (wet weight)/l5 ml of buffer. -39-III Enzyme Assays i) Fumarase The assay contained i n 1 ml: 100 umole potassium phosphate buffer pH 7.2, 17.5 umole L-malate and crude c e l l extract. The blank was prepared by omitting L-malate from the assay mixture. The reaction was followed by measuring the increase i n absorbance at 2l(.0 nanometers on a Cary 15 recording spectrophotometer at 25'C* A c t i v i t y was expressed as units/mg protein where a unit i s defined as a change of 0.001 OD^o/min (Hanson and-'0ox, 1967). i i ) Aconitase The assay contained i n 1 ml: 100 umole potassium phosphate buffer pH 7.2, 15 yraole D,L-isocitrate and crude c e l l extract. The blank was prepared by omitting D,L-isocit-rate from the assay mixture. A c t i v i t y was measured at 25'C by an increase i n 0D at 2l|0 nanometers using a Qary 15 re-cording spectrophotometer and expressed as units/mg protein. A unit i s defined as a change of 0.001 0D2^Q/min (Hanson and Cox, 1967). i i i ) Glucose-6p Dehydrogenase The assay contained i n 1 ml: 100 umole gl y c y l g l y c i n e buffer pH 7.2, 5 umole glucose-6p, 10 umolle MgCl 2, 0,1k Umole NADP+ and crude c e l l extract. The blank was prepared by omitting glucose-6p from the assay mixtu^o. A c t i v i t y was measured by an increase i n 0D at 3U0 nanometers using a Cary 15 recording spectrophotometer at 25!C and expressed as units/mg protein. A unit i s defined as 1 mUmole of -1+0-NADPH formed per min. iv) Acetohydroxy Acid Synthase The assay contained i n 1 ml: 100 jamole T r i s buffer pH 8 . 0 , 0.25 mmoles sodium pyruvate, 0 .5 ymole MgCl 2, 0 . 3 umole thiamine pyrophosphate, 0.21+ ymole FAD* and crude c e l l extract. The blank was prepared by omitting pyruvate from the assay mixture. The reaction was started by the addition of c e l l extract and the mixture was incubated at 37'C for 15 min. The reaction was stopped jby the addition of 0 .1 ml of 1+0$ t r i c h l o r o a c e t i c acid and the product of the reaction, acetolactate, was converted to acetoin by incubation of the mixture at 6o'C f o r 15 min, Acetoin was then measured as follows: 0 .1 ml of the reaction mixture was dil u t e d i n 10 ml d i s t i l l e d water. One ml of t h i s d i l u t e d mixture was then incubated with 1 ml of creatine (5$ solu-tion i n water) and 1 ml of freshly prepared 5% alpha-napthol i n 2.5N sodium hydroxide. The solution was mixed vigorously and the colour was allowed to develop for 1 hour i n the dark. The r e s u l t i n g colour was read at 51+0 nanometers using a Gary 15 spectrophotometer at 25'C. Optical density was converted to ymole acetoin by the use of a standard curve and a c t i v i t y was expressed as ymole acetoin formed/ hr/mg protein. AHAS i s a very l a b i l e enzyme i n broken c e l l preparations and was, therefore, assayed only i n freshly prepared c e l l extracts (Desai and Polglase, 1965) . v) Beta-Galactosidase Beta galactosidase was assayed according to the modified -1+1-inethod of Pardee et a l (1959). To 1 ml aliquots of culture was added toluene (1 drop) and 2 mercaptoethanol ( f i n a l concentration l5mM). The c e l l s were then lysed by agitating this mixture vigorously for 30 seconds on a Vortex s t i r r e r followed by incubation at 37'C for 30 min. The contents of the tubes were then brought to 28'G and 0 .2 ml of 0.02M ortho-nitropheny 1-beta D-galacto-pyranoside (ONPG) in 0.25M phosphate buffer pH 7.0 was added. The incubation was then continued for a measured period of time u n t i l the desired colour i n t e n s i t y was pro-duced. The reaction was stopped by the addition of 0 .5 ml of IM sodium carbonate. The l i b e r a t e d ortho-nitrophenol was measured at 1+20 nanometers on a Cary 15 spectrophotometer at 25'C. The t u r b i d i t y of the c e l l s was corrected for by subtracting from this measurement, 1.6 times the absorption at 55,0 nanometers. Under these conditions 1 mufitiole of ortho-nitrophenol has an absorbance at 1+20 nanometers of 0.0075 (Pardee et a l , 1959) . A c t i v i t y was expressed as units/mg protein where a unit of a c t i v i t y i s defined as the amount of enzyme which catalyzes^the "hydrolysis of 1 ,<mumole of ortho:-»nitrophenyl-beta-D-galactopyranoside/min at 28 'C. IV Quantitative and Qualitative Analysis i ) Protein Protein was estimated using the method of Lowry et. a l (1951). Bovine gamma-globulin (Calbiochem. Los Angeles, C a l i f . , U.S.A.) was used as a standard. -1+2-i i ) Estimation of amino acid excretion by Escherichia c o l i  cultures Cultures were harvested and 50 ml aliquots of the c e l l - f r e e medium were evaporated to dryness at 1+0'C using a rotary vacuum evaporator. The residue was dissolved i n 1.5 ml of d i s t i l l e d water and the f l a s k was washed once with an additional 1.0 ml. The washing was added to the dissolved residue and the pH was adjusted with 6N HC1 to pH 2.0. The a c i d i f i e d sample was then placed on a Dowex H + AG 50 W-X12 (100-200 mesh) column 1 cm x 9 cm which had been washed previously with 15 ml of 0.1N HAc. The column was eluted with 10 ml of 0.1N HAc to remove s a l t s , followed by the elution of amino acids, In a band, with 0.1N NHj^ OH. The eluant was concentrated to dryness under vacuum and the residue was dissolved i n 2 ml of d i s t i l l e d water i f the sample was to be analyzed by paper chromatography, or i n 2 ml of 0.2N sodium c i t r a t e buffer pH 3.1+5 for analysis on a Technicon Amino Acid Analyzer.v Qualitative i d e n t i f i c a t i o n of amino acids i n the growth medium was c a r r i e d out using descending paper chromatography. One hundred ul of the sample was applied to a Whatman #1 chromatograra along with several amino acid standards. Two solvent systems were used to i d e n t i f y the amino acids: BuOH:HAc:H20 (1+:1:1) and EtOHzMH^OHrHgO (18:1:1). The chromatograms were developed overnight, dried and sprayed with a ninhydrin-acetone spray (0.2 g ninhydrin/l00 ml acetone) and heated for 2 min at 100'C to develop the colour. -1+3-i i i ) Determination of P503 (prototetrahydroporphyrin IX) C e l l s used for the determination of P 5 0 3 were grown from an ODj^o of 0.1 to 1.0 and harvested. The washed c e l l s were used d i r e c t l y for difference spectra analysis as follows: the c e l l p e l l e t was suspended i n 0.0f>M potassium phosphate buffer pH 7.0 at a concentration of 1 g c e l l s (wet weight)/l5 ml buffer. 2.3 ml of the c e l l suspension was added to both the experimental and reference cuvettes. In addition, the experimental cuvette contained 0.2 ml of a 20$ glucose solution and the reference cuvette con-tained 0.2 ml of 0.3$ H 202. The difference spectrum was taken on a Cary 15 recording spectrophotometer at 25'C using a scale of 0.1 0D. P 5 0 3 was I d e n t i f i e d as the peak absorbing at 5 0 3 nanometers. In order to determine the r e l a t i v e quantites of P 5 0 3 i n various c e l l extracts, the peak area was determined using the formulae 1/2 height x width. iv) Extraction of Escherichia c o l l Membrane Lipids and  Fatty Acid Analysis Five hundred mis of culture were grown from an 0D^20 of 0.1 to 1.0*;and harvested. Twenty mis of MeOH and 1+0 mis of CHCl^ were added to 1 gram wet weight of c e l l s and the r e s u l t i n g suspension was s t i r r e d with a magnetic s t i r r e r for 15 min and f i l t e r e d . Eight mis of 0.9$ NaCl was then added to the f i l t r a t e and the aqueous layer was removed by aspir-ation. The methanol phase was evaporated to dryness using a vacuum rotary evaporator^ - at 37'C „and the dry weight of the l i p i d residue was determined. -kk-The l i p i d s of the membrane were then subjected to methan-o l y s i s i n order to quantitate the f a t t y acid component by gas l i q u i d chromatography (GLC)• Methanolysis was carr i e d out as follows: the l i p i d residue was dissolved i n 1 ml of MeOH:CHCl3 (1:2) and an aliquot containing 500 micro-grams of l i p i d was dried under nitrogen. The residue was then dissolved i n 3 mis of IN HC1 i n MeOH and incubated overnight at 80'C i n a test tube containing a t e f l o n - l i n e d cap. Upon completion of the incubation, the e s t e r i f i e d f a t t y acids were extracted with 3 x 5 mis of heptane. The extract was dried under nitrogen immediately p r i o r to use and dissolved i n 100 m i c r o l i t r e s of heptane. Two micro-l i t r e s of this werei then analyzed by GLC. - l iS-PART A: Iso l a t i o n and Characterization of Sm Escherichia  c o l i K12 RESULTS I. I s o l a t i o n of Smd Escherichia c o l i K12 Twenty-three Smd Escherichia c o l i K12 mutants were iso l a t e d as spontaneous mutants and characterized i n the following studies. I I . Derepression of Catabolite Sensitive Enzymes During  the Growth of Smd Escherichia c o l i K12 on Glucose The a c t i v i t y of the catabolite sensitive enzyme, fumarase, was elevated i n a l l the twenty-three Smd mutants compared to the Sm wild -type parent, i e . an average of 3.2 f o l d higher. However, the a c t i v i t y of the catabolite i n -sensitive enzyme, glucose - 6 P dehydrogenase, was generally s l i g h t l y lower i e . 0 . 