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Studies in the mechanism of action of streptomycin Autor, Dorothy Anne Pomeroy 1957

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STUDIES IN THE MECHANISM OF ACTION OF STREPTOMYCIN by DOROTHY ANNE POMEROY AUTOR B. A., University of B r i t i s h Columbia, 1956 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of BIOCHEMISTRY We accept t h i s thesis as conforming to the required standard Members of the Department of Biochemistry THE UNIVERSITY OF BRITISH COLUMBIA September 1957 I ABSTRACT Using three d i f f e r e n t streptomycin variants of Escherichia c o l i the mechanism of action of streptomycin, as dihydrostreptomycin, has been studied. The variants used were either susceptible to, resistant to or dependent upon, the presence of dihydrostreptomycin during growth. It i s a well-known fa c t that streptomycin, i n s u f f i c i e n t quantities, w i l l form a p r e c i p i t a t e with nuc l e i c acids, thus the p r e c i p i t a t e formed from dihydrostreptomycin and b a c t e r i a l c e l l contents was studied. Streptomycin also i n h i b i t s the formation of proteins, as exemplified by adaptive enzyme production, when i t i s added, during induc-t i o n , to susceptible organisms. Corresponding e f f e c t s c o r r e l a t i n g growth and synthesis have been noted, also, with the dependent and res i s t a n t variants. Using these facts and the fact that adaptive enzymes can be removed by the p r e c i p i t a t i o n of c e l l contents with dihydrostreptomycin, regeneration of enzyme a c t i v i t y from the p r e c i p i t a t e was attempted with polymethacrylic acid. This compound w i l l d issociate protamine-nucleic acid complexes but proved i n e f f e c t i v e i n regenerating enzyme a c t i v i t y . By using dihydrostreptomycin p r e c i p i t a t e s of d i s -rupted susceptible Ej. c o l i the observation was confirmed that DNase i s i n e f f e c t i v e i n depolymerizing these p r e c i p i -t a t e s . Dihydrostreptomycin p r e c i p i t a t e s of susceptible E. c o l i are p a r t i a l l y depolymerized by RNase, however. Analysis i n the ultra-centrifuge was carried out on c e l l - f r e e material from susceptible E. c o l i on the addition of RNase, DNase and low, b i o l o g i c a l l y active con-centrations of dihydrostreptomycin. RNase and dihydro-streptomycin both removed two of the three t y p i c a l components shown to be present i n susceptible E. c o l i c e l l s . DNase had no e f f e c t on these components. Purine and pyrimidine analysis of these dihydro-streptomycin pre c i p i t a t e s from E. c o l i showed an absence of u r a c i l , as such, but a r e l a t i v e l y unaffected thymine content which indicates that i t i s mainly DNA rather than RNA affected by the dihydrostreptomycin. From these and previously observed facts the follow-ing theory i s advanced. Streptomycin reacts with DNA to form a complex which i n susceptible E. c o l i i s not depoly-merized by DNase. This complex formation between dihydro-streptomycin and DNA prevents protein synthesis as exemplified by adaptive enzyme formation. Because depoly-merization of DNA by DNase i s prevented, c e l l d i v i s i o n cannot take place i n the presence of dihydrostreptomycin. And because the d i r e c t i n g e f f e c t of DNA on enzyme production i s blocked, t h i s process cannot occur. It must then follow that i n the streptomycin-dependent E. c o l i . the a n t i b i o t i c i s required f o r the depolymerization of DNA and f o r the d i r e c t i n g e f f e c t of DNA on protein synthesis. In presenting t h i s thesis i n p a r t i a l fulfilment of the requirements fo r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representative. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of cJjL^*<JL*XZg The University of B r i t i s h Columbia, Vancouver 8. Canada. Pate Qye~-£c»i* •A.fc-c/ / y ACKNOWLEDGMENT I would l i k e to thank Dr. W. J . Polglase very sincerely f o r the invaluable assistance and encouragement he has given me throughout t h i s project. I would also l i k e to thank Dr. Charles A. Dekker of the University of C a l i f o r n i a f o r a sample of highly polymerized thymus DNA, and Mr. A. T. Matheson of the University of Toronto for a sample of polymethacrylic acid. I was aided, f i n a n c i a l l y , by a research assistanceship granted by the Defense Research Board of Canada. \ INTRODUCTION Although the antibiotic activity of streptomycin, whereby the presence of this compound inhibits proliferation of susceptible gram negative bacteria, has been known for a number of years (37)> its mode of action has s t i l l not been elucidated clearly and unequivocally. Work on the chemical structure of streptomycin was carried out from 194-5 to 1948 and due to the cooperation of a number of research groups, the formula is now known (1). HIS / c v tSH H N - C - N - H C C H - R - C - N H ; , STREPTOBIOSAMINE Z | | , s\ H O - C C H C C C H \ / I I O H / \ H O H ° N K t = 0 | | 5 H C H H O H C - C H C H O H 1 \ / C WO I STREPTIDINE STREPTOSE N-METHYLGLUCOSAMINE C H 3 & H The molecule is a base by virtue of the extremely basic guanido groups and the less strongly basic N-methyl group. Neither streptobiosamine nor streptidine has any antibiotic activity. Antibiotically active streptomycin analogues have been produced by variation at the streptose grouping. -2-I^OH l / O H l / O H \ / C N C H , 0 H v / C X ^ 0 H C H 3 C H < 0 H C H ^ H DIHYDROSTREPTOMYCIN HYDROXY- DIHYDRO-HYDROXY-STREPTOMYCIN STREPTOMYCIN Streptomycin has been found to have a d e f i n i t e e f f e c t on some metabolic processes of micro-organisms, i n p a r t i c u l a r , the oxidation of many metabolites. In order to prove that a p a r t i c u l a r metabolic process i s d i r e c t l y i n h i b i t e d by the presence of an a n t i b i o t i c and i s implicated i n the mode of action of t h i s a n t i b i o t i c , the process must react p o s i t i v e l y to the following t e s t . In a micro-organism whose growth i s in h i b i t e d by a c e r t a i n concentration of the a n t i b i o t i c the questioned metabolic process must also be i n h i b i t e d by the same concentration. Also, the process i n a res i s t a n t mutant should be unaffected by the presence or absence of the a n t i -b i o t i c as i s m u l t i p l i c a t i o n . And, the dependent mutant requiring the a n t i b i o t i c f o r growth must also show a d e f i n i t e a n t i b i o t i c requirement f o r the occurrence of the process i n question. A great deal of work on the mode of action of strep-tomycin was done by W. W. Umbreit (29, 41-44). From i t , he postulated that the reaction i n h i b i t e d by streptomycin i n sensit i v e Escherichia c o l l was the oxalacetate-pyruvate con-densation of the t r i c a r b o x y l i c acid cycle. He found that -3-the streptomycin res i s tant and dependent var iant s of E . c o l i d id not possess the a b i l i t y to affect the oxalacetate-pyruvate condensation i n detectable amounts. Thus i t appeared that the a b i l i t y of the re s i s tant or dependent organism to grow i n the presence of streptomycin depended upon the a b i l i t y of the c e l l to replace t h i s condensation mechanism with another r e a c t i o n . The work of Stern and Ochoa, Ogstnn, Potter and