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The biosynthesis of N-putrescinylthymine in bacteriophage φW-14 infected Pseudomonas acidovorans Karrer, Earl 1973

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THE BIOSYNTHESIS OF N-PUTRESCINYLTHYMINE IN BACTERIOPHAGE 0W-14 INFECTED Pseudomonas acidovorans by EARL KARRER B.Sc. (Honours, Microbiology) Un i v e r s i t y of B r i t i s h Columbia, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT OF MICROBIOLOGY WE ACCEPT THIS THESIS AS CONFORMING TO THE REQUIRED STANDARD THE UNIVERSITY OF BRITISH COLUMBIA JULY, 1973 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 requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, 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 study. 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 copying 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 granted by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n of 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 allowed without my w r i t t e n p e r m i s s i o n . Department The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada ! • i ABSTRACT The biosynthesis of N-putrescinylthymine (NpT), a modified pyrimi-dine which occurs i n the DNA of phage 0W-14, was studied. P r i o r to t h i s study, the metabolism of arginine and polyamines i n the host organism, Pseudomonas acidovorans 29, was investigated. P_. acidovorans transported ornithine and arginine but not putrescine. Neither amino acid was used as a sole source of nitrogen. Thus arginine cannot be catabolized to putrescine and ornithine i s not catabolized to Y-aminobutyrate. Ornithine was synthesized from glutamate but the operation of t h i s pathway was not i n h i b i t e d by high concentrations of ar-ginine. Since ornithine was decarboxylated to putrescine, t h i s single route f o r the synthesis of polyamines i s unusual i n that ornithine b i o -synthesis i s c o n s t i t u t i v e . P_. acidovorans and 0W-14 contain an unusual complement of polyamines: spermidine, putrescine and 2-hydroxyputrescine. Their respective b a c t e r i a l concentrations (mM) were: 3-5, 50 and 45, phage i n f e c t i o n led to an i n -crease i n the proportion of putrescine. 14 14 C-Ornithine was used to l a b e l NpT i n phage DNA. C-5-0rnithine 14 ex c l u s i v e l y l a b e l l e d NpT, whereas C-l-ornithine was t o t a l l y i n e f f e c t i v e i n this capacity. Hence, the carboxyl group of ornit h i n e i s not a c o n s t i -tuent of the NpT molecule. 14 L a b e l l i n g of NpT with C-3-serine demonstrated a tetrahydrofolate 3 (THF) involvement i n i t s biosynthesis. The use of H-2,3-serine showed that N^^methylene THF was the carbon donor for the pyrimidine precursor of i i NpT. I t i s proposed that NpT i s synthesized i n the following way: 5-hydroxymethyl deoxyuridine monophosphate i s formed by the i n t e r a c t i o n of N^^methylene THF and deoxyuridine monophosphate; putrescine then con-denses with the hydroxymethyl group of the nucleotide to y i e l d N-putrescinyl-thymine. i i i TABLE OF CONTENTS Page INTRODUCTION 1 I. Arginine metabolism 1 A. Arginine biosynthesis.... 1 B. Arginine catabolism 4 C. Control of arginine biosynthesis.... 11 I I . Polyamine metabolism . .• 14 A. D i s t r i b u t i o n of polyamines 14 B. Synthesis of polyamines 16 C. Control of polyamine synthesis 17 D. Derivatives of polyamines 19 E. Catabolism of polyamines 20 F. S i g n i f i c a n c e of polyamines 24 1. polyamines and bacteriophage........... 24 2. polyamines and b a c t e r i a . . . . . ... 24 G. Polyamine: DNA in t e r a c t i o n s 26 I I I . Modified bases 27 MATERIALS AND METHODS 30 I. Organisms 30 I I . Media 30 I I I . Growth of b a c t e r i a . . . . 30 IV. Phage t i t r a t i o n 31 V. Preparation of phage stocks 31 VI.' P u r i f i c a t i o n of phage 31 iv Table of Contents (cont'd) Page VII. Osmotic sensitivity of 0W-14 31 VIII. Utilization of compounds as nitrogen source 32 IX. Uptake of radioactive compounds 32 X. Isolation of polyamines . 33 A. From bacteria. 33 B. From phage 34 XI. Preparation of dansylated polyamines 34 XII. Chromatography 34 A. Thin layer (TLC) 34 1. polyamines 34 2. dansylated polyamines 35 3. DNA hydrolysates 35 XIII. Detection of polyamines 36 XIV. Quantitation of polyamines 36 XV. Isolation of 2-hydroxyputrescine 36 XVI. NMR of 2-hydroxyputrescine 37 XVII. Isolation of DNA 37 A. From bacteria 37 B. From phage 38 XVIII. Hydrolysis of DNA 38 XIX. Measurement of radioactivity 38 A. Chromatographed polyamines or DNA hydrolysates 38 B. TCA soluble materials 38 C. TCA precipitable materials 38 V, Table of Contents (cont'd) Page D. Counting radioactivity 38 XX. Chemicals 39 XXI. Radiochemicals 39 RESULTS AND DISCUSSION I. Utilization of compounds as sole source of nitrogen... 40 II. Transport of radioactive substrates 40 III. Polyamines of P. acidovorans 40 IV. Labelling of polyamines.... 49 V. Quantitation of polyamines 53 VI. Polyamines of 0W-14 54 VII. Osmotic sensitivity of 0W-14 55 VIII. The biosynthesis of NpT 55 SUMMARY 61 LITERATURE CITED 63 v i LIST OF FIGURES Figure Page 1. Pathway A, Arginine biosynthesis i n higher organisms 2 1. Pathways B and C, Arginine biosynthesis i n b a c t e r i a 3 2. Arginine catabolism i n higher organisms and B a c i l l u s sp 5 3. The arginine dihydrolase pathway 7 4. Arginine catabolism i n S_. griseus and P_. putida 8 5. Arginine catabolism i n E_. c o l i 10 6. Spermidine biosynthesis i n E_. c o l i 18 7A&, Polyamine catabolism: putrescine 21 7B5. Polyamine catabolism: spermidine 23 8. The transport of "^C-U-arginine by P_. acidovorans 42 9. The transport of "^C-5-ornithine by P_. acidovorans.. .. 43 14 10. The transport of C-3,4-putrescine by P_. acidovorans. 44 14 11. The transport of C-3,4-putrescine by P_. acidovorans inf e c t e d with 0W-14 45 12. 100 MHz NMR spectrum of a D 20 s o l u t i o n of 2-hydroxyputrescine dihydrochloride 48 v i i LIST OF TABLES Table Page I. Common polyamines 15 I I . U t i l i z a t i o n of compounds as sole source of nitrogen 41 I I I . Thin layer chromatography of polyamines 47 IV. L a b e l l i n g of polyamines 50 14 V. D i s t r i b u t i o n of l a b e l (CPM) i n C-glutamate l a b e l l e d polyamine extracts 52 VI. Osmotic s e n s i t i v i t y of phage 0W-14 56 14 VII. L a b e l l i n g of 0W-14 DNA with C-ornithine 57 VIII. L a b e l l i n g of 0W-14 DNA with radioactive serine.. 59 v i i i ACKNOWLEDGEMENTS To Dr. R.A.J. Warren, for h i s thoughtful comments, guidance, encouragement and wit during t h i s study, I express my sincere gratitude. Tony's t r u t h f u l approach to science i s admirable and w i l l remain with me, always. Dr. R.A. K e l l n , an eternal source of stimu-l a t i n g discussion, good humour and exuberance, I owe thanks, as w e l l . I am indebted to, Dr. R.J. Bose for h i s operation of the NMR spectro-meter, advice and eagerness to p a r t i c i p a t e i n the project and also to Dr. J.B. Farmer, f o r the use of his Fluorometer. Thank you, also, Mrs. Pat Waldron, for your s k i l l f u l typing of t h i s thesis and patience i n deciphering my penhand. 1 INTRODUCTION I. ARGININE METABOLISM A. Arginine Biosynthesis C e l l u l a r routes leading to arginine are i l l u s t r a t e d i n Figure 1. Although some features of arginine biosynthesis are common to a l l c e l l s , there i s considerable v a r i a t i o n i n the o v e r a l l pathway. In higher or-ganisms, p r o l i n e i s the i n i t i a l precursor for arginine anabolism (White, Handler and Smith, 1968); and ornithine i s formed v i a A Jpyrroline-5-carboxylic a c i d and glutamic acid-y-semialdehyde. The formation of ornith i n e i n b a c t e r i a d i f f e r s , however. Escherichia c o l i (Vogel and Bonner, 1956), Proteus m i r a b i l i s (Prozesky, 1967), S e r r a t i a marcescens, other e n t e r i c s (Udaka, 1966) and B a c i l l u s s u b t i l i s (Vogel and Vogel, 1963) convert glutamic acid to N-acetylornithine, which i s then hydrolyzed to orn i t h i n e ( F i g . 1, pathway B). Micrococcus glutamicus, Pseudomonas  fluorescens (Udaka, 1966), Pseudomonas aeruginosa (Isaac and Holloway, 1972) and Pseudomonas putida (Chou and Gunsalus, 1971) also form N-acetylorni-thine from glutamate but convert i t to ornithine by tr a n s a c e t y l a t i o n of the ac e t y l group to glutamic acid (Udaka, 1966), (Fig. 1, pathway C). Acety-l a t i o n i n these two pathways, presumably, prevents an intramolecular c y c l i z a t i o n reaction. Glutamic acid-y-semialdehyde c y c l i z e s r e a d i l y to A 1pyrroline-5-carboxylic acid which i n h i b i t s the subsequent transamination step. The conversion of ornithine to arginine i n b a c t e r i a mimics the urea cycle of higher organisms (White, Handler and Smith, 1968). In the 2 CITRULLINE ASPARTATE ARGININE-* — ARGINOSUCCINATE / , ' FUMARATE Number p Enzyme 1 pr o l i n e oxidase 2 ornithine-6-transaminase 3 ornithine transcarbamylase 4 arginosuccinate synthetase 5 arginosuccinase 6 carbamyl phosphate synthetase FIGURE 1, PATHWAY A. Arginine biosynthesis i n higher organisms 3 N-ACET YL-iC-GLUtAM YL PHOSPHORIC AG ID N-ACETYLGLUTAMIC: 2f-SEMI ALDEHYDE ATP N-ACETYLGLUTAMATE-11 N-ACETYLORNITHINE GLUTAMIC ACID pathway B: pathway C: —ORN THINE 6,7,8 ARGININE Numb er 1 2 3 4 5 6,7,8 Enzyme N-acetylglutamate synthetase N-acetyl-y-glutamokinase N-acetylglutamic-Y-semialdehyde dehydrogenase acetylornithine-6-transaminase acetylornithinase as i n higher organisms acetylglutamate-acetylornithine transacetylase FIGURE 1, PATHWAYS B & C. Arginine biosynthesis i n b a c t e r i a 4 presence of ornith i n e transcarbamylase (OTCase), ornithine and carbamyl phosphate react to produce c i t r u l l i n e . Carbamyl phosphate, common to both arginine and pyrimidine syntheses, i s derived from CC^ and NH^ i n a reaction catalysed by carbamyl phosphate synthetase. This reaction, accomplished by two isozymes i n Saccharomyces cerevisiae (Lacroute et a l . , 1965) and Neurospora crassa (Bernhardt and Davis, 1972) and by a sing l e enzyme i n E. c o l i (Pierard and Wiame, 1964), i s subject to end product i n h i b i t i o n by arginine and u r i d i n e triphosphate (UTP) (Thuriaux et a l . , 1972). B. Arginine Catabolism The urea cycle i n higher organisms i s driven by the enzyme arginase, which hydrolyzes arginine to urea and ornithine (Fig. 2). B_. s u b t i l i s contains arginase and also an ornithine-6-transaminase which converts ornithine to glutamic acid-y-semialdehyde; following spontaneous c y c l i z a -t i o n to A 1 p y r r o l i n e - 5 - c a r b o x y l i c a c i d , either p r o l i n e i s formed by A 1 pyrroline-5-carboxylic acid reductase, or glutamate i s formed by a dehydro-genase catalyzed oxidation. In B. s u b t i l i s (de Hauwer, L a v a l l e and Wiame, 1964) and B a c i l l u s l i c h e n i f o r m i s (Laishley and Bernlohr, 1968), arginase, ornithine-S-transaminase and A 1pyrroline-5-carboxylic dehydrogenase are induced simultaneously by arginine. The f i r s t two enzymes appear to be ge n e t i c a l l y linked with an arginine permease i n B_. s u b t i l i s since a mutant was i s o l a t e d i n which a l l three a c t i v i t i e s are c o n s t i t u t i v e . The dehydro-genase remains inducible and i s , apparently, unlinked (de Hauwer, Lavalle and Wiame, 1964). 