3 f o l d lower, i n these mutants (Table 1). II I . Analysis of P5>03 Levels i n Smd Escherichia c o l i d. Difference spectra of whole c e l l suspensions of Sm and Sms Escherichia c o l i K12 revealed that P503 (prototetra-hydroporphyrin IX) levels were marke.dlyldower i n the de-pendent s t r a i n (Fig. 3 ) . S i m i l a r l y , P503 l e v e l s were lower in a Sm Escherichia s c o l l B mutant compared to i t s Sm wild-type parent. However, when the Sm mutant of this s t r a i n was grown with gluconate as the carbon source, the P 5 0 3 l e v e l was increased to the wild-type l e v e l (Fig. If). The e f f e c t was, however, s p e c i f i c d d for Sm Escherichia c o l i B and was not observed with Sm -1+6-Table I: Fumarase and G6PDH a c t i v i t i e s i n Sm and Sms  E s c h e r i c h i a j c o l i K12. Exp't No. Strai n Fumarase*-Relative to Sms C-6PDH** Relative to Sm Sm sKl2 Sm dKl2 #1 #2 #3 #k #5 Sifi sKi2;:^ SmdK12 #6 #7 #8 #9 Sm3K12 SmdK12 #10 #11 #12 #13 #11+ #15 #16 SmsK12 SmdK12 #17 #18 #19 #20 #21 #22 #23 1,770 1+A90 5,560 l+,790 3,930 l+,050 950 3,500 2,930 2,990 l+,000 960 l+,020 2,990 3,830 2,580 1+,100 2,91+0 1|,250 890 3,o5o 2,500 2,700 2,500 2,030 3,360 2,390 2.51+ 3.11+ 2.71 2.22 2.29 3.69 3.08 3.15 1+.20 U-.19 3.12 3.99 2.69 1+.27 3.06 U.33 3.U3 2.81 3.03 2.81 2.28 3.78 2.69 96.8 57.7 73 .2 67 .0 79.6 59.7 79.7 81+.2 60.8 77 .3 91.6 97.3 61.1+ 61.9 66.1+ 66 .2 71.2 62.9 63.7 86 .0 57 .2 62.7 65.8 53.7 61.8 5U.2 1|9.0 0.60 0.76 O.69 0.82 0.62 1.06 0.76 0.97 1.15 O.63 0.61+ 0.68 0.68 0.73 0.65 0.66 0.67 0.73 0.77 0.62 0.72 O.63 0.57 •K-units/mg protein where a unit i s defined asA0D2j,Q= 0.00l/min -«"*millimicromoles NADPH/min Average fumarase a c t i v i t y i n SmdK12 - 3 ,U56, i n SmSK12 - 1,11+3 Average G6PDH a c t i v i t y i n SmdK12 - 60.2, i n SmsK12 - 90.0 Cells were grown with vigorous shaking at 37'C on a salts medium containing 0.1+$ glucose. Smd cultures were frown i n the presence of 1000 micrograms DHSM/ml. ' ' • ' I I I I I I 1 1 1 ! I I I I L 5 0 0 5 5 0 6 0 0 W A V E L E N G T H nm F i g . 3* R e d u c e d - o x i d i s e d d i f f e r e n c e s p e c t r a of whole c e l l s o f Sms and Sm d E s c h e r i c h i a c o l i K12. C e l l s were grown w i t h v i g o r o u s s h a k i n g a t 37'C on a s a l t s •medium as f o l l o w s : , a) Sms K12 - 0.U# g l u c o s e , b) Sm K12 -0,l\% glucose. + 1000 micrograms DHSM/ml. A b s o r p t i o n a t 5&0 nm r e p r e s e n t s cytochrome b, the peak a t 5U0 nm i s the b e t a band f o r cytochrome b, the peak at 503 nm i s P503 ( p r o t o t e t r a h y d r o -p o r p h y r i n IX) and t h e t r o u g h a t UliO-^OO nm i s t h e f l a v i n t r o u g h . a b c i i i i i -i i i • ! I ' I ' ! I I 5 0 0 5 5 0 6 0 0 W A V E L E N G T H nm F i g . I4.: E f f e c t of carbon source on the form a t i o n o f P503 i n Sm d E s c h e r i c h i a c o l i B C e l l s were grown with vigorous shaking on a s a l t s medium at 37'C as f o l l o w s : a) Sms B - 0.I4J g l u c o s e , b) Sm d B - 0.1$ gluconate + 1000 micrograms DHSM/ml, c) Smd B - 0.k% glucose +• 1000 micrograms DHSM/ml. -1+9-Escherichia c o l i K12. This difference between the two strains was explained on the basis of the a b i l i t y of gluconate to restore a state d of catabolite repression i n Sm Escherichia c o l i B but not in Smd Escherichia c o l i K12 i e . fumarase l e v e l s were similar in Smd and Sms Escherichia c o l i B but were d i f f e r e n t i n Srad and Sm Escherichia c o l i K12 (Table I I ) . Restoration of catabolite repression i n Sm^ Escherichia c o l i K12 was accomplished by the use of a n t i b i o t i c - l i m i t e d c e l l s (Table I I ) . Analogous to the findings with^.Sm^ Escherichia c o l l B, i t was observed that under this condition, the P503 l e v e l was restored to the wild-type l e v e l (Table I I ) . At suboptimal concentrations of DHSM (less than 100 micrograms/ral), i t was observed that the l e v e l of P5>03 and protoporphyrinogen oxidase a c t i v i t y varied with the concentration of a n t i b i o t i c i n the medium i e . as DHSM con-centration decrease, th e i r l e v e l increased. The l e v e l of coproporphyrinogenase a c t i v i t y , however, was unaffected by the concentration of DHSM i n the medium (Fig. 5, Table I I I ) . IV. Excretion of Amino Acid3 by Sm Escherichia c o l i K12  Cultures Analysis of the c e l l - f r e e growth medium of each of the twenty-three Smd Escherichia c o l i K12 mutants by paper chromatography showed that the major amino acid excreted by these c e l l s was glutamic acid. d Quantitative analysis established that i n both Sm and Sm Escherichia c o l i K12 c e l l s , glutamic acid was the major - 5 o -Table I I : Relationship between the a c t i v i t y of catabolite s e n s i t i v e enzymes and Pf?03 formation i n Smd Escherichia c o l i Carbon Relative Relative S t r a i n Source ygDHSM/ml Fumarase-* A c t i v i t y P 5 0 3 Sms B glucose - 1,120 1.00 1.00 Smd B glucose 1,000 3,380 3.02 o.Uo Smd B gluconate 1,000 1,31+0 1.20 1.18 Sms K12 giucose - 1,21+0 1.00 1.00 Smd K12 glucose 1,000 k,k90 3.62 0.1+0 Smd K12 gluconate 1,000 3,100 2.50 0.1+8 Smd K12 glucose 10 1,300 1.05 1.00 *Fumarase a c t i v i t y i s expressed as units/mg protein where a unit i s defined as AOD2h.o = 0.001/min. Ce l l s were grown with vigorous shaking at 37'C on a sa l t s medium containing 0.1+$ glucose or 0.1+$ gluconate and DHSM as indicated. F i g , 5 : P503 f o r m a t i o n ' i n Sma E s c h e r i c h i a c o l i K12 grown with l i m i t i n g ' c o n c e n t r a t i o n s of DHSM. C e l l s were grown with v i g o r o u s shaking at 37'C on a s a l t s medium c o n t a i n i n g Q.l\% glucose and a) 1000 micro-grams DHSM/ml, b) 30 micrograms DHSM/ml, and c ) 10 micro-grams DHSM/ml. - 5 2 -Table I I I : Coproporphyrinogenase and protoporphyrinogen oxidase a c t i v i t i e s i n a n t i b i o t i c - l i m i t e d Sm Escherichia c o l i K12 DHSM Growth Protoporphyrinogen ug/ml rate*- Coproporphyrinogenase-** oxidase-**-1,000 0.337 30 0.271 10 0.186 2.1+3 2.1+8 2.17 1+.30 2.62 1.80 Assays were performed by Dr. R. Poulson ^Growth rate i s defined as In 2/doubling time i n hours * * S p e c i f i c a c t i v i t y Is expressed as nanomoles protoporphyrin IX formed per mg protein per hour -53-amino acid excreted; a f t e r 8 hours growth, the Smu culture excreted 1+0 times the glutamic acid observed i n the Sras parent. S i m i l a r l y , alanine, valine and methionine lev e l s were elevated In the Smd culture i e . 2.58, 1.78 and 3.C-6 f o l d higher respectively (Table IV). d Extension of the analysis to Sm Escherichia, c o i l B, again confirmed that the major amino acldf;excreted by these c e l l s was glutamic acid (Table V). Comparison of the amino d acid excretion pattern of Sm mutants of Escherichia c o l i B and Escherichia c o l i K12, revealed a s i m i l a r i t y of pheno-types i e . i n both cases the pattern of amino acid excretion was glutamic acid> alanine > leucine 7methionine"> isoleucine >valine and the r a t i o of the t o t a l amino acids excreted by Smd/SmS was si m i l a r : SmdK12/SmsK12 - 3.1>4-, SmdB/SmsB - 3.59. d Supplementation of the growth medium of Sm Escherichia  c o l i K12 with a metal ion solution, eliminated the excretion of glutamic, by these c e l l s (Table VI). Further analysis established that Fe*4" or F e + + + +/- citrate<ralone"could account for this e f f e c t (Table VI).. Although glutamic acid was not excreted under these conditions, valine excre-tion, growth and acetohydroxy acid synthase a c t i v i t y were unchanged (Table VII). V. Growth of Sm Escherichia c o l i K12 i n the Presence of  L-Valine The growth of a l l the Sm Escherichia c o l i K12 mutants - 3 was insensi t i v e to the presence of 10 JM L-valine i n the growth medium; the growth of the wild-type Sm parent was markedly i n h i b i t e d (Fig. 6). -5i+-d S Table IV: Excretion of amino acids by Sm and Sm Escherichia  c o l l K12 a f t e r 1+, 6, and 8 hours growth on a glucose-salts medium. (In the case of dependents the medium was supplemented with 1000 ugDHSM/ml) nanomoles amino acid/ml culture medium Smd K12 Sm3 K12 amino acid 1+ hr 6 hr 8 hr 1+ hr 6 hr 8 hr glutamic 6.80 12.1+0 326.00 0.57 12.10 8.00 alanine 0.68 1.20 1+.16 0.90 2.66 1.61 valine 0.03 0.12 2.11+ 0.15 0.1+1 1.20 methionine 0.33 0.60 0.82 0.11+ 0.29 0 . 27 isoleucine 0.06 0.1+2 0.59 0.13 0.23 0.56 leucine o.l+o 1.10 2.71+ 0.76 2.30 l+.l+o -55-Table V? Amino acid excretion patterns i n Smd and Sms  Escherichia c o l l K12 and Escherichia c o l i B aft e r 1+ hours growth on a glucose-salts medium. (For Smd c e l l s , the medium was supplemented with 1000 Ug DHSM/ml) Amino acid nanomoles Smd K12 amino ac Smd B id/ml culture medium SmS K12 Sma~B glutamic 6.80 8.93 0.57 -alanine 0.68 1.71 0.90 0.69 valine 0.03 0.15 0.15 1.91+ methionine 0.33 0.32 0.11+ trace isoleucine 0.06 0.29 0.13 0.21+ leucine O.kO 0.53 0.76 0.U5 Total : 8.30 11.93 2.65 3.32 - 5 6 -Table V I : E f f e c t of metal ions on the amino acid excretion pattern i n Srad Escherichia c o l i K12 growing on a glucose-salts medium supplemented with 1000 micrograms DHSM/ml. C e l l s were harvested a f t e r 7 hours growth. +0.003$ Fe (++) or Amino acid? control »metal ions (+++) +/- 0 .5$ c i t r a t e glutamic 257.00 absent absent alanine valine 1.75 i . 5 o 0.77 methionine 0.79 0.36 isoleucine 0.97 0.79 leucine 0.1+6 0.65 *nahomoles/ral culture medium Table VII: E f f e c t of Pe (++-+) on the doubling time of Smd  Escherichia c o l l K12 and the a c t i v i t y of AHAS during growth of the c e l l s on a glucose-salts medium supplemented with 1000 micrograms DHSM/inl. Growth conditions Doubling time*- AHAS*-*-control 78 min 3.30 + Pe (++•) 78 min 3.17 -^Doubling time i s the time required f o r the c e l l mass to double -»-5c-AHAS a c t i v i t y i s expressed as micromole acetoin formed/hr/ mg protein. 1 2 3 HRS 4 5 o f t h e g r o w t h o f S m s a n d Sm' col I, K12 t o t h e p r e s e n c e . ' o f t h e g r o w t h m e d i u m . C e l l s w e r e g r o w n W i t h v i g o r o u s s h a k i n g o n a s a l t s m e d i u m c o n t a i n i n g 00U/-^ g l u c o s e and t h e m e d i u m was s u p p l e m e n t e d w i t h 1000 m i c r o g r a m s D H S M / m l f o r t h e g r o w t h o f S m d K12. L - v a l i n e (10"3M f i n a l c o n -c e n t r a t i o n i n t h e m e d i u m ) was a d d e d a s i n d i c a t e d ( 1 ) O - O S m s K12; ©-© S m d K12. ' , P i g . 