Heidelberger i n which c i t r a t e was found to be the product of condensation of oxalacetate and pyruvate l e d Umbreit to study c i t r a t e production with regard to streptomycin tolerance i n E . c o l i . According to h i s work there was no i n h i b i t i o n of c i t r a t e production by streptomycin i n sens i t ive E . c o l i . A l s o , the res i s tant v a r i a n t , although unable to ox id ize oxalacetate and pyruvate accumulated considerable amounts of c i t r a t e . From t h i s data Umbreit stated that c i t r a t e was only a by-product and that the acetate-oxalacetate condensation could not be the ma^or pathway f o r the entrance of pyruvate in to the terminal re sp ira tory system. Th i s was disproved by other workers. Umbreit f i n a l l y has postulated a seven-carbon phosphorylated compound, 2-phospho-4-hydroxy-4-carboxy ad ip ic ac id to be the intermediate metabolite i n oxalacetate-pyruvate condensation i n E . c o l i . He found product ion of t h i s proposed compound i n susceptible E . c o l i to be i n h i b i t e d by streptomycin. In 1948, a group of workers (15) c a r r i e d out tes ts on the effect of streptomycin on metabolism of r e s t i n g b a c t e r i a and on c e r t a i n p u r i f i e d enzymes. They found that streptomycin -4-i n very high concentrations does not inhibit catalase, carbonic anhydrase, cytochrome-cytochrome oxidase, succin-oxidase carboxylase, urease or trypsin. In concentrations which are just bacteriostatic, streptomycin noncompetitively inhibits the metabolism of certain carbohydrate intermediates i n susceptible strains of Staphylococcus aureas, Bacillus cereus, and S h i g i l l a sonnei. These processes i n resistant organisms were l i t t l e , i f at a l l , affected by high concentra-tions of streptomycin. It was postulated that streptomycin either inhibits an enzyme or enzymes involved i n carbohydrate metabolism or inhibits their formation. The inhibitions studied resulted i n accumulation of acetate. Whether or not the inhibition of metabolic functions observed bears a causal relationship to the antibiotic activity of streptomycin could not be concluded by this group. Fitzgerald, Bernheim and Fitzgerald (12) were the f i r s t workers to demonstrate the inhibition by streptomycin of adaptive enzyme formation. Benzoic acid i s oxidized i n certain Mycobacteria by the formation of an adaptive enzyme. The presence of streptomycin during the inductive period inhibited formation of the enzyme and thus, oxidation of benzoic acid. Streptomycin had l i t t l e effect on the c e l l s which were preincu-bated with streptomycin and i n which the adaptive enzyme was already formed. Earlier, S. S. Cohen (5>6) observed that streptomycin w i l l form complexes with nucleic acids, but not with nucleic acids depolymerized with nucleases. These complexes are - 5 -polymeric compounds whose size depends on the combining r a t i o of bivalent base to multivalent nucleates* At some r a t i o s l a t t i c e formation continued u n t i l p r e c i p i t a t i o n occurred. Massart found that ribonucleic acid, as well as the desoxy-nucleic acid tested by Cohen, w i l l form complexes with strep-tomycin ( 2 6 ) . F i t z g e r a l d et. a l . suggested that i n view of Cohen's work i t i s probable that t h i s complex formation i s part of the mechanism by which streptomycin i n h i b i t s the formation of adaptive enzymes since nucleoproteins are apparently concerned with enzyme formation (10). Whether t h i s effect of strepto-mycin could also explain the i n h i b i t i o n of growth depends on how important the formation of adaptive enzymes i s to the normal functioning of the c e l l s . F i t z g e r a l d and co-workers have stated that i t was probable the i n h i b i t i o n of adaptive enzyme formation i s only one aspect of the mechanism of strep-tomycin action. The question of the r e l a t i v e importance of the formation of adaptive or Induced ( 4 ) enzymes to normal c e l l function i s answered when i t i s re a l i z e d that i f adaptive enzyme synthesis follows the same pathways as a l l enzyme (protein) synthesis, i t i s expected that streptomycin i f i t affects formation of adaptive enzymes would act i n the same manner f o r synthesis of any enzyme. I t has been observed (30) that a l l preparations of E. c o l i worked with contained trace amounts of / 3 - g a l a c t o s i -dase before induction with lactose. This follows from the statement made by Stanier that "given a s u f f i c i e n t l y sensitive assay, there are probably few inducible enzyme systems whose a c t i v i t y i s completely undetectable before induction n and that "the e f f e c t of induction i s to cause a great acceleration i n the d i f f e r e n t i a l rate of synthesis of the enzyme i n question" (40). Polglase, i n accordance with t h i s view has interpreted the action of streptomycin as a f f e c t i n g the d i f f e r e n t i a l rate of synthesis of enzyme. According to Gale and Folkes (13) there i s a strong positive c o r r e l a t i o n between the rate of protein synthesis and the nucleic acid content of the b a c t e r i a l c e l l , the former f a l l i n g to zero when the l a t t e r has f a l l e n to 4 per cent. Yeas and Brawerman (46) postulate that the formation of r i b o -nucleic acid and protein i s coupled because both are formed from common precursors. And f i n a l l y Reiner and Goodman recently found (34) that polynucleotides of the pentose type seem to be connected with adaptive enzyme synthesis. They state that genes may possibly control enzyme formation through the occurrence of nucloprotein intermediaries, synthesized by or under the supervision of genes. This can be r e a d i l y tested i n micro-organisms which can be induced to form new enzymes. The induced c e l l s should contain nucleoprotein f r a c t i o n s with s p e c i f i c b i o l o g i c a l a c t i v i t y not present p r i o r to induction. This was found to be true. However, most of the protein could be removed from the polynucleotide preparation without damaging i t s inducing a c t i v i t y . Some recent work has supported the hypothesis that a process i n h i b i t e d by streptomycin i n susceptible micro--7-organisms i s that of enzyme formation. I t was demonstrated that the e f f e c t of streptomycin on the p r o l i f e r a t i o n of sus-ceptible, r e s i s t a n t and dependent variants of Escherichia c o l i correlates with i t s e f f e c t on the adaptive enzyme formation of these v a r i a n t s . M i l l e r and Bohnhoff (28) f i r s t found, with meningococci, that organisms which were o r i g i n a l l y suscep-t i b l e to streptomycin could give r i s e to two d i f f e r e n t types of mutants, streptomycin dependent and streptomycin r e s i s t a n t . This was true, also, f o r any streptomycin analogue with a n t i -b i o t i c a c t i v i t y . Enzymes f o r the oxidation of L-arabinose, lactose and D-glucuronic acid are not normally found i n detectable amounts i n E. c o l i . These enzymes could be produced, however, by the addition of the inducing substrate to a non-proliferating c e l l suspension. Measurement of the enzymes was done by manometric measurement of oxygen uptake. In the presence of dihydro-streptomycin ( q u a n t i t a t i v e l y equivalent to streptomycin with respect