5 ARGININE ORNITHINE UREA PROLINE-GLUTA IVIIC ACID GLUTAMIC ACID-V-SEMIALDEHYDE A1PYRROLINE-5-CARBOXYLIC ACID Number Enzyme 1 2 3 4 argxnase ornithine-6-transaminase , Apyrroline-5-carboxylic acid reductase glutamic acid-y-semialdehyde dehydrogenase FIGURE 2. Arginine catabolism i n higher organisms and B a c i l l u s sp. 6 An a l t e r n a t i v e route for arginine catabolism i n organisms lacking arginase i s the arginine dihydrolase pathway (Fig. 3) which occurs i n C l o s t r i d i a sp. (Mitruka and Costilow, 1967), Streptococcus f a e c a l i s (Oginsky and Gehrig, 1952), Pseudomonas sp. (Stalon et a l . , 1967), and Halobacterium salinarium (Dundas and Halvorson, 1966). Arginine deiminase f i r s t deiminates arginine to c i t r u l l i n e . OTCase, i n a phosphorlyttic reaction, generates carbamyl phosphate and ornithine from c i t r u l l i n e and inorganic phosphate. In addition to an anabolic OTCase, a s p e c i f i c cata-b o l i c enzyme occurs i n P_. aeruginosa and P_. fluorescens (Stalon et a l . , 1967). In contrast, only one, amphibolic OTCase i s found i n H. salinarium (Dundas, 197,2). The two enzymes, having pH optima of 7.1-7.4 and 8.5, are e a s i l y separable by ammonium s u l f a t e f r a c t i o n a t i o n . In P_. aeruginosa, the alka-l i n e a c t i v i t y i s repressed by growth i n the presence of arginine, a s i t u a -t i o n which augments the neutral a c t i v i t y . I t was thus reasoned that the al k a l i n e a c t i v i t y represents the biosynthetic enzyme while the catabolic OTCase has the neutral pH optimum and i s controlled coordinately with arginine deiminase and carbamate kinase (Ramos et a l . , 1967). The t h i r d enzyme of the arginine dihydrolase pathway, carbamate k i - .• nase, s p l i t s carbamyl phosphate i n t o NH^ and CO2, with one mole of ATP formed v i a substrate l e v e l phosphorylation per mole of c i t r u l l i n e hydrolyzed. This reaction enables Pseudomonads, normally immotile when grown anaerobi-c a l l y i n minimal medium, to regain m o t i l i t y i f also supplied with arginine (Stanier, P a l l e r o n i and Doudoroff, 1966). The pathway of arginine degradation i n Streptomyces grisfeus i s shown i n F i g . 4, pathway A. Arginine i s f i r s t decarboxylated to Y~guanidino-7 INORGANIC PHOSPHATE Number Enzyme 1 arginine deiminase 2 ornithine transcarbamylase 3 carbamate kinase FIGURE 3. The arginine dihydrolase pathway 8 ARGININE 1 ^-GUANIDINOBUTYRIC ACID ot-KETOARGININE UREA-y-AMINOBUTYRIC ACID Enzyme arginine decarboxyoxidase arginine deaminase guanidinobutyramidase a-ketoarginine decarboxylase y-guanidinoamidino hydrolase Arginine catabolism i n _S. griseus and P_. putida. 9 butyramide (Thoai, Thome-Beau and Pho, 1962), the reaction uses one mole of O2 per mole of CO^ l i b e r a t e d and the enzyme, arginine decarboxyoxidase, needs FAD as a pr o s t h e t i c group (Thoai, Thome-Beau and O.lomucki, 1966). The enzymes of t h i s pathway are coordinately induced by arginine, but to d i f f e r e n t l e v e l s , suggesting that the genes are not linked (Thoai, Thome-Beau and Pho, 1962) . Growth of P_. putida 7^ on arginine r e s u l t s i n the induction of a s i m i l a r degradative sequence (Fig. 4, pathway B), ( M i l l e r and Rodwell, 1971). In t h i s microbe, however, the los s of NH^ precedes decarboxylation. Hence, the f i r s t product, a-ketoarginine, i s subsequently decarboxylated to y-guanidinobutyrate (yGBA). a-Ketoarginine decarboxylase (Rodwell and 2+ 2+ Gaby, 1973) requires thiamine pyrophosphate and e i t h e r Mg or Mn as cofac-t o r s . Optima for t h i s enzyme are at pH 8.5 and 70°C. Two moles of CO2, one mole of yGBA and one mole of an " a c y l o i n - l i k e " adduct are produced for every three moles of a-ketoarginine consumed (Rodwell and Gaby, 1973). yGBA accumulates i n cultures grown on arginine at pH 7.0. Synthesis of the t h i r d enzyme, y-guanidinoamidino hydrolase, i s induced by growth on arginine or yGBA (Chou and Rodwell, 1972). The p u r i f i e d enzyme (mole-2+ cular weight: 178,000 to 190,000) requires Mn and shows optima at pH 10 and 50°C. In view of the decrease i n a c t i v i t y at pH 7.0 and the accu-mulation of yGBA, Chou and Rodwell (1972) suggested that y-guanidino-amidino hydrolase a c t i v i t y may be rate l i m i t i n g i n t h i s pathway. The patience of Gale (1940) revealed that i n E_. c o l i cultures of low pH supplemented with casein digest, arginine was decarboxylated (Fig.5 ). The enzyme, termed the inducible arginine decarboxylase, has since been 10 ARG NINE CO. AGMATINE -PUTRESCINE UREA Number Enzyme 1 2 arginine decarboxylase agmatine ureohydrolase FIGURE 5. Arginine catabolism i n E. c o l i 11 p u r i f i e d and studied i n t e n s i v e l y by Boeker:, et al.=, (1971). I t i s a pentamer of molecular weight 820,000, containing ten moles of pyridoxal phosphate per mole of enzyme-- The optimum pH i s 5.2. In cultures of neutral pH, a condition i n which the inducible de-carboxylase cannot be detected, E.coli.„continues to decarboxylate arginine. Morris and Pardee (1965, 1966) solved t h i s dilemma by demonstrating the presence of a second arginine decarboxylase, the s o - c a l l e d biosynthetic enzyme,, This enzyme, a tetramer, has an absolute requirement for pyridoxal 2+ phosphate and Mg . Its pH optimum i s 8.4 (Wu and Morris, 1973). Mutants lacking t h i s enzyme have been i s o l a t e d (Morris and Jorstad, 1970 and Maas, L e i f e r and Poindexter, 1970). Preliminary mapping studies indicated that the genetic l o c i f o r the inducible and biosynthetic enzymes are widely se-parated (Maas, L e i f e r and Poindexter, 1970). C. Control of Arginine Biosynthesis Phenotypically, arginine biosynthesis i n E_. c o l i K comprises eight steps (Fig. 1). Genotypically, however, nine genes (arg A to arg I) code for these functions. Arg I and .arg F determine the synthesis of two i s o -zymes of OTCase i n E_. c o l i K12 (Glansdorff, Sand and Verhoef, 1967 and E l s e v i e r s , Cunin and Glansdorff, 1972). Only a s i n g l e OTCase i s found i n Salmonella typhimurium (Syvanen and Roth, 1972) and E_. c o l i B (Jacoby, 1971). Only four of the nine genes are clustered the sequence being: arg E, arg C, arg B and arg H (Glansdorff, 1965). However, arg E i s not c o n t r o l l e d coordinately with arg C,B,H. Nonsense mutations i n arg C or arg B are polar for arg H but nonsense mutations i n arg E are non-polar for the remaining genes (Jacoby, 1972). Jacoby (1972) and E l s e v i e r s , Cunin and Glansdorff (1972) independently proposed, therefore, that arg ECBH 12 represents two operons, arg E and arg CBH, which are transcribed d i v e r -gently from an i n t e r n a l operator-promoter complex. Arg E, together with the unlinked gene arg I, are unusual i n that they are oriented i n the opposite d i r e c t i o n to the other genes of the arginine regulon, i . e . coun-ter clockwise (Jacoby, 1971, 1972). In E_. c o l i s t r a i n s B,C,K, and W there i s a gene, arg R, whose product i s a regulatory protein (Jacoby and G o r i n i , 1969, Udaka, 1970 and Hirvonen and Vogel, 1970). In arg R + s t r a i n s of s t r a i n s K,W, or C, excess arginine represses i t s own biosynthesis ( G o r i n i , 1962). In s t r a i n B, however, ex-cess arginine has a s l i g h t inductive e f f e c t ( G o r i n i , 1962)! A form of c a t a b o l i t e repression i s also operative i n t h i s s t r a i n since glucose grown c e l l s contain much lower enzyme l e v e l s than g l y c e r o l grown c e l l s ( G o r i n i , and Gundersen, 1961). Repression i n s t r a i n K requires both the arg R p r o t e i n and arginine since arg R mutants contain derepressed enzyme l e v e l s even i n the presence of arginine ( G o r i n i , 1962). Jacoby and Gorini (1969) found that a s t r a i n K transductant receiving the arg R locus from s t a i n B has B-type regula-t i o n i n c l u d i n g low l e v e l induction by arginine, whereas a s t r a i n B r e -c i p i e n t of the arg R locus from s t a i n K becomes arginine r e p r e s s i b l e . Karlstrom and Gorini (1969) noted that s t r a i n B i s r e p r e s s i b l e by arginine when grown at temperatures above 39°C. Subsequently, Jacoby and G o r i n i (1969) reported that single amino acid changes i n the arg R protein of s t r a i n B produces K-type r e p r e s s i b i l i t y . It was proposed, therefore, that the control of arginine biosynthesis i n the two s t r a i n s i s b a s i c a l l y s i m i l a r . In s t r a i n K, arginine and the R protein i n t e r a c t to repress 13 enzyme formation. In s t r a i n B, the R pr o t e i n , alone, mediates repression and i n t e r a c t i o n of the p r o t e i n with arginine or an arginine d e r i v a t i v e decreases t h i s a b i l i t y . Presumably, point mutations i n the R pro t e i n of s t r a i n B a l t e r s the i n t e r a c t i o n with arginine so that i t only binds to the operators i n the presence of the amino acid (Jacoby and G o r i n i , 1969). Recently, Urm et al.