6: S e n s i t i v i t y Escherichia L - v a l i n e i n - 5 8 -DISCUSSION A phenotype of catabolite derepression during the growth of Smr Escherichia c o l i K12 on glucose would appear to explain the observations i n *this section. Thus, the l e v e l of a c t i v i t y of the cata b o l i t e - s e n s i t i v e d enzyme, fumarase, was 3.2 f o l d higher i n twenty-three Sxn s Escherichia c o l i K12 mutants compared to t h e i r Sm parent; the a c t i v i t y of the ca t a b o l i t e - i n s e n s i t i v e enzyme, glucose-6P dehydrogenase, however, remained r e l a t i v e l y unchanged (0.3 f o l d lower) (Table I ) . Hence, DHSM appeared to have a p r e f e r e n t i a l a f f e c t on the synthesis of catabolite sensi-t i v e enzymes (Coukell and Polglase, 196la). Moreover, the markedly lower l e v e l s of P503 (prototetrahydroporphyrin IX) d i n Sm mutants, which was o r i g i n a l l y believed t o be a de-f i c i e n c y i n these c e l l s (Kamitakahara and Polglase, 1970), also proved t o b e a manifestation of a state of catabolite derepression (Table I I ) . The finding that the a c t i v i t y of protoporphyrinogen oxidase was sen s i t i v e to "the concentration of suboptimal leve l s of DHSM i n the growth medium of Smd Escherichia c o l i K12, explained the low l e v e l of P503 observed i n these c e l l s (Table I I , F i g . 6). Hence, when protoporphyrinogen oxidase a c t i v i t y i s high, protoporphyrinogen IX, the reduced pre-cursor of P503» would be rapidly converted fto protoporphyrin IX. If , however, t h i s a c t i v i t y were reduced, f o r example under conditions of catabolite repression, protoporphyrinogen IX would accumulate and be detected as P503 (^lg. 1). -59-The excess excretion of amino acids by Smd Escherichia  c o l i K12 may be explained by the suggestion of Bragg and Polglase (I96I4.) that t h i s represents the formation of a neutral end-product of glucose metabolism, a r i s i n g as a consequence of a condition of catabolite derepression. The f i n d i n g that these mutants excreted 'excess quantities of glutamic acid, disagrees with previous observations estab-l i s h i n g valine as the major amino acid excreted (Bragg and Polglase, 1962). However, i n the present studies, q u a l i t a -t i v e and quantitative examination of amino acid excretion in a l l the mutants tested - twenty-three Smd Escherichia  c o l i K12 and one Sm Escherichia c o l i B, unquestionably established glutamic- acid as the major amino acid excreted (Table V ) . . In f a c t , V i a one case i t was observed that a Smd  Escherichia c o l i K12 excreted up to 1+0 times the quantity of glutamic acid excreted by the Sm wild-type parent (Table IV). The novel observation that Fe ions (Fe + + > or F e + + + ) eliminated glutamic acid excretion without a f f e c t i n g the growth rate of the c e l l s or the state of catabolite dere-pression, i s notreadily explainable (Tables VI and VII). However, the observation was reproducible and may possibly be explained by a s p e c i f i c e f f e c t of Fe Ions on an enzyme, for example succinate dehydrogenase. This enzyme i s knotto. to be sensitive to the concentration of Fe ions i n the growth medium (Rainnie and Bragg, 1973) and, an increase i n the a c t i v i t y of t h i s enzyme could r e s u l t i n the channeling -60-of the keto acid of glutamic acid - alpha-ketoglutarate, into fumarate. Excess carbon may then be excreted as a non-amino acid metabolite. A l l twenty-three Sm Escherichia c o l i K12 mutants exhibited a growth i n s e n s i t i v i t y to i n h i b i t i o n by L-valine (Pig. 6 ) . Evidence w i l l be presented l a t e r establishing that t h i s too i s a manifestation of a condition of catabolite derepression i n c e l l s growing on glucose. Hence, Smd Escherichia c o l i K12 mutants i n general may be characterized as displaying a relaxation of catabol-i t e repression during growth on glucose. This observation serves to reinforce the hypothesis that the requirement of these c e l l s for DHSM (or streptomycin) may be linked to the need of the c e l l s for a condition of catabolite derepres-sion i e . high CAMP l e v e l s . However, before t h i s aspect of the nature of DHSM de-pendency i n Escherichia c o l i i s examined, a more detailed investigation of the interesting phenomenon of a loss of ci growth s e n s i t i v i t y to i n h i b i t i o n by L-valine i n Sm Escherichia  c o l i K12 w i l l be presented. - 6 1 -PART B; Regulation of Acetohydroxy Acid Synthase i n Sma  Escherichia c o l l K12 RESULTS It became apparent from the r e s u l t s of Part A that the metabolism of Smd Escherichia c o l i K12 was l a r g l y explained by the lack of catabolite repression during the growth of these c e l l s on glucose. It was, therefore, reasoned that i f catabolite repression could be returned to these c e l l s , growth s e n s i t i v i t y to i n h i b i t i o n by L-valine would also return. I. E f f e c t of Carbon Source on the S e n s i t i v i t y of Growth  to" I n h i b i t i o n by L-Vallne i n Smd Escherichia c o l i K12 Three carbon sources were tested at a concentration of 0,k% i n the medium - g l y c e r o l , glutamic acid and alpha-keto-glutarate., ; The c e l l s grew well on glutamic acid and alpha-ketoglutarate i e . a doubling time of 70 min and 6 5 min re-spectively, and i n three separate experiments growth on these carbon sources was inhi b i t e d by 10 M L-valine. How-ever, the re s u l t s were not reproducible and this approach was not pursued further. I I . S e n s i t i v i t y of Growth to I n h i b i t i o n by L-Valine i n  Antibio t i c - L i m i t e d Smd Escherichia c o l i K12 It has been established that catabolite repression was returned i n a n t i b i o t i c - l i m i t e d Sm Escherichia c o l i K12 growing on glucose In the presence of suboptimal concen-trations of DHSM i e . less than 100 micrograms/ml (Table I I ) . In a n t i b i o t i c - l i m i t e d Sm Escherichia c o l i K12, growth s e n s i t i v i t y to I n h i b i t i o n by L-valine was restored and the -62-addition of 50 micrograms isoleucine/ml growth medium, re-lie v e d the s e n s i t i v i t y (Pig. 7). As would be expected, a Smd Escherichia c o l i B mutant grown under the same conditions was i n s e n s i t i v e to the presence of valine i n the growth medium. Examination of the effect of L-valine concentration on the i n h i b i t i o n of Smd Escherichia c o l i K12 growth, showed that the i n h i b i t i o n was independent of L-valine concentra-tions greater than ^ X I C T ^ M (Fig. 8 ) . d s I I I . A c t i v i t y of Acetohydroxy Acid Synthase i n Sm and Sm  Escherichia c o l i K12 Acetohydroxy acid synthase (AHAS) a c t i v i t y was 1.8 f o l d higher i n Smd Escherichia c o l i K12 grown on glucose with an optimum concentrations of DHSM (1000 micrograms/ml) compared to the Sms parent. However, the a c t i v i t y of AHAS i n the Sm mutant grown with a l i m i t i n g concentration of DHSM ( 2 5 micro-grams/ml), was the same as i n the wild-type parent. Return of AHAS a c t i v i t y to wild-type l e v e l s was accompanied by a restoration of growth s e n s i t i v i t y to i n h i b i t i o n by valine i n Smd Escherichia c o l i K12.(Table VIII). IV. End-Product I n h i b i t i o n of Acetohydroxy Acid Synthase  by L-Valine i n Smd Escherichia c o l l K12 In order to establish that a mutation to streptomycin dependence had not altered the feed-back s e n s i t i v i t y of AHAS to valine , valine i n h i b i t i o n of AHAS was tested i n Smd Escherichia c o l i K12. The s e n s i t i v i t y of AHAS to i n h i b i t i o n by L-valine was ! , r- 1 ; , T r 1 2 3 4 5 6 7 HRS P i g . 7: I n h i b i t i o n o f growth by L - v a l i n e i n a n t i b i o t i c -l i m i t e d 3md E s c h e r i c h i a c o l i K12. C e l l s were grown w i t h v i g o r o u s s h a k i n g a t 37'C on a s a l t s medium c o n t a i n i n g Q.kfo g l u c o s e and ©-© 1000 micrograms D H S M / m l or B-B 25 micrograms D H S M / m l . L - V a l i n e (10~3M f i n a l c o n c e n t r a t i o n i n the medium) and L - i s o l e u c i n e (50 micrograms/ml) were added as i n d i c a t e d 10"3M 4X iO~ 4M 10~ 4M 4X10~ 5M 1CT 5M 10~ 6 M ( L - V A L I N E ) 1 1 1 i 1 1 1 r HOURS F i g 8: E f f e c t o f v a l i n e c o n c e n t r a t i o n on t h e i n h i b i t i o n o f growth by v a l i n e i n a n t i b i o t i c - l i m i t e d Sm d E s c h e r i c h i a c o l i K12. " C e l l s were grown w i t h v i g o r o u s s h a k i n g on a s a l t s medium c o n t a i n i n g 0.kf° g l u c o s e and 10 micrograms DHSM/ml. The v a r i o u s c o n c e n t r a t i o n s o f L - v a l i n e were added as i n d i c a t e d (| ). r e p r e s e n t s the normal growth r a t e i n the absence o f L - v a l i n e . - 6 5 -Table VIII: Relationship between AHAS a c t i v i t y and the i n h i b i t i o n of growth by valine i n Sm Escherichia c o l l K12. DHSM Relative Growth i n glucose-Culture V^/ml AHAS s a l t s plus L-valine Sms K12 0 1.0 inh i b i t e d Smd K12 1,000 1 . 8 not in h i b i t e d Smd K12 2 5 1.0 in h i b i t e d C e l l s were grown with vigorous shaking at 37'C on a salts medium containing 0.1+$ glucose and DHSM as indicated. Sensitivity, of growth to i n h i b i t i o n by valine was tested using 10""3M L - v a l i n e . -66-the same i n Smd and Sms Escherichia c o l i K12 (Fig. 9 ) . V. Effect of DHSM Concentration on Enzyme Levels i n  Antibiotic-Limited Smd Escherichia c o l i K12 A marked effect of suboptimal concentrations of DHSM on the growth rate of Smd Escherichia c o l i K12 was observed (Fig. 10). From a Lineweaver-Burke plot of these results i e . l/growth rate versus l/DHSM concentration, the K^gg^ i e . the concentration of DHSM required to maintain the Half-maximal growth rate, was determined to be 11.1 micrograms DHSM/ml (Fig. 11). The amounts of AHAS, fumarase, and aconitase (catabolite-sensitive enzymes) were found to vary with the concentration of DHSM i n the medium while the amount of glucose-6P dehydrogenase (catabolite-Insensitive enzyme) remained r e l a t i v e l y unchanged (Table IX). VI. Multivalent End-Product Repression of Acetohydroxy Acid  Synthase i n Smd Escherichia c o l i K12 The a b i l i t y of Smd Escherichia c o l i K12 to grow ex-ponentially on a glucose-salts medium containing L-valine^ in;the absence of catabolite repression, presented a unique opportunity, to t e s t end-product repression of AHAS. The pattern of end-product repression was found to be highly reproducible. The i n c l u s i o n of a single amino acid i n the growth medium had l i t t l e or no a f f e c t on AHAS synthesis while any combination of two end products except the combin-ation valine plus isoleucine, resulted i n repression; the combination, valine plus isoleucine, produced derepression. Furthermore, the presence of a l l three amino acids valine, i d o _ i I I i 0.375 0.750 1.125 1.50 L - V A H N E ( M X I O 3 ) P i g . 