e f ”7 7. # , 7- 7 7 _ ”I e £$OLATEON AND CHARACTERIZATEOR OF ARGININE MUTANT‘S OF $PQNfANEOUS; NETROUS ACID-AND LAMINOPURENE-INDUCED ORIGIN EN SALMONELLA GALLINAR’UM - PULLORUM Thesis ‘or {he Degree 0‘ M. S. MICHEGAN STATE UREVERSIW Mary Judith Robinson 1962 THESIS _.-——..._‘~ .1141 ’2 _—.l:‘-'. I‘m-é: LIBRARY Michigan State University ABSTRACT ISOLATION AND CHARACTERIZATION OF'ARGININE MUTANTS OF SPONTANEOUS, NITROUS ACID-AND 2-AMINOPURINE-INDUCED ORIGIN IN SALMONELLA GALLINARUMfPULLORUM by Mary Judith Robinson The purpose of this study was to isolate and character— ize a series of arginine auxotrophs of Salmonella gallinarum- pullorum 53w suitable for further genetic analysis. Two mutagens, nitrous acid and 2—aminopurine, were employed for the induction of mutation to arginine auxotrophy. The penicillin selection method and the replica plating tech— nique were utilized for the isolation of arginine mutants. One hundred and eighteen arginine auxotrophs and brady- trophs of spontaneous, nitrous acid-and 2-aminopurine- induced origin representing blocks at all steps of the arginine cycle tested were isolated and were characterized on the basis of precursor utilization. The two mutagens were found to differ in effectiveness of inducing particular blocks to arginine synthesis. Evidence is presented which suggests that 53w contains a partial genetic block for the ornithine to citrulline conversion. ISOLATION AND CHARACTERIZATION OF ARGININE MUTANTS OF SPONTANEOUS, NITROUS ACID—AND 2-AMINOPURINE-INDUCED ORIGIN IN SALMONELLA GALLINARUM—PULLORUM By Mary Judith Robinson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1962 ACKNOWLEDGEMENTS I wish to express my sincere appreciation to Dr. Delbert E. Schoenhard for his patience, encouragement, and guidance throughout the course of this study. This study was supported in part by a National Science Foundation Summer Fellowship for Graduate Teaching Assistants. M.J.R. ii TABLE INTRODUCTION HISTORICAL REVIEW. OF CONTENTS Occurrence of Arginine Auxotrophs Arginine Biosynthesis in Microorganisms Linearity of Biochemically Related Genes Cell Regulatory Mechanisms. Mutagens. . . . . Nitrous acid Low pH . . 2-Aminopurine. MATERIALS AND METHODS Culture Methods Induction of Mutation Nitrous Acid. Induction of Mutation and Inactivation with and Inactivation with Acetic Acid Buffer pH A. 5 Preparation of a Stock Culture Free of. Arginine Dependent Mutants Isolation of Spontaneous Arginine Mutants. Induction of Mutation with 2- -Aminopurine Isolation of Arginine Auxotrophs. . . . . Penicillin screening (selection)technique Replica plating technique. Characterization of Arginine Auxotrophs by Pre— cursor Utilization. RESULTS Inactivation of S. gallinarum—pullorum by Nitrous Acid. Induction of Mutations to Arginine Auxotrophy by Nitrous Acid. Mutation Rate to Arginine Auxotrophy with Nitrous Acid. Inactivation of S. gallinarum— pullorum. by Acetic Acid Buffer pH 3. 5 Induction of Mutation with Acetic Acic. Buffer pH A. 5 iii 23 23 30 3O Page Spontaneous Arginine Auxotrophs of S. gallinarum- pullorum 53w and Spontaneous Mutation Rate to Arginine Auxotrophy . . . . . . . . . . 35 Induction of Mutation with 2- -Aminopurine and Mutation Rate to Arginine Auxotrophy with 2- -Aminopurine . . . . . . . . . 35 DISCUSSION . . . . . . . . . . . . . . . 38 SUMMARY . . . . . . . . . . . . . . . . A5 BIBLIOGRAPHY . . . . . . . . . . . . . . A6 iv TABLE II. III. IV. VI. VII. VIII. IX. LIST OF TABLES The proline, arginine, and pyrimidine bio— synthetic pathways in Neurospora . . . The proline, arginine, and pyrimidine bio- synthetic pathways in S. coli . . Plating of cells treated with nitrous acid. Plating of cells treated with acetic acid Distribution of nutritional requirements of arginine auxotrophs of S. gallinarum-pullcrum strain 53 induced with nitrous acid . . Patterns of nutritional requirements of 53W and three representative nitrous acid-induced arginine auxotrophs of S, gallinarum- pullorum . . . . . . . . . Patterns of nutritional requirements of two bradytrophs isolated from S, gallinarum- pullorum . . . . . . . . . Distribution of nutritional requirements of spontaneous arginine auxotrophs of S, gallinarum-pullorum 53W Distribution of nutritional requirements of 2-aminopurine—induced arginine mutants of S, gallinarum-pullorum 53W 1? 