to b i o l o g i c a l a c t i v i t y ) susceptible E. c o l i would neither p r o l i f e r a t e nor show adaptive enzyme production (36). Resistant E. c o l i , which m u l t i p l i e s i n the presence or absence of dihydrostreptomycin shows the same behavior with respect to formation of induced enzymes. The process of adaptation was somewhat enhanced by the addition of dihydrostreptomycin to dependent variants which require dihydrostreptomycin f o r c e l l m u l t i p l i c a t i o n . It was found to be impossible to determine the behavior of the dependent mutant with respect to adaptive enzyme formation i n the complete absence of dihydrostreptomycin - 8 -since a c e r t a i n amount i s absorbed during growth and cannot be removed. In order to confirm and extend t h i s work the e f f e c t of dihydrostreptomycin was studied on the induction of a single enzyme, /3-galactosidase, which can be measured quan-t i t a t i v e l y and s p e c i f i c a l l y ( 3 D . In susceptible E. c o l i the amount of dihydrostreptomycin ( 2 5 to 3 0 units per ml.) which i n h i b i t s c e l l m u l t i p l i c a t i o n also i n h i b i t s formation of the induced enzyme. Large amounts ( 5 0 0 units per ml.) of dihydrostreptomycin did not i n h i b i t /3-galactosidase formation i n either r e s i s t a n t or dependent E. c o l l . Dihydrostreptomycin dependent E. c o l l showed ready formation of adaptive enzyme when dihydrostreptomycin was present but formation i n dihydro-streptomycin-depleted dependent c e l l s In the absence of added dihydrostreptomycin was s l i g h t ( 3 2 ) . Addition of dihydro-streptomycin to the depleted c e l l s permitted the renewed synthesis of induced enzyme. The concentration of dihydro-streptomycin which shows an e f f e c t on induced enzyme formation i n dependent E. c o l i i s of the same order of magnitude as the concentration a f f e c t i n g c e l l m u l t i p l i c a t i o n . Reported also was the f a c t that when dependent c e l l s are incubated with lactose then disrupted by sonic o s c i l l a t i o n , the enzyme a c t i v i t y of c e l l - f r e e preparations i s not increased by the addition of dihydrostreptomycin. However, i t has been shown that with c e l l - f r e e preparations of lactose-induced sus-ceptible E. c o l i added dihydrostreptomycin w i l l reduce induced enzyme a c t i v i t y ( 3 3 ) « -9-Similar Investigations as to the c o r r e l a t i o n of streptomycin e f f e c t s on c e l l p r o l i f e r a t i o n and enzyme induction have been carried out with another variant of E. c o l i . the "resistant-dependent" organism ( 3 0 ) . Non-p r o l i f e r a t i n g suspensions of r e s i s t a n t E. c o l i grown with dihydrostreptomycin formed induced enzyme i n the presence or absence of dihydrostreptomycin. However, when the r e s i s t a n t mutant was grown i n the absence of dihydrostreptomycin the non-proliferating suspension showed a s i m i l a r reaction only i n the presence of added dihydrostreptomycin. In the absence of the a n t i b i o t i c a much longer induction period was required before enzyme production began although the amount of enzyme f i n a l l y produced was the same. This " p a r t i a l " dependence was duplicated exactly with regard to c e l l p r o l i f e r a t i o n of t h i s mutant. A study of the e f f e c t of dihydrostreptomycin on sensi-t i v e Mycobacterium p h l e i with respect to acetate oxidation was included i n the above mentioned work ( 3 0 ) . The oxidation of acetate i s , according to these workers, probably an example of a general phenomenon of synthesis of additional enzyme by addition of excess substrate to non-proliferating c e l l s . I f dihydrostreptomycin i s present during t h i s period enzyme syn-thesis i s i n h i b i t e d . This occurs at l e v e l s of dihydrostrep-tomycin which i n h i b i t s the multiplication of Mycobacterium p h l e i . Reinvestigation of the reaction of streptomycin with nucleic acids was also carried out by the group under W. J . Polglase. A f t e r the work done by Cohen (5,6) and F i t z g e r a l d 10 et a l . (12), Donovick and co-workers (9) c a r r i e d out experi-ments which indicated that there was no connection between the reaction of streptomycin with desoxynucleic acid and the a n t i b i o t i c a c t i v i t y of streptomycin. Quantitative studies of the e f f e c t s of various ions on the action of streptomycin as an a n t i b i o t i c , as well as i t s a b i l i t y to p r e c i p i t a t e desoxy-nucleic acid were carried out. Interference of these processes by ions was recorded i n both cases but the order of a c t i v i t y of the s a l t s was d i f f e r e n t i n each case. Donovick et a l . found also i n studies with desoxyribonuclease that t h i s enzyme depolymerized desoxynucleic acid even though i t was complexed with streptomycin. Desoxyribonuclease, which w i l l penetrate b a c t e r i a l c e l l walls was then added to a culture of streptomycin susceptible b a c t e r i a along with streptomycin. Desoxyribonuclease had no effect here on modifying the growth-inhibiting powers of streptomycin. Therefore the workers concluded there was no connection between the desoxynucleic acid p r e c i p i t a t i n g a b i l i t y and a n t i b i o t i c e f f e c t of streptomycin. Polglase, however, (30) showed that there was a d i f f e r -ence of nuclease reaction between b a c t e r i a l nucleic acids and commercial (animal) samples. Insoluble dihydrostreptomycin p r e c i p i t a t e s of a sample of highly polymerized thymus desoxynu-c l e i c acid were dissolved by the addition of desoxyribonuclease. Also dissolved, but to a l e s s e r extent, was the insoluble complex formed from commercial ribonucleic acid and dihydrostreptomycin plus added ribonuclease. Neither desoxyribonuclease nor ribonuclease has any effect towards di s s o l v i n g the insoluble -11-p r e c i p i t a t e formed from dihydrostreptomycin and c e l l - f r e e extracts of sonic-disrupted E. c o l i . Measurements were made i n a spectrophotometer by recording the decrease at 420 millimicrons i n t u r b i d i t y of suspensions of the dihydrostrep-tomycin-nucleie acid p r e c i p i t a t e s . In t h i s laboratory (33) too, i t was found that the induced / 3-galactosidase a c t i v i t y i n c e l l - f r e e sonicates could be reduced by about one-half either by addition of 1000 units per ml. of dihydrostreptomycin or by u l t r a c e n t r i f u g a t i o n at 50,000 x g. This was thought to be additional evidence showing that at some stage during t h e i r formation, enzymes are attached to nucleic acid material. The work presented here i s an attempt to f i n d more evidence f o r the possible connection between the a n t i b i o t i c action of streptomycin and i t s action on nucleic acids from the various streptomycin-mutants of E. c o l i . Assuming that nucleic acids are responsible i n some way f o r enzyme synthesis, any observed differences i n nucleic acids from E. c o l i variants perhaps may contribute to the knowledge of the mode of action of streptomycin. EXPERIMENTAL METHODS AND MATERIALS A t o t a l of three variants of Escherichia c o l i were used: streptomycin-susceptible (growth i n h i b i t e d by approxi-mately 30 units per ml. of streptomycin), streptomycin-resistant (growth continues i n the absence or i n the presence of 1000 units per ml. of streptomycin), and streptomycin-dependent (growth requires a minimum of 30 units per ml. of streptomycin). Growth of the dependent v a r i e t y could be d i f f e r e n t i a t e d according to the amount of streptomycin added to the growth media. Growth i n the minimum amount of streptomycin, 30 units per ml., produces an organism which appears under the microscope as extremely long gram-negative rod forms (47). I f dependent E. c o l i are grown i n adequate amounts of streptomycin (125 to 200 units per ml.) the normal gram-negative, short rod forms appear. Streptomycin was used i n aqueous so l u t i o n i n the form of dihydrostreptomycin s u l f a t e . Dihydrostreptomycin has an a n t i b i o t i c action similar to that of streptomycin but i s more stable ( 3 0 ) . One unit of streptomycin or dihydrostreptomycin i s the equivalent of one microgram of the free base. The same semi-synthetic media was used f o r growth of a l l E. c o l i cultures used. This consisted of K 2 H P O 4 (0 . 