9~ : I n h i b i t i o n o f acetohydroxy a c i d synthase by L - v a l i n e i n Smd and Sm3 E s c h e r i c h i a c o l i K12. C e l l e x t r a c t s were prepared from c e l l s grown on 0.1$ glucose H - H Sms K12, ©-© SmdK12. .8 vn I 1 1 1 I 20 40 60 80 IOQ D H S M " 9 / m l P i g . 10: R e l a t i o n s h i p between growth r a t e and DHSM c o n c e n t r a t i o n i n Smd E s c h e r i c h i a c o l i K12. C e l l s were grown \ ^ i t h vigorous shaking at 37' C on 0,\\% glucose and DHSM as i n d i c a t e d . Growth r a t e i s d e f i n e d as l / d o u b l i n g time ( h r s ) . IS) F i g . 11: Determination of K D H S M f ° r Sm Escherichia c o l i K12 growing on glucose. -70-Table IX: Ef f e c t of DHSM concentration on the amounts of enzymes i n a n t i b i o t i c - l i m i t e d Smd Escherichia  c o l i K12. DHSM Growth iig/ml rate AHAS fumarase aconitase G6PDH 100 0.556 lw 1*0 885 353 . 56 $0 o.J+55 3.51+ 720 21+8 56 20 0.352 2.9U 223 i+5 5 0.181 2.25 356 153 52 Growth rate i s expressed as In 2/doubling time i n hours Enzyme a c t i v i t i e s are expressed as units/rag protein (p. 39) Ce l l s were grown with vigorous shaking from an O D J ^ Q of O i l to 0.8 on a s a l t s medium containing 0.1+$ glucose and D H S M as indicated. -71-isoleucine and leucine, maintained that repression or s l i g h t l y enhanced i t . The maximum repression, however, was produced by the presence of pantothenate i n combination with the three branch chain amino acids r(Table X). -72-Table X: Repression of AHAS by end-products i n Srac Escherichia c o l i K12. Exp 11 Growth Rate-»~::- Additions Relative A c t i v i t y 0.531 0.531 0.531 0.531 0.531 0.531 0.531 0.595 0.627 v a l val none val le u i l e v a l +• i l e val + leu + + leu + leu leu i l e + i l e + I l e pan 1.00 0.96 1.07 1.06 1.53* 0.61 0.1+3 0.53 0.1+0 0.531 0.627 0.627 0.627 0.627 none pan i l e + pan val + pan leu + pan 1.00 0.95 0.80, 0.82 0.1+9, 0.38 0.1+5, 0.65 # Average from three separate experiments. Growth rate Is defined as In 2/doubling time i n hours. Cells were grown on a glucose (0iii+$)-salts medium containing 1000 micrograms DHSM/ml plus the end-products as indicated. Amino acids were used i n t h e i r n a t u r a l l y occurring forms and were present at a f i n a l concentration of 10~3M. Ca pantothenate (pan) when present was used at a f i n a l concentration of 1Q-4M. -73-DISCUSSION d The l e v e l of a c t i v i t y ( i e . quantity)- of AHAS i n Sm Escherichia c o l i K12 was shown to be the c o n t r o l l i n g factor i n determining the s e n s i t i v i t y of growth to i n h i b i t i o n by L-valine. Moreover, i t was established that this enzyme i s regulated by the l e v e l of catabolite repression in the c e l l . Hence, under conditions of relaxed catabolite repression, the AHAS l e v e l was elevated i n Smd Escherichia c o l i K12 compared to the wild-type parent, and the s e n s i t i v i t y of growth to valine was r e l i e v e d . I f , however, the growth conditions of the mutant were manipulated such that a state of catabolite repression was maintained i n the c e l l s i e . growth i n the presence of suboptimal concentrations of DHSM, the l e v e l of AHAS was restored to wild-type l e v e l s and the s e n s i t i v i t y of growth t o valine was returned (Table V I I I ) . This effect could be explained s o l e l y on the basis of the l e v e l of AHAS a c t i v i t y and was not a re s u l t of an altered s e n s i t i v i t y of AHAS to i n h i b i t i o n by L-valine (Pig. 9). In agreement with the findings of Umbarger and Brown (1958) the i n h i b i t i o n of growth by valine was independent of valine concentrations greater than l+xlO~^M (Fig. 8). As would be expected for a catabolite s e n s i t i v e enzyme, the l e v e l of AHA% as well as fumarase and aconitase, varied with the DHSM concentration i n the medium of a n t i b i o t i c -l i m i t e d Smd Escherichia c o l i K12; the l e v e l of the catabolite-i n s e n s i t i v e enzyme glucose-6p dehydrogenase, remained un-changed (Table IX) . The K D H S M f > o r t l i e growth D f this mutant -71+-on glucose was calculated to be 1 1 . 1 micrograms/ml which i s s i m i l a r to the K ^ g ^ (glucose) reported for Smd Escherichia c o l i B i e . 5 . 3 (Coukell and Polglase, 1 9 6 9 a ) . Previous studies on end-product repression of AHAS have produced c o n f l i c t i n g r e s u l t s because of uncontrolled and unsuspected interference by catabolite repression. This problem has been c l a r i f i e d i n the present study by the use of Smd Escherichia c o l i K12 which allowed an unambiguous determination of the end-product combinations required for repression. Hence, i t has been reported that the maximum repression of AHAS i n Escherichia c o l l K12 required the presence of valine and leucine i n the medium (Blatt e_t a l , 1 9 7 2 ) ; studies with Sm Escherichia c o l i K12, however, showed that maximum repression required the presence of a l l four end-products - valine, leucine, isoleucine and pantothenate,/in the growth medium (Table X). Hence, i n Smd Escherichia c o l i K12, AHAS i s regulated by feed-back i n h i b i t i o n , end-product repression and catab-o l i t e repression (Whitlow and Polglase, 1 9 7 5 ) . Having established that the c h a r a c t e r i s t i c of s e n s i t i v i t y of growth to i n h i b i t i o n by L-valine was affected by manip-ul a t i n g the state of catabolite repression In Sm Escherichia  c o l l K12, the investigation was extended to the wild-type parent. The res u l t s of thi s work are presented i n the following section. - 7 5 -PART C; Control of Acetohydroxy Acid Synthase In Escherichia c o l i K12 RESULTS It was reasoned that i f s e n s i t i v i t y of growth to valine was returned to Sm^ Escherichia c o l i K12 when catabolite repression was returned tto the c e l l s , then under conditions of catabolite derepression (eg. v a r i a t i o n of carbon source or addition of CAMP), the valine s e n s i t i v i t y of the wild-type parent should be decreased. I. E f f e c t of Carbon Source on the Inh i b i t i o n of Growth  bv L-Valine i n Escherichia c o l l K12 The i n h i b i t i o n of growth by L-valine was les s during the growth of wild-type Escherichia c o l i K12 on gl y c e r o l -sa l t s than on glucose-salts medium. However, during the growth of these c e l l s on acetate medium, the i n h i b i t i o n by valine was eliminated (Pig. 1 2 ) . These results were also obtained using a W310G s t r a i n of Escherichia c o l i K12. II . E f f e c t of CAMP on the S e n s i t i v i t y of Growth to Valine  i n Escherichia c o l i K12 Inclusion of 2.5mM CAMP i n the growth medium reduced the i n h i b i t i o n of growth by valine analogous to the effect observed during the growth of these c e l l s on g l y c e r o l . Inclusion of 5mM CAMP, however, completely eliminated the s e n s i t i v i t y of growth to valine i n h i b i t i o n i n Escherichia  c o l i K12,.(Fig. 13). HRS HRS .g. 1 2 : E f f e c t of carbon source on the I n h i b i t i o n o f . growth by L - v a l i n e i n E s c h e r i c h i a c o l i K12. Cu l t u r e s were grown with shaking at 3 7 ' C on a s a l t s medium c o n t a i n i n g : A - 0.k% glucose, B - O.l>f0 g l y c e r o l , O - O.o> a c e t a t e . ®-« c u l t u r e to which 1 0 " 3 M L - v a l i n e was added as I n d i c a t e d ( 1 ) ; o-o growth i n the absence of L - v a l i n e . 0.5 . . E 0.4 -cz O C\J -.sr (O.D. 0.3 -X i -i 0.2 -o or CD 0.1 2 3 HOURS P i g 1 3 : E f f e c t of GAMP on the i n h i b i t i o n of growth by L - v a l i n e i n E s c h e r i c h i a c o l i K12. C e l l s were .grown with vigorous a e r a t i o n on a s a i l medium c o n t a i n i n g 0.1+$ glucose and 5mM CAMP, A-A 2.5>mM CAMP, o-o no a d d i t i o n s . L - v a l i n e was added as i n d i c a t e d ( \ ) to a f i n a l c o n c e n t r a t i o n o f 10~ 3 M . • - © a l s o r e p r e s e n t s the growth r a t e on glu c o s e i n the absence o f L - v a l i n e . -78-I I I . Control of Acetohydroxy Acid Synthase by Catabolite  Repression i n Escherichia c o l i K12 In order to r e l a t e the l e v e l of AHAS to the s e n s i t i v i t y of growth to i n h i b i t i o n by L-valine i n Escherichia c o l i K12, AHAS was assayed and i t s s e n s i t i v i t y to i n h i b i t i o n by L-valine was determined under a variety of growth conditions. Growth of Escherichia c o l i K12 on acetate +/- 1 0 " % L-valine resulted i n the derepression of AHAS well above glucose l e v e l s i e . 3.3 f o l d higher. Inclusion of 5mM CAMP in a glucose-salts medium enhanced the a c t i v i t y l.§ f o l d whereas, the a c t i v i t y i n glycerol grown c e l l s was 1.3 f o l d higher (Table XI). The resu l t s correspond favourably to the s e n s i t i v i t y of growth to i n h i b i t i o n by valine. Inter-mediate l e v e l s , as observed for example during growth on gly c e r o l , exhibited an intermediate s e n s i t i v i t y of growth to valine while maximum i n h i b i t i o n was observed with r e -pressed levels of AHAS i e . glucose grown c e l l s . The feed-back s e n s i t i v i t y of AHAS to valine was not changed by these modifications of growth conditions (Fig.!;!