19 26 28 37 LIST OF FIGURES FIGURE 1. Inactivation of log phase cells of S. gallinarum-pullorum by 0.025 M HN02 (pH A.57 as a function of time . . . 2. Tailing effect of nitrous acid inactivation of S, gallinarum—pullorum . . . . 3. Relation between the percentage of induced arginine mutants and time of exposure of log phase cells to 0.025 M HNO2 . A. Inactivation of log phase cells of S. gallinarum—pullorum by pH A.5 acetic acid buffer as a function of time . . . . . 5. Comparison of the inactivation rates of S, gallinarum-pullorum in nitrous acid and in acetic acid buffer . . . . . . . vi INTRODUCTION Studies of arginine auxotrophs have been of consid- erable importance in the developm nt of three areas of genetic research: intermediary metabolism, linearity of biochemically related genes, and cell regulatory mechanisms. The biosynthetic pathway to arginine in Escherichia ggll_differs from that in fungi by the use of acetylated intermediates in ornithine synthesis. It may be of taxon— omic and evolutionary significance to determine wheth r the use of acetylated precursors is specific to the biosynthesis of arginine in S, ggli or whether this alteration is generally a characteristic of arginine biosynthesis in bacteria. One aim of the isolation and characterization of arginine auxotrophs of Salmonella gallinarum-pullorum was to provide mutants for the determination of the metabolic pathway to arginine in this organism. Chromosome maps of bacteria demonstrate a linkage of genes controlling the synthesis of enzymes in the same bio— synthetic pathway for many enzyme systems. The order of genes in a linkage map coincides with the order of irdivid— ual steps in the sequence of reactions they control. In the case of arginine synthesis a linkage of genes controlling metabolically related enzymes has been observed 1 2 only over small regions of the chromosome consisting of two or three gene sites. The trend in microorganisms appears to be a non-linked or only partially linked arrangement of genes controlling enzymes of the arginine pathway. Acceptance of a trend in genetic analysis rests upon its confirmation in other strains of bacteria. Well characterized arginine auxotrophs of S, gallinarum-pullorum may serve to determine the relation of gene linkage to the order of reactions in the pathway to arginine biosynthesis. Complete and partially blocked arginine mutants (bradytrophs) have been of primary importance in the study of parallel enzyme repression in S, £913. Arginine auxo— trophs of S, gallinarum-pullorum may be of value in similar studies. In the past attempts to isolate arginine dependent mutants of spontaneous or induced origin in S. gallinarum- pullorum have rarely succeeded. The following study was undertaken to isolcte and -g c. characterize a series of arginine auxotrophs of S, gallinarum~ pullorum suitable for further genetic analysis, using as inducing agents two mutagens not previously applied to Salmonella spp. for this purpose. HISTORICAL REVIEW Occurrence of Arginine Auxotrophs Arginine auxotrophs have been readily isolated in S, coli (Vogel, 1955; Maas, 1961; Gorini, l96l), Micrococcus glutamicus (Udaka and Kinoshita, 1958). Torulopsis utilis, and Neurospora crassa (Vogel, 1955). Only two arginine mutants of Salmonella spp. have been reported (Clarke,l962). Of these mutants one was spontaneous (Iseki and Kashiwagi, 1957; cited by Clarke,l962) and the other nitrogen mustard induced (Stokes and Bayne, 1958). Clarke (1962) has recently attempted to isolate arginine mutants of spontaneous, ultra- violet-~and