7 $ ) , KH2PO4 (0 . 3 $ ) , sodium c i t r a t e (0 . 0 5 $ ) , MgS0 4 (0.01$), - 1 3 -(Mh4)2S04 (0.1$), yeast ( N u t r i t i o n a l Biochemicals Corporation) (0 .5$) and glucose (0.2$).. The appropriate amount of dihydrostreptomycin was added to the medium before inoculation, i f required. B a c t e r i a l c e l l s were harvested after 2G hours growth (logarithmic growth phase (35) ) i n Roux f l a s k s at 3 7 ° C. C e l l s were washed twice with p h y s i o l o g i c a l saline (0.85$ NaCl) and suspended i n M/45 phosphate buffer pH 7«5. I f -galacto-sidase was required, the suspension of c e l l s was incubated with lactose (100 to 200 micromoles per ml.) f o r 2 hours at 3 7 ° C These c e l l s were washed with p h y s i o l o g i c a l s a l i n e and resuspended i n phosphate buffer. A l l centrifugation was carried out at temperatures of from 2° to 5°C. Treatment of the b a c t e r i a l suspension i n a sonic o s c i l l a t o r (Raytheon 9 or 10 k i l o c y c l e s ) was used to obtain c e l l contents ( 3 ) « In a l l cases the material soniced was a thick suspension of bacteria. The amount of nitrogen i n milligrams per ml. was determined by f i n d i n g the o p t i c a l density at 420 millimicrons of the b a c t e r i a l suspension. This figure was compared with a standard curve to obtain the milligrams of nitrogen present. A semi-quantitative picture was thus obtained. Treatment i n the sonic o s c i l l a t o r was carried out f o r f i v e minutes when / 3-galactosidase a c t i v i t y was to be assayed and ten minutes when nucleic acids were to be studied. In a l l cases c e l l envelopes and debris were removed by high speed centrifugation at 14,000 r.p.m. f o r twenty minutes. 14-Beta-galactosidase was analyzed by a modification of the procedure of Lederberg (22,31). To 2.5 nil.aliquots of the sonicates i n 10 mm. absorption c e l l s was added 0.2 ml. portions of a M/200 solution of ortho-nitro - / 3-phenylgalacto-side (ONPB). I f dihydrostreptomycin was used i n the assay 0.5ml. of the appropriate d i l u t i o n was combined with 2.0 ml. of the sonicate and allowed to stand at 37°C f o r one-half hour before ONPG was added. Enzymatic hydrolysis of ONPG to the colored ortho-nitro-phenol proceeded at room temperature. Readings were taken at f i v e minute i n t e r v a l s i n a Beckman Model B spectrophotometer with a wave length of 420 m i l l i m i -crons . Concentrated c e l l sonicates of susceptible E. c o l i to be used f o r o p t i c a l analysis i n the Spinco Model E u l t r a -centrifuge were prepared as described but were soniced i n 10~3 M NaCl (2). In th i s work streptomycin i n h i b i t i o n was tested by the addition of 35 units per ml. of dihydrostrepto-mycin to the sonicates and preceding the analysis t h i s mixture was allowed to stand at room temperature f o r t h i r t y minutes. A 100 microgram per ml. aqueous solution of ribonuclease (N u t r i t i o n a l Biochemicals Corporation) was added to the sonicate i n a r a t i o of 1:4 and incubated at 37°C f o r h a l f an hour. A 206 microgram per ml. aqueous so l u t i o n of desoxyribonuclease ( N u t r i t i o n a l Biochemicals Corporation), 0.05 M MgSO/j. and c e l l sonicate were mixed i n a r a t i o of 1.7*1*10 and incubated f o r h a l f an hour at 37° before analysis. -15-M. Kunitz developed a method f o r the assay of ribonu-clease (RNase) (19) based on the discovery that the digestion of yeast nucleic acid by RNase causes a s h i f t i n the u l t r a v i o l e t absorption to shorter wave lengths (not because of increasing hydrogen ion concentration since t h i s causes move-ment to longer wave lengths). This gradual decrease i n extinc t i o n i s most d i s t i n c t i n the 290 to 305 m i l l i m i c r o n range and can be r e a d i l y measured during the i n i t i a l stages of digestion. The value of E decreases l i n e a r l y with time at 300 millimicrons during i n i t i a l stages of reaction, the rate of decrease being nearly proportional to the concentration of enzyme i n s o l u t i o n . This method was modified somewhat i n order to measure q u a l i t a t i v e l y the e f f e c t of RNase on various solu-t i o n s . Dihydrostreptomycin p r e c i p i t a t e s of commercial ribonu-c l e i c acid (RNA), susceptible E. c o l i sonicate and r e s i s t a n t E. c o l i sonicate were prepared using a f i n a l concentration of 5,000 units per ml. of dihydrostreptomycin. P r e c i p i t a t e s were suspended i n 0.1 M acetate buffer pH 5» Suspended p r e c i p i t a t e (2 mis.) and RNase (2 mis. of an aqueous s o l u t i o n containing 1 microgram per ml.) were separately incubated i n a water bath at 37°C f o r 10 minutes. These were then r a p i d l y mixed, immediately transferred to quartz c e l l s and o p t i c a l density readings taken every minute at 300 millimicrons using a Beekman Model DU spectrophotometer. Kunitz also developed a method fo r the assay of desoxy-ribonuclease (DNase) based on the fact that the digestion of thymus nucleic acid by c r y s t a l l i n e DNase i s accompanied by an -16-increase i n absorption of u l t r a v i o l e t l i g h t by the nucleic acid (20). This increase i s most s i g n i f i c a n t at 260 m i l l i -microns and i s proportional to the concentration of enzyme i n soluti o n . This method, too, was adopted i n order to measure q u a l i t a t i v e l y the eff e c t of DNase on various solutions. Dihydrostreptomycin p r e c i p i t a t e s of commercial sperm desoxynu-c l e i c acid (DNA), a sample of highly polymerized DNA, and susceptible E. c o l i sonicate were prepared using a f i n a l con-centration of 5>000 units per ml. of dihydrostreptomycin. Precipitates were suspended i n 1 M acetate buffer pH 5 contain-ing 0.05 M MgSO^ .. Suspended p r e c i p i t a t e s (3 mis.) containing a t o t a l of 1.8 milligrams MgSO^ and DNase (1 ml. of an aqueous solution containing 10 micrograms per ml.) were separately incubated i n a water bath at 25°C f o r 10 minutes. These solu-tions were then r a p i d l y mixed, immediately transferred to quartz c e l l s and o p t i c a l density readings taken every minute at 260 millimicrons using a Beckman Model DU spectrophotometer. DNase studies were also c a r r i e d out on samples of material i s o l a t e d from