*.). IV. Ef f e c t of CAMP on the Excretion of the Branch Chain  Amino Acids by Escherichia c o l i K12 The presence of 5mM CAMP i n the glucose-salts medium of Escherichia c o l i K12 enhanced the excretion of a l l the branch chain amino acids. The greatest increase was observed for the excretion of valine Ie. 1+.2 f o l d increase whereas the excretion of isoleucine and leucine was increased 2.81 and 1.55 f o l d respectively (Table XII). -79-Table XI: Ef f e c t of growth conditions on the l e v e l of AHAS i n Escherichia c o l i K12 Inh i b i t i o n of Relative Growth Conditions growth by L-val AHAS Glucose (0.lj.$) +:+ 1.0 Glycerol (0.1+$) + 1.3 Glucose (O.ki) + CAMP (5mM) - 1.9 Na Acetate (0.8$) - 3.3 Na^Acetate (0.8$) + L-val (ImM) - 3.I4. C e l l s were grown with vigorous aeration at 37fC on a salts medium as indicated. -ou-F i g . ll+: I n h i b i t i o n o f AHAS by L - v a l i n e i n E s c h e r i c h i a c o l i K12 G e l l e x t r a c t s were p r e p a r e d from c e l l s grown on ©-® 0.8$ a c e t a t e , o-o 0.1+$ g l u c o s e , and A-A 0.1$ g l u c o s e + £mM GAMP. - 8 1 -Table XII: E f f e c t of CAMP on the excretion of the branch chain amino acids by Escherichia c o l i K12. micromoles amino acid/lGO ml medium Amino acid L-valine L-isoleucine L-leucine glucose-salts 0.071; 0.015 0.031+ glucose-salts + CAMP (5mM) 0.310 0.01*2 0.053 a, 1+.17 2.81 1.55 C e l l s were grown with vigorous aeration from an ODh^Q of 0.1 to 1.0 on a s a l t s medium containing (a) 0.1\.% glucose, (b) 0.k% glucose + 5mM CAMP. -82-DISCUSSION In studies presented here, i t has been shown that s e n s i t i v i t y of growth to i n h i b i t i o n by L-valine i n Escherichia c o l i K12 describes a phenotype which can be altered by changing the growth conditions (Pig. 12). Thus, growth of Escherichia c o l i K12 under conditions of minimum catabolite repression i e . on glucose-salts medium plus f>mM CAMP or on acetate medium, removed the s e n s i t i v i t y to valine; growth under conditions promoting a state of moderate catabolite repression i e . on g l y c e r o l medium or i n a glucose-s a l t s medium containing 2.j?mM CAMP, allowed a modified i n -h i b i t i o n while growth under conditions of maximum catabolite repression displayed maximum Inh i b i t i o n by L-valine. The a l l e v i a t i o n of s e n s i t i v i t y to valine was observed not only with a wild-type Escherichia c o l i K12 but also with a W3100 s t r a i n of Escherichia c o l l K12. Assay of AHAS a c t i v i t y i n c e l l s grown under a variety of growth conditions established that s e n s i t i v i t y of growth to vali n e and the l e v e l of AHAS a c t i v i t y were i n t e r r e l a t e d (Table XI). Under a l l the growth conditions which promoted i n s e n s i t i v i t y of growth to i n h i b i t i o n by L-valine i e . con-diti o n s exhibiting catabolite derepression, the i n h i b i t i o n of AHAS a c t i v i t i y by L-valine remained unchanged (Pig. II4.). The physiological consequence of a derepressed AHAS was i l l u s t r a t e d by the observed enhanced excretion of the branch chain amino acids into medium of Escherichia c o l i K12 growing on glucos.e. in. the presence of 5mM CAMP (Table XII). -83-Hence, si m i l a r to the findings with Sm Escherichia  c o l i K12 (Part B), AHAS was shown to be controlled by catab-o l i t e repression i n wild-type Escherichia c o l i K12 and the s e n s i t i v i t y of growth to valine was established to be a manifestation of the degree of catabolite repression i n the c e l l (Whitlow and Polglase, 197U). The observation that a biosynthetic enzyme i s controlled by a form of regulation recognized as catabolic has been reported previously (Gorini and Gundersen, 1961). In Escherichia c o l l K12 i t might be reasoned that c e l l s growing on a carbon source which was i n f e r i o r to glucose as an energy source, might require an enhanced l e v e l of AHAS to permit maximum growth i e . i n the case of E s c h e r i c h l a r c o l i K12 a l i m i t i n g l e v e l of AHAS produces a condition of 'isoleucine starvation' and hence i n h i b i t e d growth (Umbarger and Brown, 1958). A si m i l a r explanation might be extended to Escherichia  c o l i B i n view of the finding of Coukell (PhD thesis, p. 93) that an enhanced AHAS during growth on glycero l medium was not accompanied by excess valine excretion; only when the glycerol grown c e l l s were l a t e r exposed to glucose did the c e l l s respond with an excess excretion of th i s amino acid. Hence, growth on glycerol medium required the enhanced bio-synthesis of valine which would only be wasteful during growth on glucose. Certainly, the biosynthesis of many amino acids i s ultimately under the control of catabolite repression i e . the a v a i l a b i l i t y of essential intermediates for example - amino acids derived from intermediates of the -8U-TCA cycle, -,; from ^ pyruvate or from g l y c o l y s i s . The observation that catabolite derepression i n a n t i -b i o t i c - l i m i t e d Sm Escherichia c o l i K12 growing on glucose responded to the l e v e l of DHSM i n the growth medium (Table IX.}; Coukell and Polglase, 1969a), prompted an investigation of the effect of growth conditions on a n t i b i o t i c requirement. These re s u l t s are presented i n the next section. 1 . - 8 5 -PART D: Streptomycin Dependency i n Relation to Catabolite  Repression i n Smd Escherichia c o l i RESULTS d Sm c e l l s have been shown to exhibit a state of re-laxed catabolite repression during growth on glucose medium containing non-limiting concentrations of DHSM*'" Moreover, i n a n t i b i o t i c - l i m i t e d c e l l s , catabolite repression increased with decreasing DHSM concentrations i n the growth medium. An obvious question at this point was - i s dihydrostreptomycin (or streptomycin) required by Sm^ Escherichia c o l i s o l e l y to prevent a state of catabolite repression which vpould be s u f f i c i e n t l y severe to prevent growth? I. Substitution of CAMP f o r DHSM i n the Growth Medium of Smd Escherichia c o l i K12, Cyclic AMP (5mM) p a r t i a l l y substituted f o r DHSM i n main-taining growth on glucose (Fig. 15B) and maintained normal growth when DHSM (endogenous) was not severely l i m i t i n g (Fig. 15A). I I . Effect of Carbon Source on the Requirement f o r DHSM i n Smd Escherichia c o l i K12 Using the data of Coukell and Polglase (1969a), the r a t i o of KDgsM to V m a x (p. 2 5 ) was calculated for a variety of carbon sources (Table XIII). Since the r a t i o was not a constant, i t appeared that factors other than the decline i n growth rate influenced the requirement for DHSM. The general trend appeared to be that the poorer the energy source i e . the weaker the a b i l i t y to cause catabolite re-F i g . 1 5 : S u b s t i t u t i o n o f CAMP (5mM).for DHSM i n Srn d E s c h e r i c h i a c o l i K12. A n t i b i o t i c - l i m i t e d c e l l s were grown w i t h v i g o r o u s s h a k i n g a t 37'C on a s a l t s medium c o n t a i n i n g , ©-® 0ah.% g l u c o s e + 1000 micrograms. DHSM/ml, o-o 0 <,hf° g l u c o s e + 5mM CAMP, arid A-A Qj$ g l u c o s e , A. and B r e p r e s e n t two s e p a r a t e e x p e r i m e n t s . -87-Table XIII: E f f e c t of carbon source on the requirement of Smd Escherichia c o l i B for DHSM. Carbon Source KDHSM/Vmax (a) gluconate 0.0138 glucose 0.0080 g l y c e r o l O.OO36 l a c t a t e 0.0031 (a) Calculation was performed on data obtained from Coukell and Polglase (1969a). Method for obtaining the data i s I l l u s t r a t e d i n Pigs. 10 & 11. V m a x - maximum growth rate.. KDHSM _ concentration of DHSM required to maintain the half-maximal growth rate. -op-pression, the less DHSM was required f o r growth. Based on this observation, growth conditions were sought which would maintain a state of minimum catabolite repression i n the expectation of reducing the requirement of Smd c e l l s f o r DHSM even further. For this purpose c e l l s were adapted to growth on acetate, a very poor energy source. Sm Escherichia c o l i K12 growing on acetate were able to grow without added DHSM, and moreover, the doubling time on acetate was reduced compared to growth on glucose plus 1 0 0 0 micrograms DHSM/ml..,, i e . 5 3 min and 7 2 min respectively (Fig. 1 6 ) . In these studies, several successive transfers of the cultures were made with acetate as the carbon source to insure the c e l l s were depleted of a n t i b i o t i c . cl I I I . 'Streptomycin Dependency1 i n Sm Escherichia c o l i A l l the Smd mutants of several Escherichia c o l i strains i e . B, CRX, PCI, A and UL which were tested grew readi l y on acetate i n the absence of DHSM, but required the presence of the a n t i b i o t i c during growth on glucose. Moreover, a l l the dependent.1 strains grew i n the presence of glucose + 3 $ ethanol, but f a i l e d to grow i n the presence of glucose +• 2 0 0 micrograms paromomycin/ml medium (Table XIV). IV. Phenotype of 'Smd' Escherichia c o l i K12 Grown Without  DHSM 'Smd' Escherichia c o l i K12 grown with glucose (0 .LL$) as a carbon source i n the presence of (a) 3$ethanol, (b) 1 0 0 0 micrograms DHSM/ml or with 0 . 8 $ acetate as the carbon source, exhibited a state of catab.olj.te derepression compared to the 1 i i r 2 3 4 5 HRS P i g . 