(1973) have reported an i n v i t r o assay f o r the arg R protein. This development should now enable i t s p u r i f i c a t i o n and should also f a c i l i t a t e studies of the r e l a t i v e binding of arginine to the repressor from s t r a i n s B and K. Regulation of arginine biosynthesis i n E_. c o l i involves both i n h i b i -t i o n of enzyme a c t i v i t y and repression of enzyme synthesis. Udaka (1966) suggested that i n a l l organisms deriving ornithine by tra n s a c e t y l a t i o n , the second enzyme, N-acetyl-y-glutamokinase i s i n h i b i t e d by arginine, whereas i n a l l organisms forming ornithine by d i r e c t hydrolysis of N-acetylor-n i t h i n e , i t i s the f i r s t enzyme of the pathway, N-acetylglutamate synthetase which i s i n h i b i t e d by arginine. The regulation of enzyme synthesis i n E_. c o l i involves t r a n s l a t i o n a l as well as t r a n s c r i p t i o n a l controls. The addition of r i f a m p i c i n followed by excess arginine to previously derepressed,.culturesrof E._,c6li W r e s u l t s i n a decrease i n the rate of synthesis of the enzymes of arginine biosynthe-s i s (Vogel et a l . , 1971). This decrease i s not observed i n the presence of rif a m p i c i n alone, implying that i n the presence of the arg R pro t e i n and arginine, the t r a n s l a t i o n of mRNA from the arginine genes i s retarded. McCellan and Vogel (1972), Vogel, Knight and Vogel (1972) have shown also that mRNA from the arg ECBH c l u s t e r , decays at a faster rate during repres-sion. Furthermore, streptomycin or t e t r a c y c l i n e reduces the d i f f e r e n t i a l 14 rate of N-acetylornithine-6-transaminase synthesis only under conditions of derepression, leading Vogel et a l . , (1971) to reason that some facet of the t r a n s l a t i o n a l machinery must be altered during repression. C e l i s and Maas (1971) and L e i s i n g e r and Vogel (1969) showed that a r g i n y l tRNA i s probably not modified during repression since p u r i f i c a t i o n of tRNA species from c e l l s harvested i n the derepressed or repressed states produced i d e n t i -c a l column chromatographic e l u t i o n p r o f i l e s . T r a n s c r i p t i o n a l control over the enzymes of arginine biosynthesis has recently been shown by h y b r i d i z a t i o n experiments. The amount of arg ECBH mRNA transcribed during derepression exceeds that made during repres-sion (Kryzek and Rogers, 1972, Rogers et a l . , 1971). T r a n s c r i p t i o n a l control i n genes other than the arg ECBH c l u s t e r has not been observed because of technical d i f f i c u l t i e s . I I . POLYAMINE METABOLISM A. D i s t r i b u t i o n of Polyamines Polyamines are low molecular weight organic bases. Table I provides the systematic and common names of several polyamines (Tabor and Tabor, 1972). Systematic Name Common Name 1.3- diaminopropane -1.4- diaminobutane putrescine 1.5- diaminopentane cadaverine N-(3-aminopropyl)-1,4-diaminobutane spermidine N ,N ''-Bis (3-aminopropyl) -1,4-diaminobutane spermine TABLE I. Common polyamines 16 Polyamines are ubiquitously d i s t r i b u t e d i n nature. The highest i n t r a c e l l u l a r concentrations reported so far are i n b a c t e r i a and fungi. For example, i n E_. c o l i , spermidine and putrescine are present at con-centrations of 1-2 mM and 10-20'r..mM re s p e c t i v e l y (Cohen, 1971). Spermine, while common i h higher organisms, so f a r has been found i n two b a c t e r i a : B a c i l l u s stearothermophillus (Stevens and Morrison, 1968) and P_. aeruginosa (Weaver and Herbst, 1958). Once thought to occur i n only a few gram p o s i -t i v e b a c t e r i a , such as B_. s u b t i l i s and Staphylococcus aureus (Herbst, Weaver and K e i s t e r , 1958)-,more s e n s i t i v e detection techniques should now enable the discovery of polyamines i n other gram p o s i t i v e species. Though very high l e v e l s of spermidine are present i n L a c t o b a c i l l u s casei during la g phase, c e l l u l a r growth d i l u t e s this pool to a very low l e v e l ( E l l i o t t and Michaelson, 1969). B. Synthesis of Polyamines E_. c o l i can synthesize putrescine by either of two routes. The f i r s t , mentioned e a r l i e r (Fig. 5), involves the decarboxylation of arginine. More d i r e c t l y , putrescine can a r i s e from the decarboxylation of o r n i t h i n e . Gale (1940) described an ornithine decarboxylase which appeared i n ornith i n e supplemented E_. c o l i cultures under conditions of low pH and poor aeration. Termed the inducible o r n i t h i n e decarboxylase, t h i s p rotein i s heat l a b i l e and has a pH optimum of 5.3 (Tabor and Tabor, 1972). C e l l s of E_. c o l i grown i n unsupplemented minimal media at neutral pH also contain a b i o -synthetic ornithine decarboxylase (Morris et al.1970). This enzyme has a pH optimum of 8.4; i t i s i n h i b i t e d by putrescine and spermidine (Morris 17 et al,. ,1970), but i t s a c t i v i t y i s enhanced by guanosine or deoxy guano sine triphosphate (Holtta, Janne and Pispa, 1972). Both ornithine decarboxy-lases have an obligate requirement f o r pyridoxal phosphate. To date, mutants lacking e i t h e r a c t i v i t y have not been i s o l a t e d . The biosynthesis of spermidine i s i l l u s t r a t e d i n P i g . 6. S-Adeno:syl-methionine (SAM) decarboxylase i s an octamer of 113,000 molecular weight (Wickner, Tabor and Tabor, 1970); i t contains pyruvate not pyridoxal-2+ phosphate, as a pro s t h e t i c group, requires Mg , i s s p e c i f i c f o r L - ( - ) -SAM and i s not stimulated by putrescine (Zappia et a l . , 1969). Propylamine transferase has been p u r i f i e d from E. c o l i W (Bowman, Tabor and Tabor, 1973). The enzyme i s a dimer; i t has no cofactor require-ment and i t s pH optimum is;iO.3 with putrescine as substrate. Spermidine and cadaverine are also s u i t a b l e substrates f o r the enzyme i n vitro-. ; V Dion and Cohen (1972) detected cadaverine and a spermidine analogue, N-C3-ami.no-propyl)-l,5-diaminopentane, i n a polyamine depleted mutant of E_. c o l i K12 grown i n the presence of l y s i n e . Cadaverine, then, i s also a suitable substrate f or the enzyme i n vivo. Mutants lacking t h i s a c t i v i t y or SAM decarboxylase have not been i s o l a t e d . C. Control of Polyamine Synthesis Tabor and Tabor (1969) reported that, despite severe arginine re-s t r i c t i o n , up to 18% of the a v a i l a b l e arginine i s used f o r polyamine syn-thesis i n an E_. c o l i arg s t r a i n . Such data argue that polyamines, a l -though t h e i r function i s uncertain, are important i n c e l l u l a r metabolism. The evolution of two routes f o r t h e i r synthesis strengthens t h i s premise. A c l a s s i c a l means of regulation, however, awaits discovery. 18 METHIONINE S-ADENOSYLMETHIONINE + A T P 2 SPERMIDINE METHYLTHIOADENOSINE DECARBOXYLATED S-ADENOSYL METHIONINE PUTRESCINE Number 1 2 3 Enzyme S-adenosylmethionine synthetase S-adenosylmethionine decarboxylase propylamine transferase FIGURE 6. Spermidine biosynthesis i n E_. c o l i . 19 When growing i n minimal media, E_. c o l i derives 95% of i t s polyamines from o r n i t h i n e (Morris and Koffron, 1968). This route i s e n e r g e t i c a l l y more p r a c t i c a l , since ATP i s expended i n the anabolism of or n i t h i n e to arginine (Fig. 1). Only i f arginine i s i n excess are polyamines synthe-size d from arginine (Morris and Koffron, 1968). This suggests that poly-amine synthesis i s regulated, Tabor and Tabor (1969) demonstrated that exogenous rather than endogenous arginine was s e l e c t i v e l y catabolized to polyamines i n E_. c o l i . The genes spe A (biosynthetic arginine decarboxy-lase) , spe B (agmatine ureohydrolase) and met K (SAM synthetase) are c l u s -tered i n E_. c o l i and perhaps form an operon (Maas, 1972). Arg P, coding for an arginine permease, l i e s w ithin one minute of t h i s c l u s t e r on the E_. c o l i map (Taylor, 1970). Maas (1972) has, therefore, speculated that the channeling of exogenous arginine into polyamines may be due to a co-ordinate control of these genes. In t h e i r study, the Tabors (1969) also i n f e r r e d that a homeostatic balance e x i s t s between putrescine and spermidine. During arginine l i m i t a -t i o n of an arginine auxotroph, the putrescine concentration was reduced s i g n i f i c a n t l y while the spermidine l e v e l remained normal. D. Derivatives of Polyamines F i r s t reported by Dubin and Rosenthal (1960), N-acetylated polyamines were thought to be normal constituents of E_. c o l i . A more cautious analy-s i s , however, revealed that these derivatives are a c t u a l l y a r t i f a c t s of handling procedures, and 98% of the polyamines are not normally acetylated (Tabor, 1968). This phenomenon occurs i n c e l l s either c h i l l e d during 20 harvesting or grown i n the presence of excess putrescine, spermidine and/or spermine (Tabor and Hobbs, 1970). A Bseudomonad i s o l a t e d by Kim (1966), lacks spermidine but contains putrescine and 2-hydroxyputrescine (Tobari and Tchen, 1971). The amount 2+ of t h i s d e r i v a t i v e bound to ribosomes varies inversely with the Mg concentration, as does the spermidine bound to E_. c o l i , ribosomes (Rosano and Hurwitz, 1969). Hence, i t was proposed that 2-hydroxyputrescine, with three hydrogen bonding s i t e s , could assume the roles normally played by spermidine. 