ausceptible E. c o l l according to the DNA extraction method of A. S. Jones (17) using c e t y l t r i m e t h y l -ammonium bromide (cetavion). The p r i n c i p l e of the method involves the formation of a nucleoprotein-cetavlon complex which i s soluble i n 1 M NaCl and insoluble i n 0.3 M NaCl. Protein i s removed by a chloroform-octanol (9*1) mixture. Sep-aration of DNA from pentose nucleic acid (PNA) (11) was based on the fac t that the former complexed with cetavlon i s insoluble i n 0.5 M NaCl and the l a t t e r complexed with cetavlon i s soluble - 1 7 -i n 0 .5 M NaCl. Ethanol p r e c i p i t a t i o n , d i a l y s i s against d i s t i l l e d water and freeze-drying produced a s o l i d material with a; nitrogen/phosphorous r a t i o of 5.86 and an u l t r a v i o l e t absorption at 260 millimicrons of 24 .0 using a 1.7 milligram per ml. solution. Nitrogen was determined by the micro-Kjeldahl method (23,24) and phosphorous determined by the method of Jones, Lee and Peacock ( 1 8 ) . Protein was probably the main impurity i n t h i s preparation. Material i s o l a t e d according to the further procedure for PNA proved to be too impure f o r RNase investi g a t i o n . The action of DNase on the i s o l a t e d DNA mater-i a l alone and on the p r e c i p i t a t e formed with 5»000 units per ml. of dihydrostreptomycin was determined according to the p r e v i -ously outlined method. Three E. c o l i variants were grown f o r a n a l y t i c a l studies of purine and pyrimidine contents, susceptible E. c o l i (grown with no added dihydrostreptomycin), dependent E. c o l i (grown with 35 units per ml. dihydrostreptomycin) and dependent E. c o l l (grown with 125 units per ml. dihydrostreptomycin). Heavy suspensions of each were subjected to sonic o s c i l l a t i o n as usual. P r e c i p i t a t i o n of 260 millimicrons absorbing material was carried out using 5»000 units per ml. dihydrostreptomycin. Perchloric acid hydrolysis of the s o l i d material followed according to the standard method modified by Wyatt ( 45 ) . A f t e r hydrolysis and d i l u t i o n the hydrolyzate was neutralized with potassium hydroxide i n the cold, stored at 5°C overnight and then centrifuged to remove the potassium perchlorate p r e c i p i t a t e . The supernatant solu t i o n was placed i n a 10 ml. beaker i n a dessicator containing -18-s o l i d sodium hydroxide and concentrated s u l f u r i c acid. I t remained at 37°C u n t i l the solution was reduced to dryness. The dry material was taken up, i n a known quantity of d i l u t e hydrochloric acid and spotted on Whatman Number 1 chromato-graphy paper. The chromatographic solvent used was an aqueous solution of isopropanol (65$ v/v) and 2 N HC1 (45). The descending method of paper chromatography was used. A f t e r allowing the chromatogram to run f o r approximately twenty hours, the paper was dried, and spots located and photographed under u l t r a v i o l e t l i g h t . Spots were cut out and eluted overnight i n 2 mis. of 0.1 M HC1. These solutions were read and the o p t i c a l densities from 300 to 230 m i l l i -microns recorded with a Beckman Model DK2 spectrophotometer. -19-RESULTS A. Action of Polymethacrylic Acid on P r e c i p i t a t e s Formed  from Dihydrostreptomycin and Cell-Free Sonicates of  Escherichia C o l i Containing the Induced Enzyme, Beta- Galactosidase. Other work has shown that (33) induced enzyme a c t i v i t y can be reduced by the addition of a large concentration of dihydrostreptomycin to b a c t e r i a l sonicates containing formed or forming enzyme* Thus i t was postulated that at some formative stage the enzyme i s attached to nucleic acid material. Polymethacrylic acid, a white, s o l i d polymer of methacrylic acid, has been used to dissociate complexes of protamin and nucleic acid (27)* This experiment was an attempt to regenerate enzyme a c t i v i t y by a s i m i l a r d i s s o c i a t i o n which i t was thought may occur with the dihydrostreptomycin-nucleoprotein (enzyme) complex. Washed, non-proliferating suspensions of E. c o l i variants were induced with lactose but without exogenous nitrogen to form /$ -galactosidase. A f t e r induction the c e l l s were disrupted by sonic o s c i l l a t i o n and assayed f o r enzyme a c t i v i t y . P r e c i p i -t a t i o n was produced by addition of 5»000 units per ml. of dihydrostreptomycin and the p r e c i p i t a t e was co l l e c t e d by c e n t r i -fugation. In a l l cases, dihydrostreptomycin p r e c i p i t a t e s formed from b a c t e r i a l c e l l contents appeared white and compact when centrifuged. In contrast, dihydrostreptomycin p r e c i p i -tates of nucleic acids from animal or yeast sources, although 20 white, appeared to be more di f f u s e after centrifugation. The p r e c i p i t a t e was resuspended i n phosphate buffer (pH 7 » 5 ) and assayed f o r /3 -galactosidase a c t i v i t y . Polymethacrylic acid i n aqueous solution ( t o t a l of 21.5 micrograms) was added to resuspended p r e c i p i t a t e and enzyme a c t i v i t y again assayed. The supernatant solution a f t e r the dihydrostreptomycin p r e c i p i tate was removed was also assayed f o r enzyme a c t i v i t y . A l l concentrations of assay material used were equivalent. The res u l t s are given i n Table I. TABLE I Table I shows the reduction of induced beta-galactosidase a c t i v i t y from Escherichia c o l i sonicate upon the removal of the p r e c i p i t a t e formed by adding dihydrostreptomycin. The Source Mg.Np/mL. of sonicate mpM ONPG hydrolysed per minute by sonicate mpM ONPG hydrolysed per minute by resus-pended d i HS-sonicate precipitates mpM ONPG hydrolysed per minute by resus-pension: + 21.5 pgm. polymeth-a c r y l i c acid mpM ONPG hydrolysed per minute by super-natant from dlHS-sonicate p r e c i p i t a t i o n % Enzyme a c t i v i t y removed by diHS Susceptible E. c o l i 0.138 14560 182 182 5.460 62.5 Resistant E. c o l i (grown with 200 units per ml.diHS 0.124 9100 145.6 109.2 2548 72.1 Dependent E. c o l i (grown with 35 units per ml. diH 0.087 3 2730 91.0 0 72.8 97.0 Dependent E. c o l i (grown with 200 units per ml.diHS 0.111 1820 0 0 182 90.0 -22-B. Beta-Galactosidase A c t i v i t y of Lactose Grown Escherichia  C o l i TABLE II Material Assayed Mg. N 2 per ml. mpM ONPG Hydrolysed per minute Susceptible E. c o l i i n phosphate buffer 0.391 27300 Dependent E. c o l i i n phosphate buffer 0.388 11830 Susceptible E. c o l i i n 0.8$ saline 0.391 4550 Dependent E. c o l i i n 0.8$ saline 0.388 2730 Table II shows /3-galactosidase l e v e l s i n susceptible and dependent E. c o l i grown with the usual synthetic medium but with 0.2% lactose instead of 0.2% glucose as the carbon source. In t h i s case the enzyme was induced i n growing rather than i n resting c e l l s . The dependent E. c o l l was grown with 200 units per ml. dihydrostreptomycin. -23-C. A n a l y t i c a l Ultracentrifuge Studies of Susceptible  Escherichia C o l l Sonicates with Low Concentrations  of Dihydrostreptomycin, Ribonuclease and Desoxy- ribonuclease TABLE III Table I I I shows the v a r i a t i o n of components as detected i n the ultracentrifuge of sonic extracts of E. c o l i Solutions Analysed X Gravity S Tempera-ture °C Heavy Component Medium Component Light Component E. c o l i Sonicate 187,100 2.64 1.98 .39 l8 .5°-20 .4° E. c o l i Sonicate + RNase 187,100 - - .33 20.1°-22.3° E. c o l i Sonicate + DNase 187,100 K>3. 