16: Non-dependency o f 'Sm ' E s c h e r i c h i a c o l i K12 growing on a c e t a t e . C e l l s were grown w i t h vigorous s h a k i n g a t 37'C on a s a l t s medium c o n t a i n i n g © - 9 O.8/0 a c e t a t e +/-1000 micrograms DHSM/rr!, o r 0-0 0»h% g l u c o s e + 1000 micrograms DHSM/ml. - 9 0 -Table XIV: Growth of Sm Escherichia c o l i i n r e l a t i o n to carbon source and drug supplement. Growth Conditions carbon source: glucose glucose acetate acetate glucose supplement : ethanol paromomycin none DHSM none Smd s t r a i n . ; K12 (#1-23) + - 4 + B + - + 4 CRX 4 4 4 PCI 4 - + 4 A 4 - 4 4 UL + - 4 4 C e l l growth (4) was recorded after overnight incubation and, in each case, the c e l l growth was checked for p u r i t y by establishing that the c e l l s required DHSM (1000 micrograms/ml) during growth on glucose. -91-wild-type parent grown on glucose. Under a l l the growth conditions, 'Smd' Escherichia c o l i K12 exhibited i n s e n s i t i v i t y of growthto i n h i b i t i o n by L-valine, elevated fumarase levels-compared to the wild-type parent and a lack of P 5 0 3 (Table XV) t The growth rate on glucose 4 3% ethanol was very much less than that on glucose + DHSM ;ie. doubling time of 3 8 7 min compared to 7 2 min. V. Examination of Catabolite Repression i n Sm3 Escherichia  c o l i K12 Growing i n the Presance of Sublethal  Concentrations of DHSM Dihydrostreptomycin and ethanol shared the. general property of promoting a condition of catabolite derepression i n Escherichia c o l i K12 growing on glucose i e . AHAS was de-repressed compared to a glucose control and the s e n s i t i v i t y of growth to i n h i b i t i o n by L-valine was r e l i e v e d . The sen-s i t i v i t y : of AHAS to i n h i b i t i o n by L-valine remained unchanged (Table XVI). VI. Effect of Ethanol and DHSM on Cafeabolite Repression i n Sm1* Escherichia c o l i K12 Growing on Glucose It was found that the growth of Smr Escherichia c o l i K12 on glucose with (a) ethanol, (b) DHSM, and (c) no additions, was sensitive to i n h i b i t i o n by L-valine. The c e l l s were capable of exhibiting a state of catabolite derepression i e . had an intact CAMP system, since the i n h i b i t i o n of growth by valine was rel i e v e d i n c e l l s growing on acetate or on glucose + 5mM CAMP. The s e n s i t i v i t y of AHAS to i n -h i b i t i o n by L-valine was similar under a l l the growth con-diti o n s i e . 7 1 - 7 U $ (Table XVII, F i g . 1 7 ) . - 9 2 -Table XV: Effe c t of c'arbon source and supplement on catabolite repression i n 'Smdl Escherichia c o l i K12 Carbon Supple- I n h i b i t i o n Doubling S t r a i n source ment growth by Fumarase-* P$03 time i L-val (min) SmdK12 "glucose DHSM - 2,008 - 7 2 glucose EtOH - 1,21+2 - 387 acetate none - 1,887 - 51+ SmsK12 glucose none + 871+ + 7 2 *Fumarase a c t i v i t y i s expressed as units/mg protein (p. 39). Ce l l s were grown with vigorous shaking at 37 fC on a salts medium containing (a) 0.1+$ glucose + 1000 micrograms DHSM/ml, (b) 0.1j.$ glucose + 3% ethanol, (c) 0.8$ acetate, (d) 0.1+$ glucose. - 9 3 -Table XVT: AHAS a c t i v i t y i n E s c h e r i c h i a c o l i K12 grown i n the presence of a s u b l e t h a l c o n c e n t r a t i o n o f DHSM. $ i n h i b i t i o n i n h i b i t i o n of Growth C o n d i t i o n s AHAS*- by L - v a l i n e growth by v a l TlO~3M) glucose U.91 73 + glucose + 3 yg/ml- DHSM 7.92 73 C e l l s were grown with vigorous shaking at 37'C on a salts-medium c o n t a i n i n g (a) 0.1|$ g l u c o s e and (;fo) 0*h$> glucose + 3 micrograms DHSM/ml. ->AHAS a c t i v i t y i s expressed as micro-moles a c e t o i n formed/hr/mg p r o t e i n . - 9 V Table XVII: E f f e c t of growth conditions on the l e v e l of AHAS i n Smr Escherichia c o l i K12. % i n h i b i t i o n by i n h i b i t i o n of Growth Conditions AHAS*- L-valine growth by _ L-valine (lo" JM) glucose + ethanol 6 . 8 2 7 1 + glucose + DHSM 6 , 8 5 7 2 + glucose 6 . 7 5 7k + acetate 8.1+3 7 3 C e l l s were grown with vigorous shaking at 37'C on a salts-medium containing (a) 0,1$ glucose + 3% ethanol, (b) 0.k% glucose + 1000 micrograms DHSM/ml, (c) 0.1$ glucose (d) 0.0% acetate. *AHAS a c t i v i t y i s expressed as mlcromoles acetoin formed/hr/rag protein. - 9 5 -• 8_ E - 6 -i 1 r H O U R S F i g . 17: E f f e c t o f growth c o n d i t i o n s on the i n h i b i t i o n of growth by L - v a l i n e i n Sm r E s c h e r i c h i a c o l i K 1 2 . C e l l s were groxm w i t h v i g o r o u s s h a k i n g 'at. 37'C on a s a l t s medium c o n t a i n i n g ©-© 0.8$ a c e t a t e , o-o 0»l\% g l u c o s e . +- 5mM CAMP and A—A O . u . o g l u c o s e . L - v a l i n e wa3 added as i n d i c a t e d ( \ ) t o a f i n a l c o n c e n t r a t i o n o f 10"3M. - 9 6 -DISCUSSION A l l the Sm Escherlchla c o l l mutants described i n this section can be c l a s s i f i e d as 'drug dependent' according to the d e f i n i t i o n of Gorini et a l ( I 9 6 7 ) i e . a l l the mutants would grow on glucose with 3$ ethanol substituting f o r the a n t i b i o t i c (Table XIV). The cross dependency of these mutants has been explained on the basis of a simi l a r action of streptomycin (or DHSM) and e'thanol on protein synthesis i n ribosomal extracts (Gorini et a l , 1 9 6 7 ) . However, the finding i n this work that a l l Smd mutants obtained from six strains of Escherichia c o l i i e . K12, B, A, UL and CRX, did not require any drug supplement during growth on acetate, i s not e a s i l y explained on the basis of the hypothesis of a single s i t e of action of the a n t i b i o t i c at the l e v e l of the ribosome. Previous studies (Part A) established that Smd  Escherichia c o l i K12 c e l l s exhibit a state of catabolite derepression during growth on glucose i n the presence of an optimal concentration of DHSM. S i m i l a r l y , growth on glucose medium supplemented with ethanol, or on unsupplemented acetate medium, promoted a state of catabolite derepression i e . loss of s e n s i t i v i t y of growth to Inhibition by L-valine, elevated fumarase a c t i v i t y and decreased P 5 0 3 levels (Table XV). Furthermore, noninhibiting concentrations of DHSM or ethanol produced a state of catabolite derepression during the growth of Sm3 Escherichia c o l i K12 on.'glucose i e . -97-eliminated the s e n s i t i v i t y of growth to i n h i b i t i o n by L-valine by derepressing AHAS (Table XTL)i This derepression might be explained on the basis of a stress produced i n the Sm3 c e l l s by these drugs, possibly through membrane ef f e c t s , r e s u l t i n g i n an i n t r a c e l l u l a r increase i n CAMP. SuF Escherichia c o l i K 1 2 growing i n the presence of 3$ethanol or 1 0 0 0 micrograms DHSM/ml on a glucose medium, s t i l l retained a state of catabolite repression (Table XVII). Cataholite repression was reli e v e d , however, i f the c e l l s were grown on glucose i n the presence of 5mM CAMP or on acetate (Pig. 17). The degree of Inh i b i t i o n of growth by L-valine was less i n Smr Escherichia c o l i K 1 2 compared to the wild-type Sms parent (Figs. 17 & 12). This corresponds to the d i f f e r e n t l e v e l s of AHAS i n these c e l l s , i e . 6.75 f o r the Smr and 1+.91 f o r the Sm3 strain-..(Tables XVI & XVII). A p a r a l l e l can be observed between the a b i l i t y of DHSM and ethanol to promote a state of catabolite derepression i n c e l l s growing on glucose and the reported a b i l i t y of d s r -these drugs to cause suppression i n Sm , Sm and Sm c e l l s . Several amino acid auxotrophg . are eliminated by streptomycin (Chakrabarti and Maitra, 1971+); t h i s , however, may possibly be the r e s u l t of a l i m i t i n g l e v e l of an enzyme a c t i v i t y which responds to a condition of catabolite derepression. For example, ornithine transcarbamylase i s under the control of catabolite repression i n Escherichia c o l i B (Gorini and Gundersen, 1961) and a l i m i t i n g l e v e l of this a c t i v i t y produced a 'conditionally Smd' mutant displaying an arginine -98-auxotrophy when grown i n the absence of streptomycin (Gorini, 1969). By an analogous argument," : the reduced a b i l i t y of streptomycin and ethanol to cause suppression i n Smr mutants might be related to the i n a b i l i t y of these drugs to promote a relaxation of catabolite repression i n these c e l l s . It i s possible, therefore, that the l e t h a l effects of streptomycin (or DHSM) are exerted at the level: of the ribo-some and that resistance i s incurred through ribosomal muta-tions, or through mutations at the l e v e l of the membrane which precludes the - entry of the a n t i b i o t i c into the cells,-or through an episomal factor. Dependency might be explained, not on the basis of a requirement for protein synthesis, but on the basis of a requirement for a state of catabolite de-repression with the additional property that these c e l l s are also resi s t a n t to high l e v e l s of streptomycin. Certainly, at t h i s point i t i s d i f f i c u l t to explain the eff e c t of carbon source on the requirement f o r DHSM and the elimination of this requirement during the growth of Sm mutants on acetate, on the basis of a ribosomal dependency. 