2-Hydroxyputrescine has now been found i n B a c i l l u s megatherium, S^. aureus (Tehen, personal communication) and P_. acidovorans (Karrer, Bose and Warren, 1973). Studies on the hydroxylation reaction have not been reported. At the end of logarithmic growth, E_. c o l i normally converts a l l of i t s spermidine and 50% of i t s glutathione to glutathionylspermidine (Tabor and Tabor, 1972). The molecule has the following sequence: Y - g l u t a m y l -cysteinylglycylspermidine. When stationary phase c e l l s are d i l u t e d i n t o fresh media (pH 7.0), there i s a rapid loss of t h i s d e r i v a t i v e coincident with an increase i n free spermidine. Since l i t t l e i s known of the func-tions of spermidine or glutathione i n vivo, i t i s not s u r p r i s i n g that the adduct i s even more enigmatic. E. Catabolism of Polyamines Putrescine may be o x i d a t i v e l y deaminated to y-aminobutyraldehyde by putrescine oxidase i n Mycoplasma (Evelyn, 1967) and Micrococci (De Sa, 1972). (Fig. 7A, pathway A). In Micrococcus rubens, putrescine oxidase i s a fl a v o p r o t e i n containing FAD. The enzyme i s unusual among fla v o p r o t e i n o x i -dases i n that i t contains only one mole of FAD per mole of enzyme (De Sa, 21 PYRROLINE pathway A: pathway B: PUTRESCINE °2 \ / 1 ©C-KETOGLUTARATE GLUTAMATE 2T-AMINOBUTYR ALDEHYDE ^-AMINOBU 4 SUCCINIC S +NAD YRIC ACID MIALDEHYDE <*KG •GLU +NADP SUCCINIC ACID Number 1 2 3 4 5 Enzyme putrescine oxidase putrescine-a-ketoglutarate transaminase Y-aminobutyraldehyde dehydrogenase Y-aminobutyrate-a-ketoglutarate transaminase su c c i n i c semialdehyde dehydrogenase FIGURE 7A: Polyamine catabolism: putrescine 22 1972). In ce r t a i n pseudomonads and E_. c o l i , however, putrescine i s catabolized non-o'xidatively to Y - a m i n ° b u t y r a l d e h y d e (Bachrach, 1970) (Fig. 7A, pathway B). The product of both the oxidative and non-oxida-t i v e reactions, Y - a mi n°butyraldehyde, i s unstable and c y c l i z e s spontan-eously to A 1 p y r r o l i n e . Y-Aminobutyraldehyde dehydrogenase i s induced i n P_. f luorescens, by growth on putrescine (Jakoby and Fredericks, 1959). Kim (1963), i s o -l a t e d a mutant of E_. c o l i B which u t i l i z e s putrescine as the sole source of carbon and nitrogen. In t h i s mutant, putrescine-a-ketoglutarate transaminase i s c o n s t i t u t i v e while the enzymes converting Y _ a m i n ° b u t y r a l -dehyde to s u c c i n i c acid are inducible. These enzymes occur also i n P_. aeruginosa (Nakamura, 1960) and E_. c o l i K12 (Dover and Halpern, 1972). In the l a t t e r organism, the enzymes are eit h e r repressed or induced co-ordinately under a v a r i e t y of growth conditions, implying a common control (Dover and Halpern, 1972). When Ne i s s e r i a per f lava, S. marsescens and M.' rub ens are grown i n the prescence of polyamines, spermidine i s oxidized (Tabor and Tabor, 1972) (Fig. 7 B, reaction A). The S e r r a t i a enzyme has been p u r i f i e d by Campello et al-.,(1965) and requires FAD as cofactor. Pseudomonads and Mycoplasma also oxidize ^spermidine. In doing so, putrescine and not 1,3-diaminopropane i s the ultimate diamine formed (Fig. 7B, reaction B). The i n a b i l i t y of these organisms to oxidize the primary amino groups of spermidine undoubted-l y r e f l e c t s the b a c t e r i a l t o x i c i t y of the mono and dialdehyde products (Cohen, 1971). 23 SPERMIDINE N H 2 ( C H 2 ) 3 N H ( C H 2 ) 4 N H 2 ^-AMINOBUTYR ALDEHYDE 6 -AMINOPROPI ON ALDEHYDE reaction A: reaction B: FIGURE 7B: Polyamine catabolism: spermidine 24 F. Significance of Polyamines 1. Polyamines and bacteriophage In 1957, Hershey noted that bacteriophage T2 contains two ninhydrin p o s i t i v e , non amino acid compounds. Ames, Dubin and Rosenthal (1958) i d e n t i f i e d these substances as putrescine and spermidine and showed that they account for 40-50% of the i d e n t i f i a b l e cations w i t h i n the phage p a r t i c l e s . Ames and Dubin (1960) stated that almost a l l of the polyamines are displaced from a permeable 0 mutant of T4 by washing. Although t h i s r e s u l t implied that polyamines are unnecessary for i n f e c t i v i t y , phage reproduction i s strongly influenced by these bases (Cohen, 1971). As i s we l l known, T4 r l l mutants are unable to multiply i n E_. c o l i K12( A) (Benzer, 1957) unless spermidine i s present e x t e r n a l l y ( F e r r o l u z z i -Ames and Ames, 1965). Since i n f e c t i o n leads to a leakage of putrescine, the addition of spermidine, presumably, restores the i n t r a c e l l u l a r poly-amine balance. O r d i n a r i l y , when T4DD mutants i n f e c t E_. c o l i B, the onset of phage DNA synthesis i s delayed (Dion and Cohen, 1971). I n f e c t i o n i n the pre-sence of spermidine but not i n the presence of putrescine, shortens t h i s delay considerably (Dion and Cohen, 1971). T4 i n f e c t i o n of E_. c o l i mutants depleted i n polyamine content r e s u l t s i n very retarded phage development (Dion and Cohen, 1972). The addi t i o n of putrescine, cadaverine or sper-midine j u s t p r i o r to i n f e c t i o n , however, promotes successful phage repro-duction (Dion and Cohen, 1972). From these data, i t i s clear that i f T4 i n f e c t i o n of E_. c o l i i s to be normal, polyamines are v i t a l . 2. Polyamines and ba c t e r i a Herbst and Sn e l l (1948) demonstrated that Haemophillus parainfluenziae 25 i s unable to grow unless supplied with polyamines. The i s o l a t i o n of co n d i t i o n a l polyamine auxotrophic s t r a i n s of E_. c o l i has been achieved; the i r growth under r e s t r i c t i v e conditions i s very slow (Morris and Jorstad, 1970 and Maas, L e i f e r and Poindexter, 1970). In one s t r a i n , the c e l l s form snakes (Maas, L e i f e r and Poindexter, 1970). A stronger connection : •:• between polyamines and c e l l d i v i s i o n was established by Inouye and Pardee (1970) : growth of an arginine auxotroph of E_. c o l i blocked before orni t h i n e i s synchronous following the ad d i t i o n of a r g i -nine to an arginine-starved culture. In contrast, another arg mutant, blocked a f t e r ornithine divides asynchronously when arginine i s added to a starved culture. I t was suggested that an adequate polyamine pool i s a p r e r e q u i s i t e for c e l l d i v i s i o n i n E_. c o l i . Synchrony may also be ob-tained by heat shock (Smith and Pardee, 1970). Smith and Pardee (1970) noted the existence of a heat l a b i l e p rotein i n E_. c o l i and postulated that i t s s e n s i t i v i t y might create synchrony. I t was l a t e r reported that an enzyme concerned with methionine biosynthesis i n E_. c o l i i s heat l a b i l e (Lomnitzer and Ron, 1972). I t i s p o s s i b l e , therefore, that heat shock may d i s t o r t the polyamine pool (see F i g . 6). In b a c t e r i a , spermidine i s p h y s i o l o g i c a l l y more a c t i v e than putre-scine. During polyamine depletion i n E. c o l i , a homeostatic balance maintains a spermidine pool at the expense of putrescine (Tabor and Tabor, 1969). In Myxococcus' • xanthus, microcyst formation i s blocked by spermi-dine but enhanced by putrescine (Witkin and Rosenburg, 1970). The synthe-s i s of spermidine and RNA appear to be linked i n E_. c o l i . The addition of methionine to E. c o l i stimulates both RNA and spermidine syntheses (Raina 26 Jansen and Cohen, 1967). When a relaxed s t r a i n of E_. c o l i TAU i s starved for arginine, RNA synthesis continues and the spermidine l e v e l but not the putrescine l e v e l r i s e s during t h i s time (Cohen et a l . , 1967). Spermidine, however, does not accumulate when the organism i s starved f o r u r a c i l . S u r p r i s i n g l y , i n a stringent s t r a i n of E_. c o l i TAU, the addition of exo-genous spermidine during arginine s t a r v a t i o n e f f e c t s an increase i n RNA s y n t h e s i s — as i f the s t r a i n were relaxed (Raina, Jansen and Cohen, 1967). Moreover, spermidine, at p h y s i o l o g i c a l concentrations, stimulates glucose-6-phosphate dehydrogenase (Sanwal, 1970) which catalyzes the f i r s t step of the phosphogluconate pathway and ribose i s derived mainly v i a t h i s path-way i n E_. c o l i (Lanning and Cohen, 1954). It would seem, then, that sper-midine or the spermidinerputrescine r a t i o i s important i n RNA synthesis and b a c t e r i a l metabolism generally. G. Polyamine: DNA Interactions The discovery of. polyamines i n T-even b a c t e r i a l viruses indicated that polyamines could bind to DNA i n vivo (Hershey, 1957). At the concen-t r a t i o n s observed, polyamines could n e u t r a l i ze one t h i r d to one h a l f of the T4 DNA (Ames, Dubin and Rosenthal, 1958). The nature of t h i s binding i s i o n i c and polyamines can be displaced i n solutions of high osmotic strength. Mahler and Mehrotra (1963) showed that polyamines increase the Tm of DNA and that of those tested, cadaverine produces the l a r g e s t increment, with putrescine being almost as e f f e c t i v e . The X-ray c r y s t a l l o g r a p h i c studies of L i q u o r i et a l . , (1967) led them to propose that spermidine and spermine form i o n i c bridges across the narrow groove of the DNA helixivA Mandel (1962) Vtheori'zed^tfr spermine 27 r e f l e c t s the DNA base composition, A-T r i c h DNA molecules were thought., to p r e f e r e n t i a l l y bind spermine more strongly than A-T poor DNA (Liquori et a l . , 1967). In disputing t h i s proposal, Hirschman et a l . , (1967), found that the net charge of spermine varied with temperature. When the DNA:spermine i n t e r a c t i o n was measured by equilibrium d i a l y s i s , no corre-l a t i o n between spermine and DNA base composition was observed (Hirschman et a l . , 1967). The demonstration of polyamine:nucleic acid i n t e r a c t i o n s i n vivo has yet to be made. The i s o l a t i o n of polyamines and nu c l e i c acids together, therefore, could merely be an a r t i f a c t of extra c t i o n . I I I . MODIFIED BASES Though common i n tRNA, modified bases of DNA, i n comparison, are un-usual..5-Hydroxymethylcytosine (HMC) which occurs i n the DNA of T-even phages as a glucosylated d e r i v a t i v e , was f i r s t discovered i n 1952 (Wyatt and Cohen, 1952). HMC t o t a l l y replaces Cytosine i n T-even phage DNA. This i s brought about by two enzymes: cytosine deaminase, which deaminates deoxycytidine monophosphate (dCMP) to deoxyuridine monophosphate (dUMP) and cytosine hydroxymethylase (Cohen, 1968). HMC, then, e x i s t s i n the pool and i s polymerized as such. A further modification, glucosylation, occurs only a f t e r the T-even DNA i s synthesized (Cohen, 1968). Certain B_.. s u b t i l i s phages, such as 0e, SPOr-1 and SP8, contain 5-hydroxymethyluracil (HMU) i n place of thymine (Roscoe and Tucker, 1966). The biosynthesis of HMU i n phage infected c e l l s also occurs at the nu-cleot i d e l e v e l . The incorporation of thymine into phage DNA i s prevented by a thymidine triphosphate nucleotidohydrolase which hydrolyzes deoxythy-midine triphosphate (dTTP) to dteoxythymidine monophosphate (dTMP) (Roscoe, 1969). In i t s absence, thymine i s incorporated i n t o 0e DNA without a 28 loss of i r i f e c t i v i t y (Marcus and Newlon, 1971). PBS-1, another B_. s u b t i l i s phage, contains u r a c i l instead of thymine (Tomita and Takahashi, 1969). A novel enzyme, deoxycytidine triphosphate deaminase, which deaminates dCTP to dUTP, i s responsible for the l a t t e r i n the pool (Tomita and Takahashi, 1969). The question of j u s t how cyto-sine appears i n PBS-1 DNA has not been answered but regulation of dCTP deaminase i s indicated. The B y s u b t i l i s phage, SP-15, has recently been shown to contain d i — hydroxypentyluracil (DHPU.) (Marmur et a l . , 1972 and Brandon et a l . , 1972). The base only p a r t i a l l y replaces thymine and e x i s t s i n the DNA i n a glu-cosylated form. Since t h e i r biosyntheses can be complex (Cohen, 1968) , these modified bases presumably evolved f o r a reason or reasons. One major function of modification i s probably the protection of DNA from nuclease digestion i n viv o . In the T-even phages, HMC serves as a d i s t i n c t i v e s i t e on the DNA molecule at which glucosylation can occur. That phages containing non-glucosylated DNA are unable to multiply i n r e s t r i c t i v e or nuclease contain-ing hosts supports t h i s idea. Often, work leading to the i d e n t i f i c a t i o n of modified bases was i n i -t i a t e d by a common c u r i o s i t y . That i s , the Tin and the buoyant density did not correspond to the same percent GC value. In characterizing the DNA of the P_. acidovorans phage, 0W-14, Kropinski (1970; made a s i m i l a r obser-vation. 