1.98 .33 20.1°-22.3° E. c o l l Sonicate + 35 units per ml. diHS 187,100 - - .39 17.3°-18.8° The sedimentation constant, S, i s defined as the rate of sedimentation per unit f i e l d of force. The d e f i n i t i o n assumes convection-free sedimentation and applies s t r i c t l y only to d i l u t e solutions which are large compared with the molecules - 2 4 -of the medium and are e l e c t r i c a l l y neutral with regard to the medium. The sedimentation constant i s calculated according to the formula: S = logs x 2 ^ x l * 1 X 2 _ * T t 2 - t x = 2.303 ( 20 )2 ( l o g 1 0 x i ) r ' p ' m - x 7 r t o - fa where <i i s the angular v e l o c i t y of rot a t i o n ; x 2 and x-^  are the distances from the axis of ro t a t i o n to a point i n the c e l l where the solution has the maximum concentration gradient, the boundary, at time t 2 and t ^ respectively. Figures I, I I , III and IV are tracings of ultracentrifuge photographs. -25-3 3 , 3 . , 3 3 3 Figure 1 3 Figure II The arrow indicates the d i r e c t i o n of r a d i a l migration. Figure 1 shows the sedimentation of E. c o l i (susceptible) sonicate; figure I I , the sedimentation of E. c o l i (susceptible) sonicate with RNase added. 3 -26-3 1 3 1 3 3 Figure III Figure IV The arrow indicates the d i r e c t i o n of r a d i a l migration Figure III shows the sedimentation of E. c o l i (susceptible) sonicate with DNase added} Figure IV, the sedimentation of E. c o l i (susceptible) sonicate with dihydrostreptomycin (35 units per ml.) added. - 2 7 -Reaction of Dlhydrostreptomycin-Nucleic Acid P r e c i p i t a t e  with Nucleases TABLE IV Reaction with DNase : Material Assayed Increase i n Optical Density Units at 260 m\i a f t e r 15 minutes treatment with DNase Commercial Sperm DNA + DlHS pre c i p i t a t e 0.020 Highly Polymerized DNA • DiHS pre c i p i t a t e 0.024 Susceptible E . c o l i Sonicate + DiHS pr e c i p i t a t e 0 Cetavlon Isolated "DNA" material 0.029 Cetavlon Isolated "DNA" Material + DiHS pr e c i p i t a t e 0.013 -28-T A B L E V Reaction with RNase : Material Assayed Decrease i n Optical Density Units at 300 mp Aft e r 15 minutes treatment with RNase Commercial RNA + DiHS pr e c i p i t a t e 0.014 Susceptible E . c o l i + DiHS pr e c i p i t a t e 0 Resistant E. c o l i + DiHS p r e c i p i t a t i o n 0.020 E. Purine and Pyrimldine Analyses on Dihydrostreptomycin Precipitates of Three Escherichia C o l i Mutants TABLE VI P r e c i p i t a t i o n by dihydrostreptomycin of 260 millimicron absorbing material from sonicates Mutant Analyzed Mg. N2/M1. or Sonicate To t a l Mg.K2 U l t r a v i o l e t Absorption at 260 my of c e l l - f r e e Sonicate % of U l t r a v i o l e t Absorption at 260 my removed by p r e c i p i t a t i o n of Sonicate with 5,000 units per ml.DiHS Hydrolysate (1 Mg. s o l i d DiHS p r e c i p i -tate per ml.) U l t r a v i o l e t absorption at 260 mp . Susceptible E. c o l i 0.435 47.85 8.93 24.8$ 0.86 Dependent E. c o l i Tgrown with 35 units per ml. diHS.) 0.435 37.84 7.58 56.1$ 1.35 Dependent E. c o l i Tgrown with 125 units pe ml. diHS) 0.447 r 122.9 5.75 18.6$ 1.61 Analysis after chromatographic separation! TABLE VII Location of Spot on Chromatogram Standard Susceptible E. c o l i Dependent E. c o l i (grown with 35 units per ml. DiHS) Dependent E. C o l i (grown with 125 units per ml. DiHS) Base Rf Rf |jM base per ml. of 0.1 N HCl eluate . Ratio of U.V. absorption maxima at 260 my Rf pM base per ml. of 0.1 N HCl eluate Ratic of U. V. ab-sorp-t i o n maxi-ma at 260nu Rf ptlbase per ml. of 0.1 N HCl eluate Ratio of U.V. absorp-t i o n maxima at 260 mu Spot 1 Guanine 0.21 0.25 0.059 1.00 0.2: 0.029 1.00 0.19 0.062 1.00 Spot 2 Adenine 0.32 0.35 0.053 1.09 0.32 0.023 0.935 0.29 0.050 0.953 Spot 3 Cytosine 0.42 0.45 0.055 0.86 0.42 0.022 0.715 0.39 0.037 0.545 Spot 4 U r a c i l 0.63 0.60 - 0.296 at 273 mu - - 0.256 al 273 mu - -0.162 at 273 mp 0.262 at 26l my 0.295 at 261 mu 0.179 at 261 mp Spot 5 Thymine 0.71 0.70 0.078 0.84 0.72 0.028 0.655 0.65 0. 061 0.314 Table VII shows the analysis by u l t r a v i o l e t absorption of bases is o l a t e d chromatographically from E. c o l i mutants -31-Figure V <• Figure V shows chromatograms of hydrolysates of dihydrostrep-tomycin precipitates from sonicates. Photographs were taken under u l t r a v i o l e t l i g h t of t y p i c a l chromatograms of samples analyzed. - 3 2 -DISCUSSION The attempt to regenerate a c t i v i t y of the induced enzyme, / 3-galactosidase, from dihydrostreptomycin-bacterial sonicate precipitates using polymethacrylic acid was unsuccessful. This re s u l t could show only that polymethacrylic acid does not have the a b i l i t y to dissociate the proposed nucleoenzyme-dihydrostreptomycin complex but not that t h i s complex does not e x i s t . It i s known that streptomycin w i l l p r e c i p i t a t e nucleo-protein as well as DNA and RNA but i t w i l l not pr e c i p i t a t e free proteins (7>8). Thus i t seems l o g i c a l that the p r e c i p i t a t e formed from dihydrostreptomycin and b a c t e r i a l sonicate which results i n loss of enzyme a c t i v i t y should be some kind of com-bination of nucleic acid and enzyme. It was noted that even with extremely d i l u t e solutions of polymethacrylic acid, a white filmy p r e c i p i t a t e occurred on i t s addition to combinations of b a c t e r i a l sonicate and dihydro-streptomycin. The cause of t h i s i s not known. Resting c e l l suspensions of dependent jS. c o l l . grown either with 35 or with 200 units per ml. of dihydrostreptomycin, then induced with lactose to form /3-galactosidase are shown to form much les s enzyme than does the susceptible E. c o l i under the same conditions. This i s also true f o r / 3-galactosidase pro-duced from these variants under maximal growth conditions (presence of a nitrogen source) with lactose as the carbon source. Overall amounts of / 3-galactosidase i n both variants -33-are greater when lactose i s the carbon source i n a maximal media than when i t i s added as an inducer to resting, non-p r o l i f e r a t i n g c e l l s . During a procedure f o r the i s o l a t i o n of /3-galactosidase, Monod (16) used streptomycin to pr e c i p i t a t e and remove nucleic acids from E. p o l l extracts. Contrary to our results he reported no loss of enzyme a c t i v i t y on p r e c i p i t a t i o n with strep-tomycin. However, Monod produced /3-galactosidase by growth with lactose i n a nitrogen containing medium instead of induction of resting c e l l s with added lactose. This, plus the fac t above shown, that more enzyme i s formed by growing c e l l s than by resting c e l l s may account f o r the difference. I t i s possible that i n growing c e l l s l i t t l e of the protein remains attached to nucleic acid material whereas i n the res t i n g c e l l s with no exogenous nitrogen source the greater part of the enzyme remains attached and i s thus p r e c i p i t a b l e by streptomycin. U l t r a c e n t r i f u g a l analysis of the e f f e c t s of RNase, DNase and low ( a n t i b i o t i c a l l y active) concentrations of dihydrostreptomycin showed that RNase completely removed two of the peaks t y p i c a l of susceptible E. c o l i sonicates whereas DNase had no