Mn012_induced origin in Salmonella typhimurium. One arginine auxotroph was isolated. The rarity of arginine mutants in Salmonellae suggests a genus-wide refractoriness to the occurrence of this particular class of mutants. Arginine Biosynthesis in Microorganisms Intermediary metabolism occupies a prominent place in current biochemistry and microbial genetics. It is the aim of intermediary metabolism to discover individual steps in the synthesis of metabolites and to determine their arrange- ment in biosynthetic pathways. Two techniques are commonly employed for the study of metabolic pathways with mutants: intermediate accumulations and precursor utilization. 4 Use of these techniques had led to the observation that requirements of certain microorganisms for pyrimidines and proline as well as a requirement for arginine can be overcome by the addition of arginine to a growth medium (Davis, 1962). The pathway of arginine synthesis was pre- sumed to be intermediate in the synthesis of proline and pryimidines. The biosynthetic pathways to arginine, proline, and pyrimidines have been described in Neurospora. The path- ways were found to include the sequence of reactions given in Table I (Vogel and Kopac, 1959;8rb and Horowitz, 19AA; Srb gt_§i,, 1950;\Davis, 1962). Glutamic semialdehyde is shown twice to emphasize the probability that the semialde— hyde as proline precursor is distinct from the semialdehyde as ornithine precursor. The gultamic—proline-ornithine interrelation in Torulopsis utilis is identical to that of Neurosopora (Abelson and Vogel, 1955). The interrelated biosynthetic pathways to proline, arginine, and pyrimidines in bacteria have been described for S, 2222: The sequence of reactions given in Table II was determined (Maas, 1961). Of particular significance is the utilization of acetylated compounds in ornithine synthesis. Vogel (1955) has postulated a possible taxonomic and evolutionary significance in the difference between the pathway to arginine in fungi and bacteria. mcflaomd ofiom OHHSGHLD a ocflcflwnm opmpomo mpdahxonmmoum a. H mafiaomnha opmcfioosmocflcflwnm moMCwooSmoonms onmnooamfiEmm % mpmzdmond A, anmQSHw ocflaazmpflo ahEmnpmo .IIIW mpdpmmmm< A commoUHmHEom .H .H mmeMpDHm III¢. onEdpSHw .lIIIImV mcfinpflnno mmz + moo + mam .mmoamomzoz CH mzmznpma OHumzpcmeHn mcfiowefinhd ocm smcficflwmm «ocflaoma chua.H mqmH mamas 20 volume of saline. Approximately 102 twice—washed cells were transferred to 10 m1 basal medium. The culture was incubated with aeration at 370 until maximum turbidity developed. The cells were immediately centrifuged away from the synthetic medium, washed twice, and resuspended in an equal volume of saline. Since arginine dependent mutants are incapable of growth in basal medium not supplemented with arginine, a stock culture free of arginine dependent mutants was obtained. ’ Isolation of Spontaneous Arginine Mutants Approximately 101 cells of a stock suspension of 53w 1 free of arginine auxotrophs were inoculated into a tube of sf BHI broth. The culture was incubated with aeration until maximum turbidity developed. The culture was washe twice to remove extracellular metabolites and the final cell count was determined. This suspension was used to inoculate tubes of basal medium plus penicillin according to the penicillin screening techniques of Lederberg and Zinder (1948). Arginine mutants were isolated by the replica- plating tec.nique (Lederberg and Lederberg, 1952). Induction of Mutation with 2~Aninopurine 2 Approximately 10 cells of a stock culture of 53W free of arginine dependent mutants were inoculated into three tubes containing BHI broth plus 200 mg per ml of 2—amino-pur1ne (Hartman, 6t 1~, 1962). The tubes were incubated overnight at 37 C with aeration. Five ml samples from each culture 2l were washed twice and the cell count was determined. One- tenth ml aliquots of 10-3, 10‘4, 10‘5, dilutions were inocu- lated into basal medium plus penicillin according to the penicillin selection technique. Arginine auxotrophs were detected by replica plating. Isolation of Arginine Auxotrophs Pencillin screening (selection)technique.