0W-14 contains, i n addition to the regular four bases, N-putrescin^ ylthymine (NpT) (Kropinski, Bose and Warren, 1973), which replaces almost h a l f of the thymine residues i n the phage DNA. 29 An obvious question which arose was the biosynthetic o r i g i n of NpT i n phage infe c t e d c e l l s . I t was the goal of t h i s t h e s i s , then, to esta-b l i s h the o r i g i n of the putrescine moiety of the base. An e s s e n t i a l pre-liminary to t h i s work was the e l u c i d a t i o n of arginine and polyamine meta-bolism i n the uninfected host. 30 MATERIALS AND METHODS .1. ORGANISMS Pseudomonas acidovorans 29, obtained from R.Y. Stanier, was used e x c l u s i v e l y throughout t h i s study. I t was maintained on minimal agar plates supplemented with disodium succinate. A fresh plate was prepared every two months. Bacteriophage 0W-14 stocks^; were stored i n complex media at 4°C over chloroform. I I . MEDIA Mannitol L u r i a broth (MLB) contained (g/1): tryptone, 10.0; yeast extract, 5.0; NaCl, 2.5; and d-mannitol, 1.0. D i l u t e mannitol L u r i a broth (DMLB) was MLB d i l u t e d ten f o l d . 007 minimal medium (Clark, 1968) was modified to contain (g/1): (NH^SO^ , 2.0; Na 2HP0 4, 6.0; KH 2P0 4, 3.0; MgCl 2, 0.4; FeCl 3-6H 20, 0.0008; and C a C l 2 ; 0.017. Disodium succinate (4 g/1) was added separately as a s t e r i l e 20% s o l u t i o n . For s o l i d media, Bacto-Agar (Difco) was added to the appropriate medium to give a f i n a l concentration of 1.5%. I I I . GROWTH OF BACTERIA A l l cultures were grown at 30°C. Routinely, l i q u i d cultures were agitated on a Metabolyte G-77 shaker water bath (New Brunswick S c i e n t i f i c Co., New Brunswick, N.J.) set at 250 revolutions per minute (RPM) . Ten 1 cultures were grown i n a fermenter (Fermentation Design, Allentown, Pa.), adjusted to 250 RPM and a flow rate of 5000 cc of a i r per minute. C e l l 31 density was determined with a Klett-Summerson colorimeter (Klett Manufac-t u r i n g Co., New York, N.Y.) equipped with a number 54 f i l t e r . K l e t t units were converted to o p t i c a l density (OD) units by reference to a standard curve. IV. PHAGE TITRATION The agar layer technique of Adams (1959) was used to assay plaque forming units (PFU)/ml of l y s a t e . Using MLB plates, the top and bottom layers contained 0.75 and 1.5% agar, re s p e c t i v e l y . V. PREPARATION OF PHAGE STOCKS Q Cultures were grown to 2.5-5.0 x 10 c e l l s / m l and s u f f i c i e n t phage added to give a m u l t i p l i c i t y of i n f e c t i o n (m.o.i.) of 0.1. Incubation was continued u n t i l l y s i s was complete". T i t e r s of 10^PFU/ml were generally obtained. VI. PURIFICATION OF PHAGE Deoxyribonuclease (DNase) 1 was added to lysates at 1 ug/ml and i n -cubation continued f o r 60 min. B a c t e r i a l debris was removed by centifuga-t i o n (10,000 x g f o r 10 m i n ) . The supernatant was centrifuged at 14,000 x g for 2 hrs and the p e l l e t resuspended i n DMLB. A l l centrifugations were performed at 4°C. VII. OSMOTIC SENSITIVITY OF 0W-14 An 0.1 ml sample of phage i n DMLB was transferred to 0.2 ml 5 M NaCI. Af t e r 16 hrs, the suspension was d i l u t e d one thousand f o l d i n water. For p l a t i n g , further d i l u t i o n s were also made i n water. As a c o n t r o l , a s e r i e s of d i l u t i o n s was made i n 5 M NaCI. PFU's were determined as i n section IV. 32 VIII. UTILIZATION OF COMPOUNDS AS NITROGEN SOURCE Minimal agar plates were used except that MgSO^ and Ionagar No. 2 (Oxoid) replaced MgC^ and Bacto-Agar, re s p e c t i v e l y , (NH^^SO^ was omitted. Bacteria were washed with 0.05 M KH^PO^ buffer (pH 7.0).,prior g to p l a t i n g . P l a t i n g was performed by spreading 2.0 x 10 c e l l s with a flamed glass rod. Approximately 1.0 mg of the test compound was then placed i n the center of each p l a t e . A compound was considered non u t i l i z a -ble i f growth was not observed within three days. IX. UPTAKE'OF RADIOACTIVE COMPOUNDS g Cultures were grown to a density of 2.5 x 10 c e l l s / m l i n (.007 modi-f i e d medium. Then the appropriate compound (non-radioactive) was added to give a concentration of 20 ug/ml. Incubation was continued and when the g c e l l density was 5.0 x 10 c e l l s / m l , the radioactive compound was added to give 0.25 uc/ml ( i . e . 0.25 uc/20ug of compound). 14 For the transport of C-putrescine by phage infected c e l l s , s u f f i -8 cient phage was added to a culture at 5 x 10 c e l l s / m l to give a m.o.i. of 14 10. A f t e r allowing 1.0 min for adsorption, C-putrescine was added as described above. Ten seconds a f t e r adding the radioactive test substrate, a 1.0 ml sample was removed; further samples were taken every 5 min thereafter for 60 min for uninfected cultures. Infected cultures were sampled for 80 min. One h a l f ml of each sample was passed through a 0.45 urn f i l t e r ( M i l l i p o r e F i l t e r Corp., Bedford, Mass.)pre-^wetted with minimal medium; the f i l t e r was then washed with 2.0 ml of minimal medium. The remaining 0.5 ml was added to 0.5 ml i c e cold 10% t r i c h l o r o a c e t i c acid (TCA); a f t e r 20 min. on i c e , the TCA p r e c i p i t a b l e material was c o l l e c t e d on a 0.45 um f i l t e r previously 33 moistened with 5% TCA; the tube was rinsed with 2.0 ml of cold 5% TCA which was also passed through the f i l t e r . X. ISOLATION OF POLYAMINES A. From b a c t e r i a The method of Raina (1963.) was adopted for the routine extraction of polyamines. C e l l s were centrifuged and washed i n a half volume of 0.05 M KR^PO^ (pH 7.0) buffer. During these procedures, c h i l l i n g was avoided. The washed p e l l e t s were resuspended i n cold 5% TCA and l e f t on i c e for 20 min. Following centrifugation at 4°C, the TCA supernatant was extracted three times with an equal volume of ether. The pH of the extracted super-natant was adjusted to 12-13 with 5 N NaOH. To 10 ml of supernatant were added 2.5 g of a Na^O^: Na 3P0 4»12H 20 s a l t mix (62.5 g:9.0 g) and an equal volume of Ji^-butanol. The mixture was shaken at room temperature for 30 min. , a f t e r which the butanol phase was removed and a c i d i f i e d to pH 2 with 6N HC1. The butanol extraction was repeated three times. The butanol extracts were pooled and evaporated to dryness using a rotary f l a s h evapor-ator. The residue was washed i n d i s t i l l e d water and f i n a l l y dissolved i n a small volume of water. The polyamine solutions obtained were stored at o.:?cv.. The extraction method of Dion and Cohen (1972) was used for quantita-9 t i v e studies. A t o t a l of 10 c e l l s was c o l l e c t e d on 0.45 urn f i l t e r s 8 (5 x 10 c e l l s / f i l t e r ) pre-wetted with 007 medium. The f i l t e r s were washed with 2.0 ml 007 medium then transferred to 1.0 ml of i c e cold 0.2 N p e r c h l o r i c acid (PCA). A f t e r 20 min on i c e , the PCA solutions were f i l t e r e d using 0.45 um f i l t e r s of 13 mm diameter ( M i l l i p o r e F i l t e r Corp.). PCA f i l t r a t e s were stored at 0°C. 34 B. From Phage 3.0 x l O ^ p u r i f i e d phage p a r t i c l e s were washed (3x) with T2 bu f f e r which contained (g/1):Na 2HP0 4•12 H 20, 7.4; VNaH^PO^, 1.5; NaCI, 4.0; K 2S0 4, 5.0; MgS04-7H2G, 0.50; CaCl 2'2H 20, 0.0194 and g e l a t i n ; 0.01. The p e l l e t was extracted with 0.5 ml of i c e cold 0.2 N PCA. PCA f i l t r a t e s containing polyamines were obtained i n a manner i d e n t i c a l to that des-cribed f o r b a c t e r i a . XI. PREPARATION OF DANSYLATED POLYAMINES To 0.2 ml PCA f i l t r a t e were added 50 mg Na 2C0 3 and 1.0 ml of dansyl chloride (2 mg/ml i n acetone). A f t e r 16 hrs i n the dark at room tempera-ture, 0.1 ml of a p r o l i n e s o l u t i o n (100 mg/ml) was added and incubation continued f o r another 30 min. The s o l u t i o n was then extracted 3x i n sub-dued l i g h t with 0.5 ml benzene. The pooled extracts were evaporated with a gentle stream of a i r ; the aqueous residue was taken to complete dryness i n vacuo over NaOH. The residue was dissolved i n 1.0 ml benzene. As re-ferences, 0.2 N PCA solutions of putrescine (0.00056 M) and spermidine (0.00034 M) were treated i n an i d e n t i c a l manner. XII. CHROMATOGRAPHY A. Thin Layer (TLC) 1. Polyamines Solvents f o r development were: A (Hammond and Herbst, 1968), diethylene glycolmonoethyl ether/propionic acid/H 20.(70/15/15, V/V/V) saturated with NaCI; B (Holder and Bremer, 1966), isopropanol/concentrated HC1/H20 (80/30/ 20, V/V/V), C (Tobari and Tchen, 1971), methanol/concentrated NH^OH (7/3, V/V) and D (Stahl, 1969), acetone/diethylamine/H 20 (30/6/15, V/V/V). 35 C e l l u l o s e sheets (Eastman Chromogram Sheets, 6064, without fluorescent i n d i c a t o r , Eastman Organic Chemicals, Rochester, N.Y.) were used with solvents A, B and D. S i l i c a gel sheets (Eastman Chromogram Sheets, 6061, without fluorescent indicator) were used with solvent C_. 