1 Hence, although the twenty-eight mutants tested i n this section were is o l a t e d under the condition of requiring DHSM during growth on glucose, they should actually be c l a s s i f i e d as 'conditionally dependent' on the a n t i b i o t i c according to the d e f i n i t i o n of Gorini and Kataja (1961+a). : Examination of the growth of Sms, Smd andSm r Escherichia  c o l i on acetate revealed several interesting phenomena which -99-suggested another possible s i t e of action for DHSM i n the b a c t e r i a l c e l l . These r e s u l t s are examined i n the next section. -100-PART E: E f f e c t of Acetate as a Carbon Source on 'SmQ« and  Sm3 Escherichia c o l i K12 RESULTS The observation that dependency on DHSM was relieved i n 'Smd| c e l l s growing on acetate (Part D), prompted an investigation of the eff e c t of this carbon source on the metabolism of these c e l l s . During the course of this i n -vestigation several i n t e r e s t i n g e f f e c t s , which are presented i n t h i s section, were noted indicating that growth on acetate had altered the character-.of the membrane i n these c e l l s . I. Beta-galactosidase Induction Beta-galactosidase could not be induced i n tSm*** Escherichia c o l i K12 growing on acetate (•/- 1000 micro-grams DHSM/ml) i n the presence of the gratuitous inducer TMG or i n the presence of lactose. Moreover, this enzyme could not be induced by TMG i n the wild-type parent growing on acetate. Induction by TMG was established, however, i n the Smd mutant and the wild-type parent growing on glucose (Fig. 18). I I . Growth on a Limiting Concentration of Acetate •Smd' Escherichia c o l i strains B, PCI, UL, CRX, A and K12, as well as wild-type SmS Escherichia c o l i K12, exhibited an inte r e s t i n g growth pattern i n medium containing a l i m i t i n g concentration of acetate (0.02$) supplemented with a non-l i m i t i n g concentration of lactose (0.2$) or glucose (0.2$). There was a precipitous drop in o p t i c a l density at 1+20 nano-HRS HRS F i g 18: E f f e c t o f c a r b o n s o u r c e o n b e t a - g a l a c t o s i d a s e i n d u c t i o n b y TMG i n S m d . a n d S m s E s c h e r i c h i a c o l l K12.. C e l l s w e r e g r o w n w i t h v i g o r o u s s h a k i n g a t 37'C o n a s a l t s m e d i u m c o n t a i n i n g lrnM TMG a n d ' S r n 3 : A - 0.8fo a c e t a t e : B - O.I4./6 g l u c o s e ; S m d : C~- 0.8% a c e t a t e •*•/- 1000 m i c r o g r a m s D H S M / m l ; D - O.k-% g l u c o s e + 1000 m i c r o g r a m s D H S M / m l . 0-0 b e t a - g a l a c t O ' s i d a s e ( P . l[0), o-® growth. -102-meter s a f t e r the medium was exhausted of acetate (Pig. 1 9 ) . Microscopic examination of the c e l l s , revealed that the drop i n o p t i c a l density was due to a clumping of the c e l l s ; Furthermore, the normally rod shaped c e l l s were now coccoid while s t i l l r etaining gram-negative staining properties. Subsequent growth on glucose required a long induction period (several hours) but restored the rod shaped morphol-ogy. The clumping effect could not be explained on the basis of an ion deficiency, since supplementation of the medium with a metal ion solution (p. 36) and i n the case of 'dependents' with DHSM, did not prevent the clumping. However, i f the acetate-depleted medium was supplemented with acetate, the c e l l s disassociated and the o p t i c a l density at 1+20 nanometers increased followed by growth u n t i l the medium was once again depleted of acetate (Fig. 20). The precipitous f a l l i n o p t i c a l density observed when the medium was exhausted of acetate, was not observed I f the c e l l s were grown into stationary phase on non-limiting acetate. However, when c e l l growth had stopped under these conditions, the pH of the medium was i n h i b i t i n g i e . 8.5 and i f the medium was subsequently neutralized, growth continued indicating the carbon source had not been depleted. I I I . A n t i b i o t i c S e n s i t i v i t y Wild-type Escherichia c o l l K12 and Escherichia c o l l B, which are normally sensitive to DHSM concentrations greater than 1 0 micrograms/ml medium, were able to grow on acetate at an uninhibited rate i n the presence of 1000 micrograms -103-HOURS HOURS P i g 19: Growth of s e v e r a l Sm d E s c h e r i c h i a c o l i s t r a i n s and Sms E s c h e r i c h i a c o l i K12 on l i m i t i n g a c e t a t e supplemented w i t h n o n - l i m i t i n g l a c t o s e . Sm and Sm 3 E s c h e r i c h i a c o l i were grown w i t h v i g o r o u s s h a k i n g a t 37'C on a s a l t s medium c o n t a i n i n g 3m d - 0.02% a c e t a t e and 0.2$ l a c t o s e +/- 1000 micrograms DHSM/ml, Sm s -0.03$% a c e t a t e and 0,2$ l a c t o s e . -10L+-• 4 J H O U R S F i g . 20: Growth of a Sm E s c h e r i c h i a c o l i K12 with s u c c e s s i v e a l i q u o t s of a l i m i t i n g concen-t r a t i o n of a c e t a t e . C e l l s were grown wit h vigorous shaking at 37'C on a s a l t s medium c o n t a i n i n g 6.02$ a c e t a t e . Succes-s i v e a d d i t i o n of acetate to a f i n a l c o n c e n t r a t i o n o f 0.02$ i s i n d i c a t e d by ( } ) . -io5-DHSM/ml medium (Pig. 21B). Furthermore, the onset of i n -h i b i t i o n of c e l l growth by the a n t i b i o t i c chloramphenicol (10 micrograms/ml medium) was s i g n i f i c a n t l y delayed i n c e l l s growing on acetate (Pig. 21A), IV. Analysis of the Fatty Acids of the C e l l Membrane A relationship has been reported between 'the l i p i d content of a b a c t e r i a l c e l l and the s e n s i t i v i t y of the c e l l to a n t i b i o t i c s (p. 11). Analysis of the f a t t y acid component of the membrane d s r l i p i d s of Sm , Sm and Sm Escherichia c o l i K12 revealed a marked effect of carbon source on the r a t i o of ol e i c acid (18:l)/palmitic acid (16:0) (Fig. 22, Table XVIII). In a l l cases, the degree of unsaturation increased i n c e l l s adapted to jgrowth on acetate compared to growth on glucose. Moreover, the r a t i o of unsaturated/saturated f a t t y acid varied amongst the dependent, s e n s i t i v e 5 , and re-sista n t c e l l s under both growth conditions. The most s t r i k i n g difference was observed with the Smr mutant where the predominance of palmitic acid was very much exaggerated compared to'the Sm and Sm st r a i n s . In addition 18:0 (stearic acid) was much more prominant i n the Smr mutant -grown on glucose compared to the other strains (Pig. 22). -10b-P l g . 21: E f f e c t of carbon source on the s e n s i t i v i t y o f Sms E s c h e r i c h i a c o l i K12 to the a d d i t i o n o f chloramphenicol or d i h y d r o s t r e p t o m y c i n to the growth medium. C e l l s were grown with vigorous shaking at 37'C on a s a l t s medium c o n t a i n i n g N ® - « 0.8$ a c e t a t e , or A - A 0,1+$ "glucose. Chloramphenicol (10 micrograms/ml) (A) and d i h y d r o s t r e p t o m y c i n (1000 micrograms/ml) (B) were added as i n d i c a t e d ( \ ). Sm GLUCOSE 16:0 S m r ACETATE Z o a s -108 Table XVIII: Analysis of membrane f a t t y acids i n Sm , Sm and Smr Escherichia c o l l K12 grown on acetate and glucose. Growth Conditions flucose acetate : l / l 6 : 0 * 18:1/16:0-: SmS K12 0.237 0.920 Smr K12 0.065 0.839 Smd K12 0.1+30 0.722 -*18:1 - o l e i c acid 16:0 - palmitic acid C e l l were grown with vigorous shaking at 37'C on a salts medium containing (a) 0,U% glucose and (b) 0,8$ acetate. The medium of Sm K12 c e l l s growing on glucose was supplemented with 1000 micrograms DHSM/ml. -109-DISCUSSION d s The observation that 'Sm * and Sm Escherichia c o l i K12 grown on acetate medium could not be induced to form beta-galactosidase by the addition of the gratuitous inducer TMG or lactose and that subsequent growth on glucose was delayed (Pig. 18), indicated that the permeability character-i s t i c s of the membrane may have changed. A change i n membrane structure was implied also by the.rf,inding that Escherichia  c o l i K12 which had been adapted to growth on acetate had assumed a coccoid morphology. A change i n the surface prop-e r t i e s of the membrane of acetate-adapted b e l l s was indicated by the observation that i n medium depleted of acetate, the c e l l s clumped together (Pig. 19); subsequent addition of acetate reversed the clumping (Pig. 20). SmS Escherichia c o l i K12 and Escherichia c o l i B which are normally i n h i b i t e d by DHSM le v e l s greater than 10 micro-grams/ml medium, were shown to be insensitive to 1000 micro-grams DHSM/ml medium (Pig. 21B) and the s e n s i t i v i t y of Escherichia c o l i K12 to chloramphenicol (10 micrograms/ml medium) was s i g n i f i c a n t l y delayed during growth on acetate (Pig. 21A). These findings are compatible with the hypo-thesis that the permeability of the membrane to these a n t i -b i o t i c s had changed. Membrane l i p i d s are known to have a considerable i n -fluence not only on the physical properties of the membrane, for example f l u i d i t y , but also on the s e n s i t i v i t y of bacteria to a n t i b i o t i c s (Cronan and Vagelos, 1972; Brown, 1971). The 110-major f a t t y acids observed i n an analysis of Escherichia  c o l i K12 membranes were o l e i c acid (18:1) and palmitic acid (16:0). In a preliminary analysis, the ra t i o of these f a t t y acids i n the membranes of Sms, Smr and Smd Escherichia  c o l i K12 was found to vary, not only according to the strep-tomycin phenotype, but also with carbon source (Table XVIII; Pig. 22). This l a t t e r finding i s surprising in l i g h t of the b e l i e f that carbon source does not influence the character of the membrane l i p i d s (Cronan and Vagelos, 1972). Growth of c e l l s on acetate resulted i n an increase i n the degree of fat t y acid unsaturation i n the membrane. This i s known to increase the f l u i d i t y of the membrane (Cronan and Vagelos, 1972) and may account for the observed change i n morphology of Escherichia c o l i c e l l s growing on acetate*; E s p e c i a l l y remarkable i s the predominance of palmitic acid (16:0) in Smr Escherichia c o l i K12. This may be related to the i n a b i l i t y of DHSM and ethanol to promote a condition of catabolite derepression i n these c e l l s i e . the membrane •A s d may be very r i g i d ; compared to that of the Sm and Sm s t r a i n s . Changes i n a n t i b i o t i c s e n s i t i v i t y and membrane perme-a b i l i t y i n Sms Escherichia c o l l K12 adapted to growth on acetate may be due to the change i n the character of the fa t t y acids in the membrane. The requirement f o r DHSM i n (3. 'Sm ' Escherichia c o l i grown on glucose may be to maintain membrane function. The modified membrane of acetate grown c e l l s may then account f o r the reversion of streptomycin dependence to Independence. On the other .hand, the pre-- I l l -dominant effect of carbon source on a n t i b i o t i c requirement may be related to the phenomenon of catabolite repression. In either case, the observations do not support a theory of dependency based on a ribosomal s i t e of action for the a n t i b i o t i c . 