—-One-tenth ml portions of 10'3, lO'u, 10-5 dilutions of twice—washed treated or untreated cultures were transferred to three tubes containing 3 ml basal medium plus 250 units of penicillin per ml. The tubes were incubated at 37 C without aeration for about 2A hours. Penicillin inactivates growing cells by preventing cell wall synthesis. Wild-type cells are able to grow in unsupplemented basal medium and are destroyed by the penicillin (Davis, 1949; Lederberg (D t a ., 1945). Arginine dependent mutants which arise as a result of mutagenic action or which arise spontaneously in a culture are incapable of growth in unsupplemented basal medium and are spared from the action of penicillin. After 24 hours incubation in the presence of penicillin ten 0.1 ml aliquots were removed from each tube and spread on BHI agar plates. Plates were incubated 48 hours. Plates containing ten to one hundred colonies were replicated. Replica plating technique.-—Colonies on a BHI agar plate were imprinted onto a piece of sterile velveteen spread 22 over the surface of a wooden cylinder by pressing the BHI plate gently against the velveteen. Cells from each colony retained on the fine fibers of the fabric were transferred to a plate containing basal medium and to a plate containing basal medium supplemented with arginine by pressing these plates against the imprinted velveteen. Plates were com- pared for growth after 24 and 48 hours incubation at 37 C. Colonies which grew on basal medium with arginine but did I) not grow on unsupplemented basal medium were classified as arginine auxotrophs. All such colonies were transferred from the original BHI plate to BHI agar slants for mainten~ 1 ance prior to characterization. Characterization of Arginine Auxotrophs by Precursor Utilization Each arginine auxotroph and 53w was grown overnight in BHI broth. Cultures were washed twice and resuspended in the original volume of saline. One-tenth ml of a 10‘2 dilution of each saline suspension was inoculated into a series of 16 x 150 mm test tubes containing 2 ml of media as follows: unsupplemented basal medium, basal medium sup- plemented with proline, with ornit.ine, with citrulline, with argininosuccinic acid, or with arginine. Tubes were incubated at 37C. The amount of growth in each tube was recorded approximately every twelve hours according to turbidity estimates (+1, +2, +3, +4) for six to seven days. Imam-‘2" ‘ A .."W '..\A.. ‘_- RESULTS Inactivation of S. gallinarum-pullorum by Nitrous Acid Nitrous acid inactivation of bacterial cells was measured as a decrease in the ability of a suspension to form colonies. The inactivation of S, gallinarum-pullorum strain 53 cells in the log phase by 0.025M HN02 followed a two-hit curve (Fig. 1). Starting with about 108 cells per ml the rate of inactivation was determined over a five log decrease in viable cell count. The inactivation rate was determined to be .l26/sec. A tailing effect was observed after a six log decrease in viable cell count of S. gallinarum—pullorum in 0.025M HN02 (Fig. 2, page 25). This tailing effect may represent a selection for cells in the culture which are resistant to the action of nitrous acid. Induction of Mutations to Arginine Auxotrophy by Nitrous Acid The spectrum of arginine auxotrophs induced with nitrous acid is given in Table V on page 26. Nutritional requirements of the arginine auxotrophs isolated were tested in basal medium unsupplemented and in basal media supplemented with proline, with ornithine, with citrulline, with argininosuccinic acid, or with arginine. 23 Fraction of Surflnn |°-°¢. l I11!” 1 lll'lll I llllll I0-4 24 l l I l l Fig. 1. no no no 40 4 so 00 Tina of Exposure to "no; In Seconds Inactivation of log phase cells of S. qallinarum- pullorum by 0.025M HNO2 (pH A.5) as a function of time. ‘ * 1n.