2. Dansylated Polyamines Solvent E_ (Dion and Cohen, 1972), ethylacetate/cyclohexane (1/2, V/V) and s i l i c a gel G sheets (glass backed, 1011, Analtech Inc., Newark, Dele-ware) were used to e f f e c t separation of dansylated polyamine extracts and dansylated polyamine standards.Chromatography was performed i n the dark. 3. DNA Hydrolysates DNA hydrolysates and reference standards, were applied to c e l l u l o s e sheets (Eastman Chromogram Sheets, 6064, without fluorescent i n d i c a t o r ) . Solvents for development were: F_ (Bendich, 1957), isopropanol/concentrated HC1/H20 (65/17/18, V/V/V/); G (Kropinski et a l . , 1973), isopropanol/concen-trated NH40H/H20 (70/10/20, V/V/V) and H (Cline et a l . , 1959), t-butanol/ methyl ethyl ketone/concentrated HC1/H20 (40/30/10/20, V/V/V/V). A Chromato Vue ( U l t r a - V i o l e t Products Inc., San G a b r i e l , C a l i f . ) was used to detect u l t r a - v i o l e t absorbing areas on developed chromatograms. B. Column chromatography Polyamines were separated on Dowex 50W-X2 (100-200 mesh) (Bio-Rad Laboratories, Richmond, C a l i f . ) . Polyamines were eluted with a l i n e a r gra-dient of HC1 (0-2.5 N.HC1; t o t a l volume 600 ml) (Tabor, Rosenthal and Tabor, 1958). A flow rate of 1.0 ml/min was maintained and 3.0 ml f r a c -tions were c o l l e c t e d . 36 XIII. DETECTION OF POLYAMINES Free polyamines were detected on developed chromatograms by spraying with ninhydrin as follows.:" the following solutions were prepared: (a) 50 ml of 0.25% ninhydrin i n absolute ethanol, 10 ml of g l a c i a l a c e t i c acid and 2.0 ml of 2,4 ,6-trimethylpyridine, (b) 1% Cu(N0 3) 2•3H 20 i n absolute ethanol. Just p r i o r to use, 50 parts (a) were mixed with 3 parts (b). Afte r spraying, chromatograms were heated at 100°C f o r 5 min. Freshly prepared periodate spray (Lemieux and Bauer, 1954) was used to detect 2-hydroxyputrescine. The chromatagram was sprayed with a mixture of 4 parts aqueous sodium metaperiodate and 1 part 1% KMnO^ dissolved i n 2% Na2C0.j. A f t e r the periodate p o s i t i v e spots developed, the chromatograms were washed with water. XIV. QUANTITATION OF POLYAMINES Developed chromatograms of dansylated extracts and standards were scanned using a Turner Model .11-1 Flubrometer -equipp.ed-with'-a TLC-scanner and a recorder (G.K. Turner Associates, Palo A l t o , C a l i f . ) . Dansylated polyamines present i n extracts were quantitated by reference to a standard curve prepared from scans of the reference, dansylated polyamines. XV. ISOLATION OF 2-HYDROXYPUTRESCINE The polyamines were extracted from the c e l l p e l l e t obtained from a 10 1 culture, harvested at a density of 10 c e l l s / m l , by Sharpies c e n t r i f u -gation. The polyamines were separated by chromatography on a column (2 cm x 12 cm) of Dowex (see Materials Methods XIIB). Amine p o s i t i v e f r a c t i o n s were detected by spotting aliquots on Whatman no. 1 f i l t e r paper and 37 spraying with ninhydrin. Thin layer chromatography (solvent C) was used to determine f r a c t i o n s containing pure 2-hydroxyputrescine. Impure 2-hydroxyputrescine f r a c t i o n s were pooled, reduced i n volume and applied to a second column (0.9 cm x 13.5 cm) and the p u r i f i c a t i o n repeated. XVI. NMR OF 2-HYDROXYPUTRESCINE An NMR spectrum was obtained of a D^O s o l u t i o n of 2-hydroxyputrescine using an HA-100 instrument (Varian Associates, Palo Alto., C a l i f . ) . Chemi-ca l s h i f t s were recorded r e l a t i v e to an external tetramethylsilane (TMS) standard. XVII. ISOLATION OF DNA A. From b a c t e r i a DNA was i s o l a t e d by the sodium dodecyl s u l f a t e (SDS) - phenol method of Kozinski and L i n (1965). Bacteria were centrifuged, washed once with 0.05 M KH 2P0 4 buffer (pH 7.0) and resuspended i n 2.0 ml 0.01 M TRIS-HC1/ 0.15 M NaCl/0.15 M ethylenediamine t e t r a a c e t i c acid (EDTA) bu f f e r , pH 7.6 (TNE b u f f e r ) . L y s i s was effected by adding SDS to a concentration of 1% then heating at 45°C for 15 min. The volume was increased to 5.0 ml by the addition of 0.15 M NaCl/0.015 M trisodium c i t r a t e , pH 7.0 (standard s a l i n e c i t r a t e , SSC). DNA was extracted by adding an equal volume of water-saturated phenol to the suspension. The emulsion was separated by cen t r i f u g a t i o n at 4°C, the aqueous layer removed, and re-extracted with water-saturated phenol. Residual phenol was removed from the aqueous layer by c a r e f u l extraction with ether (3x). I f the DNA was ra d i o a c t i v e , i t was dialysed against 2,2:1 volumes 0.1 M SSC for 24 hrs. The DNA so l u t i o n was c h i l l e d and 2 volumes of i c e cold 95% ethanol were added. Nucleic acid 38 p r e c i p i t a t e s were c o l l e c t e d on a glass rod by s t i r r i n g . P r e c i p i t a t e s were washed i n acetone, dried and transferred to glass ampoules. B. From phage Phage were p u r i f i e d according to section VI. I f the phage were radio-a c t i v e , they were washed three times with DMLB. The phage p e l l e t was re-suspended i n 2.0 ml TNE buffer p r i o r to the addition of 3.0 mis of SSC. Phage l y s i s and DNA extraction were achieved simultaneously by adding an equal volume of water-saturated phenol. The DNA was p u r i f i e d further as described f o r b a c t e r i a l DNA. XVIII. HYDROLYSIS OF DNA Aft e r 0.2 ml of 6N HC1 was added to the glass ampoule containing the DNA, the ampoule was sealed under reduced pressure and the DNA hydrolyzed at 100°C f o r 90 min. The so l u t i o n was cooled and evaporated to dryness i n vacuo over NaOH. The residue was dissolved i n 0.1 N HC1. XIX. MEASUREMENT OF RADIOACTIVITY A. Chromatographed polyamines or DNA hydrolysates Chromatograms containing radioactive material were s l i c e d into sec-tions and each section then transferred to an i n d i v i d u a l s c i n t i l l a t i o n v i a l . B. TCA soluble materials Aliquots of acid soluble material were spotted on glass f i b e r f i l t e r s (Reeve. Angel, C l i f t o n , N.J.) and dried thoroughly. The f i l t e r s were then placedci i n s c i n t i l l a t i o n v i a l s . C. TCA p r e c i p i t a b l e materials Acid p r e c i p i t a b l e materials were c o l l e c t e d on M i l l i p o r e f i l t e r s (0.45 um) and dried thoroughly. The f i l t e r s were then transferred to s c i n t i l l a -t i o n v i a l s . 39 D. Counting r a d i o a c t i v i t y A f t e r t r a n s f e r of the samples to s c i n t i l l a t i o n v i a l s , 5.0 ml of s c i n -t i l l a t i o n f l u i d ( L i q u i f l u o r , New England Nuclear, Boston, Mass.) was added and the r a d i o a c t i v i t y measured with an Isocap/300 l i q u i d s c i n t i l l a t i o n spectrometer (Nuclear Chicago Corp., Des Plaines, 111.). XX. CHEMICALS Putrescine dihydrochloride, spermidine t r i h y d r o c h l o r i d e , guanine cytosine, thymine and adenine were obtained from Calbiochem (San Diego, C a l i f . ) . Dansyl chloride was purchased from N u t r i t i o n a l Biochemical Co. (Cleveland, Ohio). Worthington Biochemical Corp. (Freehold, N.J.) supplied DNase 1. XXI. RADIOCHEMfCALS " 3 A l l radiochemicals, with the exception of H-2,3-serine (Schwarz Mann, Orangeburg, N.Y.) were purchased from New England Nuclear. 40 RESULTS AND DISCUSSION I. UTILIZATION OF COMPOUNDS AS SOLE SOURCE OF NITROGEN Table II shows the u t i l i z a t i o n of various compounds as the sole source of nitrogen. P_. acidovorans did not u t i l i z e arginine, which sug-gested that the organism could neither deaminate arginine to a-ketoarginine nor deiminate i t to c i t r u l l i n e . P_. acidovorans s t r a i n s , however, were known to lack a dihydrolase pathway (Stanier, P a l l e r o n i and Doudoroff, 1966). Since urea and Y - a m i n o b u t y r a t e (yABA) were e f f e c t i v e l y u t i l i z e d , arginine could not have been catabolized to these compounds, e i t h e r . This means that, i n contrast to E_. c o l i and B a c i l l i , P_. acidovorans cannot derive putrescine or o r n i t h i n e from arginine. This reasoning, however, assumed that the organism was permeable to arginine. I I . TRANSPORT OF RADIOACTIVE SUBSTRATES The demonstration of an arginine transport system i s shown i n F i g . 8. Since saturation occurred at 25 min, the process appeared to be c a r r i e r f a c i l i t a t e d . F i g . 9 demonstrates that ornithine was also taken up by P_. acidovorans; since saturation was not observed, e i t h e r the c a r r i e r had a very large Km or ornithine was taken up by passive d i f f u s i o n . Putrescine, on the other hand, was transported neither by the uninfected nor i n f e c t e d host (Fig. 10 and 11). I I I . POLYAMINES OF P. ACIDOVORANS As a prelude to d i r e c t l y studying the o r i g i n of NpT, the polyamine composition of the host was determined. Three TLC systems were used to show the presence of three polyamines: spermidine, putrescine and a t h i r d 41 Compound (NH 4) 2S0 4 p r o l i n e glutamic acid arginine ornithine c i t r u l l i n e putrescine spermidine spermine urea Y-aminobutyric acid (YABA) Used as Nitrogen Source + + + + + TABLE I I . U t i l i z a t i o n of compounds as sole source of nitrogen. P_. acidovorans was plated on nitrogen-free minimal agar p l a t e s . Approximately 1 mg of the test substrate was placed i n the center of each plate. Compounds were considered negative i f growth was not observed a f t e r three days. 42 10 20 30 40 SO 60 MINUTES FIGURE 8. The transport of C-U-arginine by P_. acidovorans. .;>•' ; Incorporation i n t o whole c e l l s , o ; incorporation into TCA p r e c i p i t a b l e m a t e r i a l , a . See Materials and Methods (Section IX), for p r o t o c o l . Since cultures were a c t i v e l y growing, the CPM/ml were calculated by d i v i d i n g the ac t u a l CPM/ml values by a factor proportional to the growth rate. 0 10 20 30 40 50 60 MINUTES 14 FIGURE 9. The transport of C-5-ornithine by P. acidovorans., Incorporation i n t o whole c e l l s , e ; incorporation into TCA p r e c i p i t a b l e m a t e r i a l , • . See Mater i a l s and Methods (Section IX) f o r protocol. Since c u l -^ tures were a c t i v e l y growing, the CPM/ml were c a l c u -l a t e d by d i v i d i n g the actual CPM/ml values by a f a c t o r proportional to the growth rate. 1 0 10 20 30 40 MINUTES 50 60 14 i FIGURE 10. The transport of C-3,4-putrescine by P. acidovorans. Incorporation into whole c e l l s , • ; incorporation i n t o TCA p r e c i p i t a b l e m a t e r i a l , a . See Mater i a l s and Methods (Section IX) f o r protocol. Since c u l -tures were a c t i v e l y growing, the CPM/ml were ca l c u -lated by d i v i d i n g the actual CPM/ml values by a fact o r proportional to the growth rate. MINUTES FIGURE 11. The transport of C-3,4-piitrescine by P_. acidovorans i n f e c t e d w i t h 0W-14. In c o r p o r a t i o n i n t o whole c e l l s , •©., i n c o r p o r a t i o n i n t o TCA p r e c i p i t a b l e * material. , See M a t e r i a l s and Methods (Section IX) f o r p r o t o c o l . 