eff e c t on these peaks. T h i r t y - f i v e units per ml. of dihydrostreptomycin had the same eff e c t as RNase i n that i $ completely removed these charac-t e r i s t i c peaks from the b a c t e r i a l sonicate. The reason f o r this i s not known but the e f f e c t of RNase may come about from the rapid depolymerization of the heavier two peak components thus resulting i n a disappearance of these peaks. The action of - 3 4 -dihydrostreptomycin may occur by the rapid compiexing of these same p a r t i c l e s which cause the t y p i c a l peaks. This may mean that they are just as quickly removed by sedimentation and do not appear on analysis. Thus RNase and dihydrostreptomycin may act on the same type of material i n the c e l l since they give the same a n a l y t i c a l p i c t u r e . Since low, b i o l o g i c a l l y active concentrations of dihydrostreptomycin were used, t h i s information may be s i g n i f i c a n t i n r e l a t i o n to the action of the a n t i b i o t i c . The work on nucleases and dihydrostreptomycin-nucleic acid precipitates seems to confirm previous work ( 3 0 ) that nucleases d i f f e r e n t i a t e between pre c i p i t a t e s of dihydrostreptomycin-nucleic acid material from animal sources and from b a c t e r i a l sources. And i t seems to disagree with the work of Donovick et a l . ( 9 ) previously outlined i n which i t was concluded that the a b i l i t y of streptomycin to p r e c i p i t a t e DNA has l i t t l e to do with the a n t i - b a c t e r i a l action of the a n t i b i o t i c . However, i t i s appropriate here to mention work done by Massart ( 2 6 ) i n which i t was found that nucleoproteins of yeast combined with streptomycin as electroabsorptive complexes are not attacked by nucleases. Gros and Rybak (14) confirmed t h i s using d i f f e r e n t techniques. They suggest the cause i s the i n h i b i t i o n of RNase by streptomycin. However, they used a much greater concentration of streptomycin, 2 0 , 0 0 0 units per ml., than was used i n t h i s work. Thus the results may not be s t r i c t l y comparable. - 35 -R e s u i t s from p y r i m l d i n e and p u r i n e a n a l y s i s of d i h y d r o -streptomycin p r e c i p i t a t e s of E . c o l i v a r i a n t s o n i c a t e s showed t y p i c a l peaks f o r guanine, adenine, c y t o s i n e and thymine. Each of these bases showed u l t r a v i o l e t a b s o r p t i o n maxima on the DK2 spectrophotometer at the c o r r e c t wave l e n g t h . The spot l o c a t e d i n the p o s i t i o n i n which u r a c i l i s g e n e r a l l y found on the chromatogram and w i t h the same Rf v a l u e (O.6O-O.63) as the standard u r a c i l , d i d not show a t y p i c a l u r a c i l a b s o r p t i o n maximum. Instead of showing 257^5 m i l l i m i c r o n s , the m a t e r i a l from the Rf O .63 zone o f the h y d r o l y z a t e from s u s c e p t i b l e E . c o l i showed a maximum at 273 m i l l i m i c r o n s and s i m i l a r zones from h y d r o l y z a t e s of n u c l e i c a c i d s from dependent organisms showed maxima at 2 6 l m i l l i m i c r o n s . I n a l l t h r e e cases the amount of m a t e r i a l recovered from t h i s p o s i t i o n was a great d e a l l e s s than from zones of the o t h e r bases. The u l t r a v i o l e t a b s o r p t i o n r a t i o s f o r the p u r i n e s d i d not v a r y e s s e n t i a l l y among the t h r e e organisms. C y t o s i n e showed a s l i g h t decrease from s u s c e p t i b l e to dependent (35 u n i t s ) and from dependent (35 u n i t s ) to dependent (125 u n i t s ) E. c o l i . The m a t e r i a l from spot 4 was e s s e n t i a l l y unchanged from mutant to ffiutant. The g r e a t e s t change, however, was observed w i t h thymine, the component o f DNA. The amount of thymine from d i h y d r o s t r e p t o -mycin p r e c i p i t a t e s of dependent E. c o l i (35 u n i t s ) was lower than that from s u s c e p t i b l e E . c o l i . And the amount o f thymine recovered from d i h y d r o s t r e p t o m y c i n p r e c i p i t a t e s of dependent E. c o l i (125 u n i t s ) was c o n s i d e r a b l y lower than these f i r s t two. - 3 6 -Comparison with the figures of Marshak (25) on molar ratios of recovered purines and pyrimidines of E. c o l i hydrolyzates w i l l give some i n d i c a t i o n of dihydrostreptomycin p r e c i p i t a t i o n effects on these bases. The purine and pyrimidine contents of three strains of Escherichia c o l i have been determined by Marshak ( 25 ) ; parent E. c o l i s t r a i n B, s t r a i n B/r (more resistant to X-rays and u l t r a v i o l e t l i g h t than the parent) and s t r a i n K^2 (capable of genetic recombination) Table VIII. Analysis was ca r r i e d out on cold t r i c h l o r a c e t i c acid extracts of the organisms and the dry powder obtained was hydrolyzed with perchloric acid. The y i e l d of the nitrogenous bases was determined by spectro-photometry. TABLE VIII (25) E. c o l i S t r a i n Molar Ratios Adenine Guanine Cytosine U r a c i l Thymine B 1 1 .27 1.01 0 .58 0.22 B/r 1 1 .67 1 .16 0.46 0.20 K12 1 1 .63 1.21 0.45 0.24 - 3 7 -Dihydrostreptomycin p r e c i p i t a t i o n seems to reduce the amount of guanine recovered. This i s also true f o r the amounts of cytosine. Assuming that the material from spot 4 (on the chromatograms) i s uracil-containing material (mononucleotides or nucleosides etc.) i t i s l e s s , also, comparatively speaking i n a l l three E. c o l i types. The amount o f thymine recovered by d i h y d r o s t r e p t o m y c i n p r e c i p i t a t i o n i s greater, however, r e l a t i v e to the purines recovered, the greatest being from the susceptible organ-ism. The t o t a l effect of t h i s dihydrostreptomycin p r e c i p i t a t i o n p r i o r to hydrolysis may have the e f f e c t then of removing a l l thymine from the organism but not a l l of the rest of the purines and pyrimidines, the least affected being u r a c i l . Less thymine, however, i s affected with the dependent than with the susceptible organisms. Whether t h i s i s because of content or d i f f e r e n t i a l dihydrostrepto-mycin ef f e c t s on RNA and DNA cannot be said yet. Other work done i n t h i s laboratory (21) indicates that perhaps i t i s a matter of t o t a l DNA content. Analyses of the RNA to DNA r a t i o s of E. c o l i variants grown under varying conditions of dihydrostreptomycin con-centration have just been completed i n t h i s laboratory by Mr. Woo-Pbck Lay. - 3 8 -TABLE IX (21) Material Assayed RNA-Phosphate per 100 mg. c e l l s DNA-Phosphate per 100 mg. c e l l s R M- P/DNA-P Susceptible E. c o l i 1.09 mg. 0.400 mg. 2 .7 Resistant E. c o l i grown with 125 units per ml, DiHS 1.59 0.434 3 .7 Resistant E. c o l i grown i n absence of DiHS 1.60 0.452 3.6 Dependent E. c o l i grown with 125 units per ml. DiHS 1.65 0.233 7.1 Dependent E. c o l i grown with 35 units per ml. DiHS 2.41 0.318 7.5 The analyses, carried out by the methods of Schmidt and Thannhauser (38) were based on dried, l i p i d - f r e e acid extracted c e l l s . - 3 9 -The r a t i o of RNA to DNA i s much greater i n dependent organisms (35 units and 125 units) than i n sus-ceptible organisms. This change seems to be more the result of greater decreases i n DNA content than increases i n RNA content. However, dependent E. c o l i (35 units) shows a greater increase i n RNA and less decrease i n DNA contents than does the dependent E. c o l i (125 units) as compared with the normal susceptible organism. A marked r i s e i n RNA content, brought about by use of a special media, occurs with the increase of streptomycin resistance i n Haemophilus pertussis . This was shown by Smolens and Vogt ( 3 9 ) . I t was found that from a s t r a i n of H. pertussis susceptible to 0.001 milligram per ml. strepto-mycin to a s t r a i n resistant to 10 milligrams per ml. streptomycin, RNA content increased from 5.1$ to 9«8$ whereas DNA content decreased from 10.6$ to 8 . 