112-GENERAL DISCUSSION Twenty-three spontaneous 'Sm ' mutants of Escherichia co l i . K12 were i s o l a t e d and a l l exhibited relaxed catabolite repression during growth on a glucose-salts medium. The P 5 0 3 (prototetrahydroporphyrin IX) content was low; growth was i n s e n s i t i v e to i n h i b i t i o n by L-valine; catabolite sen-s i t i v e enzymes were derepressed, and the c e l l s excreted excess quantities of amino acids. A l l of these observations may be explained by a state of relaxed catabolite repression. In contrast to previous findings, i t was observed that in place of valine (Bragg and Polglase, 1962; Tirunarayanan et a l , 1962), 'Sm ' mutants of Escherichia c o i l B and Escherichia c o l i K12 excreted glutamic acid. Quantitative analysis established that the mutants excreted up to f o r t y times as much glutamic acid as the wild-type parent. This excretion was eliminated by the addition of traces of iron s a l t s . However, the addition of iron to the medium did not a l t e r the l e v e l of the catabolite sensitive enzyme AHAS, nor did i t r e s u l t i n the alternate excretion of any other amino acid. The p o s s i b i l i t y that some other metabolite such as pyruvate was excreted, was not eliminated. ' The very low l e v e l of P503 previously observed i n 'Sm ' mutants of Escherichia c o l i B and Escherichia c o l l K12 was Interpreted as i n d i c a t i n g a deficiency of this pigment i n these c e l l s (Kamitakahara and Polglase, 1970). However, restoration of catabolite repression i n these o e l l s through a n t i b i o t i c l i m i t a t i o n or change of carbon source, restored the normal l e v e l of P503. It was also established that -113-protoporphyrinogen oxidase synthesis was normally derepressed in these c e l l s thus accounting for the low l e v e l of P503; repression of this a c t i v i t y accompanied an increase i n P503 to wild-type l e v e l s . v The i n h i b i t i o n of growth by L-valine waa shown to depend upon the l e v e l of catabolite repression i n Sm and 'Sm ' Escherichia c o l i K12 and more d i r e c t l y on the l e v e l of a c t i v i t y of AHAS (Whitlow and Polglase, 197U; Whitlow and Polglase, 1975). In contrast to previous reports, it^was found that the d synthesis of AHAS i n 'Sm ' Escherichia c o l i K12 was re-pressed by the presence of four end-products - valine, leucine, isoleucine and pantothenate, i n the growth medium. Bl a t t et a l (1972) and O ' N e i l l and Preundlich (1972) reported that the maximum repression of AHAS required the combination of end-products - valine plus leucine, i n the growth medium. However, under the conditions used i n th e i r experiments, i t was necessary to supplement the medium with O.Of>mM L-iso-leucine (7.3 micrograms/ml) to prevent growth i n h i b i t i o n by L-valine. Hence, 'Sm ' Escherichia c o l i K12 presented an opportunity to test the effect of the indi v i d u a l end-products i n a variety of combinations without the complication of i n h i b i t i o n of growth by L-valine. The data of Coukell and Polglase (1969a) showed that the quantity of DHSM required f o r the growth of »Srad' Escherichia c o l i B, depended upon the carbon source. The general trend was interpreted as suggesting that the re--111+-quirement for DHSM diminished as the a b i l i t y of the carbon source to promote a relaxation of catabolite repression, increased. Using lactate as a carbon source, Coukell and Polglase (1969a) estimated the KQHSM i e . the concentration of DHSM required to maintain the half-maximal growth rate, was as low as 1.5 micrograms/ml. Extension of these studies using a very poor energy source, acetate, as a carbon source showed that the 'Smd' mutants of six strains of Escherichia c o l i i e . B, A, PCI, UL, CRX and K12, did not require DHSM for growth. These results are d i f f i c u l t to reconcile with the current theory of dependency based on a ribbsomal s i t e of action f o r DHSM (Modolell and Davis, 1969b; Garvin et a l , 1973). The l e t h a l i t y of DHSM (or streptomycin) may be explained through an i n h i b i t i o n of protein synthesis. Dependency, however, may r e l y on the requirement f o r a state of catabolite repression. H S Growth of 'Sm 1 and Sm Escherichia c o l i c e l l s on acetate, produced several interesting e f f e c t s . Escherichia c o l i K12 and Escherichia c o l i B demonstrated an i n s e n s i t i v i t y to DHSM (1000 micrograms/ml) and a delayed s e n s i t i v i t y to f ' . chloramphenicol (10 micrograms/ml). Preliminary observa-tions indicated that the membranes of these c e l l s had been altered, i e . gram negative rods were transformed to gram negative c o c c i , permeability appeared to have changed, and the acetate-adapted c e l l s clumped upon exhaustion of the acetate supply i n the medium. - 1 1 5 -This would explain the altered s e n s i t i v i t y of Escherichia  c o l i to a n t i b i o t i c s . However, dependency might also be explained on the basis of an altered membrane. Reports i n the l i t e r a t u r e have established that the membrane l i p i d s of both Smr and »Sm d' mutants d i f f e r e d from'the Sms parent (Laurence and Scruggs, I966; Yaraagami et a l , 1970). In thi s work, preliminary studies with the membranes of acetate-adapted SmS, Smr and 'Sm ' Escherichia c o l i K12 indicated that the r e l a t i v e concentrations of saturated and unsaturated f a t t y acids i n the membrane had changed i n these c e l l s com-pared to the same c e l l s grown on glucose. In agreement with this observation i t has also been observed that the proteins found i n the membrane of Escherichia c o l i . changed the i r r e l a t i v e concentrations under conditions of catabolite derepression (personal communication from R. Reithmeier). Hence, at least two changes can be noted i n Sm and 'Sm ' mutants i e . altered ribosomal protein and altered membrane l i p i d s . Although It i s d i f f i c u l t at this point to define strepto-mycin (or DHSM) dependency i n 'Sm ' Escherichia c o l i , these r e s u l t s have served to establish the possible duality of streptomycin (or DHSM) action i e . a ribosomal e f f e c t , and an effect possibly at the l e v e l of the membrane which results in a state of catabolite derepression during the growth of s d r Sm and 'Sm ' but not Sm c e l l s on glucose. As has been noted previously, not a l l 'Sm ' mutants exhibit a state of catabolite derepression i n the presence -116-of DHSM. Coukell and Polglase ( 1 9 6 9 a ) observed that growth of a 'Smd' Escherichia c o l i . B mutant on gluconate restored a state of catabolite repression. The nature of the de-pendency on DHSM under these conditions i s d i f f i c u l t to explain on the basis of a requirement to maintain a state of catabolite derepression. However, these c e l l s did lose t h e i r dependency on DHSM when grown on acetate. Furthermore, the observation of Coukell and Polglase (1969a) was made on a single representative of this s t r a i n . Hence, i t i s not known i f this i s a general c h a r a c t e r i s t i c of this s t r a i n or an.isolated incidence. That this i s not a general phenomenon i s established by the finding reported here, that 'Sm ' Escherichia c o l i K12 exhibits a state of catabolite derepres-sion during growth on gluconate. Hence, examination of the nature of dependency i n 'Smd' Escherichia c o l i suggests that a state of catabolite dere-pression i s required f o r a function necessary to the v i a b i l i t y of the c e l l . Whether this function is linked with the membrane, the l e v e l of a c t i v i t y of a catabolite sensitiveV enzyme or i s ribosomal in nature, i s unknown. However, the results do show that the normal assembly of the ribosome and- normal ribosomal function, do not require the physical presence of streptomycin (or DHSM) i n 'Smdt c e l l s growing on acetate. -117-SUMMARY OF MAJOR FINDINGS •Smd' Escherichia c o l i K12 cultures exhibit a general state of catabolite derepression during growth on glucose i n the presence of non-limiting concentrations of DHSM ( i e . greater than 100 micrograms/ml). Various phenotypic c h a r a c t e r i s t i c s i e . i n s e n s i t i v i t y of growth to i n h i b i t i o n by L-valine, reduced accumulation of Pj?03, excess excretion of amino acids, and derepressed catab-o l i t e sensitive enzymes can be explained on this basis. Acetohydroxy aeid synthase i s controlled by catabolite j s repression i n 'Sm ' and wild-type Sm Escherichia c o l i £12. Moreover, the s e n s i t i v i t y of growth to i n h i b i t i o n by l - v a l i n e depends on the degree of catabolite repression i n the c e l l Six strains of 'Smd' Escherichia c o l i - K12, B, A, PCI, UL and CRX l o s t t h e i r dependency on DHSM during growth on acetate. Furthermore, those Sms- Escherichia c o l i strains tested i e . Escherichia c o l i K12 and Escherichia  c o l i B, were shown to lose t h e i r s e n s i t i v i t y to DHSM under these growth conditions. The l a t t e r results may possibly be interpreted on the basis of membrane ef f e c t s . The r a t i o of unsaturated/saturated f a t t y acids i n the membrane of Escherichia c o l i . varies with the strepto-mycin phenotype and the carbon source. - 1 1 8 -SUOGESTIONS FOR FUTURE WORK The r e s u l t s i n this thesis suggest that examination of a few of the condi t i o n a l l y ' Sm d | mutants be per-formed i n order to determine i f the suppression of the mutations could be accomplished by an a l t e r a t i o n of growth conditions as opposed to the controlled mis-reading e f f e c t attributed to t h i s a n t i b i o t i c . The effect of iron ions on eliminating the excess excretion of glutamic acid i n 'Smr1 Escherichia c o l i was not f u l l y explained. Further investigation might determine an Interesting aspect of regulation of the biosynthesis of this amino acid. 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