46 compound which, because of i t s Rf values (Table III) could have been ei t h e r 1,3-diaminopropane or 2-hydroxyputrescine. I t was decided to ascertain conclusively the i d e n t i t y of t h i s unknown substance. F i r s t l y , the unknown polyamine reacted with ninhydrin to give a purple color; 1,3-diaminopropane, i n comparison, produced a red color. As expected of an amine with a v i c i n a l hydroxyl group, i t also reacted with periodate.1,3-Diaminopropane was oxidized only s l i g h t l y by periodate. 14 The t h i r d polyamine could be l a b e l l e d by C-6rnithine (Table IV). The only radioactive product that could a r i s e from the oxidation of 14 C-omithine l a b e l l e d spermidine would be Y - a mi nobutyraldehy.de'. F i g . 7B, reaction A); 1,3-diaminopropane would be non-radioactive v i a such a cleavage. Rigorous proof of the compound's i d e n t i t y was obtained by NMR spectroscopy. Approximately 22 mgs of the dihydrochloride s a l t of the compound were p u r i f i e d from a ten 1 batch culture. The NMR spectrum of t h i s material i s presented i n F i g . 12. Signals at: 2.25 ^H.-CE^) , 3.49 (4H, -CH2N-) and 4.37 (IH, -CH0-) confirmed that the compound was 2-hydroxyputrescine (Tobari and Tchen, 1971). The occurrence of 2-hydroxyputrescine, putrescine and spermidine to-gether, i s to my knowledge, the f i r s t reported case of such a combination. Kim's (1966) Pseudomonad contained 2-hydroxyputrescine and putrescine but lacked spermidine. Rosano and Hurwitz (19'69), i n th e o r i z i n g that spermi-dine may not occur i n c e l l s containing 2-hydroxyputrescine, suggested also that these polyamines may have s i m i l a r metabolic functions (see Introduc-t i o n , Section IID) . The occurrence of both compounds i n P_. acidovorans suggests that they may play d i s t i n c t r o l e s . 47 Compound Rf Values A B spermidine 0.24 0.25 0.10 putrescine 0.40 0.38 0.22 P_. acidovorans 0.20, 0.32 0.29,0.39 0.11, 0.24 extract 0.39 0.38 TABLE I I I . Thin layer chromatography of polyamines. See Materials and Methods (Section XIIA1) for a d e s c r i p t i o n of s o l -vents A, B and C, and the conditions employed. 48 49 IV. LABELLING OF POLYAMINES In an attempt to create an i n t e r n a l pool of rad i o a c t i v e putrescine, various compounds were tested as p o t e n t i a l polyamine precursors (Table 14 IV). The i n a b i l i t y of C-putrescine to l a b e l the polyamines r e i t e r a t e d the concept that P_. acidovorans could not transport t h i s diamine. The small amount of r a d i o a c t i v i t y (156 CPM) that was confined to the putre-scine area was probably due to the presence of contaminating input l a b e l which was present i n the polyamine extract. 14 The i n e f f i c a c y of C-arginine i n l a b e l l i n g the polyamines also strengthened the notion that P_. acidovorans could not obtain urea from arginine. Presuming that arginine i s degraded at a l l , two catabol i t e s are po s s i b l e : agmatine or y-guanidinobutyramide. Enzymatically, the formation of these two compounds d i f f e r s , so that enzyme assays could determine i f arginine can be degraded and i f so, to which c a t a b o l i t e . 14 r-CnQrnithine e f f e c t i v e l y l a b e l l e d the polyamines. The organism, therefore, must possess an a m i thine decarboxylase. Since o r n i t h i n e was not u t i l i z e d as a sole source of nitrogen (Table I I ) , putrescine was apparently not catabolized to yABA (Fig. 7A). Stanier, P a l l e r o n i and Doudoroff (1966) have reported that, of o r n i t h i n e and yABA, only the l a t t e r i s s u i t a b l e as a sole source of carbon. Hence, i n s t r a i n 29, as i n other P_. acidovorans s t r a i n s , o r n i t h i n e i s not converted to yABA. 14 C-Glutamic acid also l a b e l l e d the polyamines s i g n i f i c a n t l y (Table IV) i n d i c a t i n g that a glutamate to o r n i t h i n e pathway i s operative. In 14 using C-glutamate, a large proportion of r a d i o a c t i v i t y was detected i n the 2-hydroxyputrescine area. Since arginine and 2-hydroxyputrescine have s i m i l a r Rf values i n solvent C, the presence of radioactive arginine Precursor Supplement ,Phage Infection Label (CPM) i n Polyamines spermidine putrescine 2-hydroxyputrescine 14 C-3,4-putrescine 14 C-U-arginine 14 C-5-ornithine 14 C-5-ornithine 14 C-3,4-glutamate 14 n i l n i l n i l n i l n i l + C-3,4-glutamate arginine (1 mg/ml) 32 20 67 45 83 64 156 31 631 1216 244 228 79 50 641 441 627 637 TABLE., I V . ^ L a b e l l i n g of polyamines. Label (0.25 uc/20 ug precursor and 20 ug precursor/ml) was added to 25 mi ciilLures; cultures were harvested at a density gf 10 c e l l s / m l . Where appropriate, phage was added at a m.o.i. of 10 to cultures at a desity of 5 x 10 c e l l s / m l ; the infected cultures were harvested 20 min a f t e r i n f e c t i o n . Polyamines were extracted by the Raina method. Aliquots of polyamine extracts were applied to c e l l u l o s e sheets and chromatography performed i n solvent C. Areas containing polyamines were detected by ninhydrin, excised and counted i n d i v i d u a l l y . o 51 was suspected. The polyamine extracts were, therefore, chromatographed i n solvent D, which e f f e c t i v e l y separated arginine from putrescine, 2-hydroxyputrescine and spermidine. In t h i s system, the arginine region 14 was e s s e n t i a l l y non-radioactive, thus a large percentage of the C-glutamate l a b e l l e d polyamines was t r u e l y due to 2-hydroxyputrescine (Table V). Whether or not P_. acidovorans forms ornithine v i a t r a n s a c e t y l a t i o n or not i s unknown. It would seem, however, that the organism possesses 14 only one pathway for polyamine synthesis. Since C-glutamate l a b e l l e d the polyamines even i n the presence of arginine (1 mg/ml), exogenous ar-ginine did not repress o r n i t h i n e biosynthesis (Table IV). Such a r e s u l t was somewhat unexpected, because i t means that the regulation of arginine synthesis i s unusual. On the one hand, arginine biosynthesis could be t o t a l l y unregulated. On the other hand, i f ornithine i s synthesized v i a the t r a n s a c e t y l a t i o n route, then perhaps, through mutation, the f i r s t and not the second enzyme (Fig. 1) i s i n h i b i t e d by arginine. The pathway would thus continue to cycle i n the presence of t h i s amino ac i d . A l t e r n a t i v e l y , regulation of arginine biosynthesis may be s i m i l a r to that seen i n E_. c o l i B. Arginine, therefore, would have a s l i g h t inductive e f f e c t on the enzymes concerned with i t s biosynthesis. This i s mere speculation for i t i s not even known i f an arg R locus e x i s t s i n P_. acidovorans. Also f e a s i b l e i s a s i t u a t i o n i n which the enzymes of ornithine biosynthesis are uneffected by arginine but the ornithine to arginine steps are i n h i b i t e d by arginine. Enzyme assays could e a s i l y prove or disprove t h i s point. Since there i s apparently only one route for polyamine biosynthesis 52 Precursor Supplement D i s t r i b u t i o n of Label (CPM) arginine polyamines 14 C-3,4-glutamate n i l 32 955 14 C-3,4-glutamate arginine (1 mg/ml) 22 923 14 TABLE V. D i s t r i b u t i o n of l a b e l (CPM) i n C-glutamate -l a b e l l e d polyamine extracts. Aliquots of polyamine extracts were applied to c e l l u l o s e sheets and chromatography performed i n solvent D. Ninhydrin p o s i t i v e areas were excised and counted i n d i v i d u a l l y . 53 i n t h i s bacterium, the c o n s t i t u t i v i t y of arginine synthesis may be r a t i o n a l i z e d as follows: polyamines appear to be e s s e n t i a l f o r normal c e l l u l a r metabolism and an i n h i b i t i o n of arginine and polyamine biosyn-theses would be detrimental to metabolism. V. QUANTITATION OF POLYAMINES The very s e n s i t i v e dansyl technique (Dion and Cohen, 1972) was used to quantitate the polyamines of P_. acidovorans. The i n t e r n a l m i l l i m o l a r concentrations of spermidine, putrescine and 2-hydroxyputrescine were 3-5, 50 and 45, r e s p e c t i v e l y , assuming that the volume of P_. acidovorans equalled that of E_. c o l i and the i n t r a c e l l u l a r water content of P_. acidovorans was 70%. The absolute polyamine concentrations corresponded to a r e l a t i v e spermidine, putrescine and 2-hydroxyputrescine r a t i o of 1: 10:9; which approximated the r a t i o of 1: 9.4:9.6, obtained by l a b e l l i n g i n f e c t e d c e l l s 14 with C-ornithine (Table IV). Hence, the use of a putrescine standard curve f o r the determination of 2-hydroxyputrescine (due to a lack of a synthetic standard) seemed reasonable. K e l l n (1973) ascertained that phage DNA synthesis begins at 20 min 14 post i n f e c t i o n . To examine the e f f e c t ( s ) of phage i n f e c t i o n on C-orni-thine l a b e l l e d polyamine pools, i n f e c t e d c e l l s were harvested j u s t p r i o r to the s t a r t of phage DNA synthesis (Table IV). The increased proportion of putrescine suggested that phage DNA synthesis imposed a greater demand for t h i s diamine. In other words, i t implied that putrescine was a pre-cursor of NpT i n phage DNA. 54 Phage i n f e c t i o n could d i s t o r t the polyamine pool i n any one of several ways. The phage genome might code for an ornithine decarboxylase so that, simply, a gene dose e f f e c t would be operative. Phage i n f e c t i o n might lead to the formation of a p o s i t i v e e f f e c t o r f or the host's o r n i -thine decarboxylase. Conversely, a negative e f f e c t o r of t h i s enzyme might be n e u t r a l i z e d as a r e s u l t of phage i n f e c t i o n . Lysates of 0W-14 are extreme by viscous; an i n d i c a t i o n that host DNA i s not degraded dur-ing i n f e c t i o n . The phage, therefore, could synthesize ai; rj-like factor which s p e c i f i c a l l y enhanced the t r a n s c r i p t i o n of the host's gene f o r ornit h i n e decarboxylase. One or more of these means, then, could account for the elevated pool of putrescine observed p r i o r to the synthesis of phage DNA. VI. POLYAMINES OF 0W-14 The dansyl method (Dion and Cohen, 1972) was also used to analyze the polyamines present i n p u r i f i e d phage p a r t i c l e s . The dansyl derivatives of spermidine, putrescine and 2-hydroxyputrescine were resolved by chroma-tography (solvent E). Though not quantitated, the phage polyamine extract contained a q u a l i t a t i v e l y l arger proportion of spermidine than observed i n the uninfected or inf e c t e d host. Since the b a c t e r i a l spermidine con-centration i s much lower than the concentrations of putrescine or 2-hydroxy-putrescine, this suggested that, of the three polyamines, the triamine had the greatest a f f i n i t y for 0W-14 DNA. The presence of polyamines, even a f t e r thorough ." washing of the phage p a r t i c l e s , implied that the phage was osmotically s e n s i t i v e . 