8 $ . CONCLUSION In t h i s work i t has been confirmed that dihydro-streptomycin i n high concentration (about 100 times the concentration necessary f o r demonstration of a n t i b i o t i c a c t i v i t y ) precipitates induced / 3-galactosidase from extracts of E. c o l i variants i n a complex from which enzyme a c t i v i t y cannot be regenerated by polymethacrylic acid, the compound which i s e f f e c t i v e i n d i s s o c i a t i n g protamine-nucleic acid complexes. The nature of the dihydrostreptomycin p r e c i p i t a t e deserves further study since i t appears, from t h i s study and from other work done i n th i s laboratory, to contain nucleic acid, as well as a n t i b i o t i c and inactivated enzyme. It has been observed, i n th i s work, that complexes of dihydrostreptomycin with 260 millimicron-absorbing material from E. c o l i are not depolymerized by DNase although complexes of dihydrostreptomycin with animal DNA are ra p i d l y dissolved and depolymerized by DNase. This resistance of the b a c t e r i a l complexes to depolymerization by DNase may be a factor i n the mechanism of action of the a n t i b i o t i c . Dihydrostreptomycin complexes of E. c o l i 260 millimicron-absorbing material are altered somewhat by RN ase i n the d i r e c t i o n of depolymerization so thst i t would seem RNA might have a less d i r e c t relationship to the a n t i b i o t i c action of streptomycin than has DNA. The fact that u r a c i l could not be i d e n t i f i e d i n hydrolysates of dihydrostreptomycin-precipitated complexes from E. c o l l while -41 thymine could be r e a d i l y detected indicates that these complexes are r i c h e r i n DNA than i n RNA. While i t i s not possible at t h i s time to draw de f i n i t e conclusions regarding the mode of action of streptomycin i t i s not i n contradiction with the observed facts to postulate as follows: streptomycin reacts with DNA to form a complex which i n susceptible E. c o l i i s not depolymerized by DNase. This complex formation between dihydrostreptomycin and DNA prevents protein synthesis as exemplified i n adaptive enzyme formation. C e l l d i v i s i o n cannot take place i n the presence of dihydrostreptomycin because depolymerization of DNA by DNase i s prevented. Enzyme formation cannot occur because the d i r e c t i n g e f f e c t of DNA on t h i s process i s blocked. One must then postulate that i n the streptomycin-dependent E. c o l i . the a n t i b i o t i c i s required f o r the depolymerization of DNA and f o r the d i r e c t i n g e f f e c t of DNA on protein synthesis. BIBLIOGRAPHY 1. Brink, N. G., and Folkers, K., i n Waksman, S. A., ed., Streptomycin. Baltimore, The Williams & Wilkins Co., 1949, p. 5 5 . 2 . C a v a l i e r i , L. F., Rossoff, M., and Rosenberg, B. A., J. Am. Chem. S o c , 78 :5239. 1956. 3 . Chambers, L. A., and Flosdorf, E. W., J . B i o l . Chem., 114 :75. 1936. 4. Cohen, M., Monod, J . , Pollock, M. R., Spiegelman, S., and Stanier, R. Y., Nature, 172:1096. 1953. 5 . Cohen, S. S., J . B i o l . Chem., 166:393. 1946. 6. Cohen, S. S., J . B i o l . Chem., 168 :511 . 1947. 7. Deken-Grenson, M. de, Arch. Internat. P h y s i o l . Biochem., 63:256. 1955. 8 . Deken-Grenson, M. de, Arch. Internat. Physiol. Biochem., 63:257 . 1955-9 . Donovick, R., Bayan, A. P., Canales, P., and Pansy, F., J. B a c t e r i d . , 56 :125 . 1948. 10 . Dounce, A. L., Enzymplogia, 15 :251 . 1952. -43-11. Dutta, S. K., Jones, A. S., and Stacy, U., Biochim. et Biophys. Acta, 10:613. 1953. 12. F i t z g e r a l d , R. J., Bernheim, F., and F i t z g e r a l d , D. B., J. B i o l . Chemv, 175:195. 1948. 13. Gale, E. F., and Folkes, J. P., Biochem. J., 53:483. 1953. 14. Gros, F., and Rybak, B., Helv. Chim. Acta, 31:1855. 1948. 15. Henry, J . , Henry, R. J . , Housewright, R. D., and Berkman, S., J . B a c t e r i d . , 56:527. 1948. 16. Hogness, D. S., Cohen, M., and Monod, J . , Biochim. et Biophys. Acta, 16:99. 1955. 17. Jones, A. S., Biochim. et Biophys. Acta, 10:607. 1953. 18. Jones, A. S., Lee, W. A., and Peacock, A. R«, J . Chem. S o c , 150:623. 1951. 19. Kunitz, M., J. B i o l . Chem., 164:563. 1946. 20. Kunitz, M., J . Gen. Physiol., 33:349. 1950. 21. Lay, WooPok, University of B r i t i s h Columbia, personal communication. 22. Lederberg, J . , J . B a c t e r i o l . , 60:381. 1950. - 4 4 -23.. Ma, T. S., and Zuazaga, G., Ind. Eng. Chem. Anal. Ed., 14:280. 1942. 24. Markham, R., Biochem. J . , 36:790. 1942. 25. Marshak, A., Science, 113:181. 1951. 26. Massart, R., Experientia, 3:288. 1947. 27. Matheson, A. T., University of Toronto, personal communication. 28. M i l l e r , C. P., and Bohnhoff, M., Science, 105:620. 1947. 29. Oginsky, E. L., Smith, P. H., and Umbreit, W. W., J . B a c t e r i o l . , 58:76l. 1949. 30. Peretz, S. and Polglase, W. J . , A n t i b i o t i c s Annual. 1956-1957, Medical Encyclopedia, Inc., New York, N. Y. p. 533. 31. Polglase, W. J., Canad. J . Biochem. and Physiol., 34:555. 1956. 32. Polglase, W. J . , Peretz, S., and Roote, S. M., Canad. J . Biochem. and Physiol., 34:558. 1956. 33. Pomeroy, D. A., Bachelor of Arts Thesis, University of B r i t i s h Columbia, 1956. 34. Reiner, J . M., and Goodman, F., Arch. Biochem. Biophys., 57:475. 1955. -45-35. Roberts, R. B., Cowie, D. B., Abelson, P. H., Bolton, E. T., and B r i t t e n , R. J., eds., Studies of Biosynthesis  i n Escherichia C o l i , Carnegie I n s t i t u t i o n of Washington Publication 607, Washington, D. C , 1955, p. 6. 36. Roote, S. M., and Polglase, W. J . , Canad. J . Biochem. and Physiol., 33:792. 1955. 37* Schatz, A., Bugle, E., and Waksman, S. A., Proc. Soc. Exper. B i o l , and Med., 55:66. 1944. 38. Schmidt, G., and Thannhauser, S. J . , J . B i o l . Chem., 161:83. 194-5. 39. Smolens, J . , and Vogt, A. B., J . B a c t e r i o l . , 66:140. 1953. 40. Stanier, R. Y., i n Neilands, J . B. and Stumpf, P. K., eds., Outlines of Enzyme Chemistry. New York, John Wiley, & Sons, Inc., 1955, p. 285. 41. Umbreit, W. W., J . B a c t e r i o l . , 66:74. 1953. 42. Umbreit, W. W., Trans. N. Y. Acad. S c i . , 15:8. 1952. 43. Umbreit, W. W., Smith, P. H. and Oginsky, E. L., J . B a c t e r i o l . , 61:595. 1951. 44. Umbreit, W. W., and Tonhazy, N. E., J . B a c t e r i o l . , 58:769. 1949. - 4 6 -4 5 . Wyatt, G. R., Biochem. J . , 48 :584 . 1951. 46. Yeas, M., and Brawerman, G., Arch. Biochem. Biophys., 68:118. 1957-47 . Youmans, G. P., and Fisher, M . W., i n Waksman, S. A., ed., Streptomycin. Baltimore, The Williams & Wilkins Co., 1949, p. 104. INDEX Page Abstract Introduction 1 Experimental 12 Methods and Materials 12 Results 19 Discussion 32 Conclusion • 4-0 Bibliography 42 

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