55 VII. OSMOTIC SENSITIVITY OF 0W-14 The e f f e c t of an osmotic shock on the v i a b i l i t y of 0W-14 i s shown i n Table VI. The s i g n i f i c a n t drop i n t i t e r as a r e s u l t of osmotic shock indicated that 0W-14 contained a semi-permeable membrane so that the occurrence of free polyamines i n the p a r t i c l e s i s f e a s i b l e . I f the phage were not exposed to 5M NaCI f o r 16 hrs p r i o r to the shock, a much lower drop i n t i t e r was observed (C. Spencer, personal communication). VIII. THE BIOSYNTHESIS OF NpT Of the possible polyamine precursors (Table IV), o r n i t h i n e was deemed the most appropriate for l a b e l l i n g NpT. C e l l s were infe c t e d i n 14 the presence of C-5-ornithine, the progeny phage recovered and t h e i r 14 DNA p u r i f i e d . R a d i o a c t i v i t y from C-5-ornithine was detected only i n NpT (Table VII), so that the p u t r e s c i n y l moiety of NpT could, t h e o r e t i c a l l y , come from ornithine or putrescine. Since P_. acidovorans was impermeable to putrescine, t h i s problem was not resolved. The s e l e c t i o n of a mutant permeable to putrescine seemed hopeless. Any s e l e c t i o n procedure would require the organism to u t i l i z e putrescine as a sole source of nitrogen. As discussed e a r l i e r , t h i s i s not possible i n P_. acidovorans, thus the s e l e c t i o n of a multiple mutant was considered highly improbable. The use of phage i n f e c t e d , toluenized c e l l s may provide an answer, however. To r e i n f o r c e the concept that putrescine, rather than o r n i t h i n e , pre-14 cursed NpT, C - l - o r n i t h i n e was u t i l i z e d . The i n t e r n a l pool of putrescine should be non-radioactive, i n t h i s instance, and s i m i l a r l y , so should NpT. Analysis of the phage DNA revealed a l l bases to be non-radioactive (Table VII). This suggested that the carboxyl group of o r n i t h i n e did not S i t u a t i o n T i t e r (PFU/ml x 10 ) + NaCI (control) 14.5 - NaCI 1.0 TABLE VI. Osmotic s e n s i t i v i t y of phage 0W-14. Phage were placed i n 5M NaCI for 16 hrs d i l u t e d i n water and plated. A c o n t r o l , i n which d i l u t i o n s were performed i n 5M NaCI, was also done. Labelled Compound Added Label (CPM) i n the Nucleic Acid Bases of Progeny Phage A C G T NpT C-5-ornithine 24 20 42 31 1680 C-l-ornithine 38 66 30 49 43 TABLE VII. L a b e l l i n g of 0W-14 DNA with C-ornithine. ^ C - o r n i t h i n e was added to 50 ml cultures at a density of 2.5 x 10^ ce l l s / m l such that there were 0.25 uc/20 ug ornithine and 20 ug ornithine/ml. Cultures were i n -fected at a density o f 10 9 c e l l s / m l with a m.o.i 10. Progeny phage were recovered 6 hrs post i n f e c t i o n . Phage DNA was p u r i f i e d and hydrolyzed. Aliquots of DNA hydrolyzates were applied to c e l l u l o s e sheets and chromatography performed i n s o l -vent F. Areas containing bases were detected by u l t r a - v i o l e t l i g h t , excised and counted i n d i v i d u a l l y . 58 contribute to the structure of NpT but did not eliminate the p o s s i b i l i t y that ornithine was a precursor to NpT. The carboxyl group could function to increase the r e a c t i v i t y of the adjacent secondary amine, thereby f a -c i l i t a t i n g the attachment of that amino group to the pyrimidine precursor. K e l l n (1973) showed the pyrimidine h a l f of the novel base to be derived from deoxyuridine; neither thymine nor thymidine were involved i n t h i s capacity. The consequences of t h i s r e s u l t were two-fold: 0W-14 DNA was synthesized from f i v e bases and the methylene group, which l i n k s the putrescine moiety to the r i n g of NpT, must a r i s e from a metabolic donor of carbon atoms. A tetrahydrofolate (THF) involvement was immediately suspected. Serine transhydroxymethylase transfers the hydroxymethyl group of serine to THF, forming N 1 0methylene THF. K e l l n (1973) confirmed the THF 14 involvement by s u c c e s s f u l l y l a b e l l i n g NpT with C-3-serine (Table V I I I ) . Moreover, the NpT/T l a b e l l i n g r a t i o (0.88) was the same as the NpT/T r a t i o usually observed for phage DNA. The o r i g i n of dTMP and the NpT seemed r e l a t e d — b u t how, exactly? Was the oxidation state of the THF carbon 3 donor the same i n both cases? The use of H-2,3-serine c l a r i f i e d the s i -tuation somewhat. In t h i s case, N^^formyl THF, f o r example, would contain one le s s t r i t i u m atom than N"^methylene THF. Therefore, i f the former compound was used i n the biosynthesis of NpT, then the NpT/T l a b e l l i n g r a t i o would be about 0.45, i f N^^methylene THF were used for both compounds, the r a t i o would be about 0.90. Labelled Compound Label (CPM) i n the Nucleic Acid Bases Ratio Added of Progeny Phage A C G T NpT NpT/T 3624 20 10493 2133 1796 0.88 1095 16 800 639 579 0.90 Experiment performed by R.A. K e l l n ( K e l l n , 1973). 14 * C-3-serine 3 H-2,3-serine TABLE VIII. L a b e l l i n g 3 o f 0W-14 DNA with radioactive serine. H-2,3-Serine was added to 20 ml cultures supplemented with 20 ug methionine/ml at a density of 5 x 10^ c e l l s / m l such that there were 2.5 uc/20 ug serine and 20 ug serine/ml. At a density of 7.5 x 10^ c e l l s / m l , the cultures were infe c t e d with a. m.o.i of 10. Progeny phage were recovered 6 hrs post i n f e c t i o n . Phage DNA was p u r i f i e d and hydro-lyzed. Aliquots of DNA hydrolyzates were applied to c e l l u l o s e sheets and chromato-graphy performed i n two dimensions (one: solvent H, two: solvent G). Areas containing bases were detected by u l t r a - v i o l e t l i g h t , excised and counted i n d i v i d u a l l y 1 60 Analyses of hydrolysates of 0W-14 DNA l a b e l l e d with H-2,3-serine yielded an NpT/T r a t i o of 0.90 (Table V I I I ) . Therefore, N 1 0methylene THF i s an intermediate i n the biosynthesis of the novel base. The accur-acy of t h i s i n t e r p r e t a t i o n i s supported by the d i s t r i b u t i o n of ra d i o -a c t i v i t y i n adenine and guanine (Table V I I I ) . These values are p r e c i s e l y 3 those expected i n l a b e l l i n g the phage DNA with H-2,3-serine. To speculate, the following sequence can be constructed: N^^methylene THF i n t e r a c t s with dUMP to y i e l d 5-HMdUMP which condenses with putrescine to produce NpT. In the biosynthesis of HMC i n T4 inf e c t e d E_. c o l i , N 1 0methylene THF i s the donor f o r the hydroxymethyl group (Cohen, 1968). Thiamine biosyn-thesis i n microbes involves the reac t i o n of an amine with a hydroxymethyl group to y i e l d a C-N bond (White, Handler and Smith, 1968). These reac-t i o n s , then, p a r a l l e l those proposed for the biosynthesis of NpT, and lend f e a s i b i l i t y to the proposed sequence. 61 SUMMARY The metabolism of arginine and polyamines was investigated as a preliminary to examining the biosynthesis of NpT. F_. acidovorans, though permeable to arginine, i s l i m i t e d i n i t s a b i l i t y to catabolize t h i s amino acid. That i s , arginine was unsuitable as a sole source of nitrogen. Ornithine was converted to putrescine but not to yABAr A glutamate to ornithine pathway was demonstrated but i t s operation was unusual because arginine, the ultimate end product, did not block o r n i -thine biosynthesis. This seems reasonable, however, since the organism appears to have only one route for the synthesis of polyamines ( i . e . arginine, as i n E_. c o l i , cannot be catabolized to putrescine). An unusual combination of polyamines occurs i n P_. acidovorans and 0W-14: spermidine, putrescine and 2-hydroxyputrescine. The respective, b a c t e r i a l concentrations (mM) were: 3-5, 50 and 45. 2-Hydroxyputrescine was i d e n t i f i e d conclusively by NMR spectroscopy. Only the putrescine pool expanded a f t e r phage i n f e c t i o n , perhaps ensuring an adequate supply for the synthesis of NpT. An i n t e r n a l pool of radioactive putrescine was necessary for study-ing the o r i g i n of the novel base. Unfortunately, since P_. acidovorans 14 i s impermeable to putrescine, C-ornithine had to be used to l a b e l the 14 polyamines. When c e l l s were infe c t e d i n the presence of C-5-ornithine and the progeny phage DNA analyzed, NpT was the only radioactive base. 14 In a p a r a l l e l experiment, C-l-ornithine was i n e f f e c t i v e i n l a b e l l i n g the phage DNA, so that the carboxyl group of t h i s amino acid was not d i r e c t l y 62 involved i n the synthesis of NpT. The suspicion that the biosynthesis of the novel base involved a THF carbon donor was confirmed by l a b e l l i n g the NpT i n phage DNA with "^C-3-serine. The use of ^ H-2,3-serine showed N"*"^methylene THF to be a l i k e l y intermediate. The proposed sequence of events leading to the f o r -mation of NpT i s as follows: N^methylene THF and dUMP i n t e r a c t to y i e l d 5-HMdUMP which condenses with putrescine to form N-putrescinylthymine. 63 LITERATURE CITED Adams, M.H. 1959. 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