- v.1 Fraction of Survivon Io-C: loJ _ Fig. 25 9 O \ t O .. ‘ I l I l I J l l L J O 80 CO 90 I20 ISO IOO IIO Tlno of Exposuro to OJ”! "no; ("I 4.5) In Socoodo 2. Tailing effect of nitrous acid inactivation of S. gallinarum-pullorum. 26 ocflaoma o m mllllll. opmempSHm omHHomm omflmfiwmm o H ATIII: cummfiooSmocflcflwmm ocficawmg omeHoozmocHQHmmm ofiod H m AIIIIII ocfiaasmpflo oflmfioozmocficflmm< oQHHHSmpHo s am. Tl massage sadistic : : omfinpflmno Alllllll. omeMpSHm ocfizpflcmo wopmaomfi mzaompmcmmn oopmfiomfi xQOHQ oHHoQMpoz meoEomHSUom mo monEsz mndompoxsm ocflcflwmm Hmsoflpfimpzz mo honezz .Ufiom mzompflm Spas ooosozfl mm :Hmmpm Esmoadmmlasnmsflaadw am mo mnaompoxsm omficflmwm mo mucosomfisoom Hmcofipfimpzc mo compsnfippmfimau.> mumme 27 Growth was recorded every twelve hours for six to seven days. Wild type strain 53 gave a +4 reaction in all testing media after 36 hours. Mutants which demonstrated a growth dif— ferential of 48 hours or more among the testing media after an initial incubation period of 36 hours were classified as arginine auxotrophs. The genetic block of a mutant was deter- mined according to the supplement earliest in the arginine cycle which would support its growth. Examples of growth r patterns of three representative nitrous acid-induced argin— r ine auxotrophs and of 53w are presented in Table VI. s Proline was included in tests for the nutritional requirements of arginine auxotrophs to separate proline mutants whose metabolic block can be overcome in the presence of arginine from mutants deficient in arginine biosynthesis. The primary requirement of mutants able to grow on proline appears to be proline rather than arginine (Vogel and Kopac, 1959) while those unable to grow on proline exhibit genetic blocks in the arginine cycle. The exact nutritional requirements of mutants with metabolic blocks priorto ornithine were not determined since N-acetylated intermediates to ornithine synthesis are not commercially available. Mutants which demonstrated a 36 to 48 hour growth dif- ferential among the various media but which did not show growth in unsupplemented basal medium until the fifth or sixth day of incubation and mutants which consistantly mm . 1 3+ 3+ 3+ 3+ 1% I I 3+ 3.3+ 3+ 3+ ms 1 . 3+ 3+ 3+ 3+ om n I 3+ 3+ m+ 3+ w: .l I. NIT 3+ I. 3+ 1 \JII . mm - - 3+ 3+ - 3+ we ml o.Muomemu33m “xooam owpocow s33 - - - 3+ 3+ 3+ woa u n a 3+ 3+ 3+ mm - - - 3+ 3+ 3+ 3w - - - 3+ 3+ 3+ ms - - - 3+ m+ 3+ om - - - 3+ - 3+ w3 - - - 3+ - 3+ - mm 1 u n 1 a 3+ mumoz AVMTo ”rooafi capomow mm. 3+ 3+ 3+ 3+ 3+ 3+. TN l l +11 +1.. “4+ 41: NH : a : m+ u m+ mms coauMQSomH UopcoE oGHHomm ocfizuficmo oCHHHStho opmcfiooSm omficfiwmg mo mmsom IoHQQSmQD nocflqfiwmm unofioaaasm .Esmoaasmuesmwcfiaamm am mo mQQOLpoxsm omacfiwpm voodocfincfiom mSompH: o>apdpcomomdom oops» Ugo 3mm no mpqoaomfisvoh HMQOHpHmpSC mo mcpoppmmnu.H> mum<fi '29 @OH I 3+ 3+ 3+ 3+ 3+ ca - 3+ 3+ 3+ 3+ 3+ 3m - 3+ 3+ 3+ 3+ 3+ mm s 3+ 3+ 3+ 3+ 3+ m3 - 3H 3+ 3+ 1 3+ mm 1 + I 3+ : 3+ waumum pmmrsa omflfiomm uxcoan oflponmw coapmn:omfl copuoa ocfifiomm ouflnpwmmo onflflasmpflo opwmflooSm onflcfimm¢ mo mmsom :oHQQSmCD nomflcwwm3 pcofiofiaqsm UmDQHpGOOII.H> mqm mamme x :02 Log Mutant. Survivor: 32 Illln1 Io° IO-'L -- - Inn-nu I 2 8 4 5 O 7 IOIO Log Numb» of Mlnutoo Fig. 3. Relationship between the percentage of induced arginine mutants and the exposure of log phase cells to 0.025M HNOE. Em... ' .Jn ‘ 'a NU'L w.rv.t 'I Function of Suruluon 33 Io-° ' IoJ 0 O '0... l n l 1 4 I 1 4 I I 8 4 l O 7 0 Tune of Contoot um: Aooflo Acid am» In Hours. Fig. 4. Inactivation of log phase cells of S, gallinarum-pullorum by acetic acid buffer as a function of time. 34 IOJOI ‘ ‘ > I Aosflo oold buffor ’ (pH 4.5) I 0 no“ I llllll Nitrous oold fraction of Survivors o I D i" (pH 4.5) IO»8 :_- L. ‘ ”.4 I I I I I 0 IO 20 IO 40 IO 60 Thus of Esposurs In Ssoouds Fig. 5. Comparison of the inactivation rates of S. qallinarum— pullorum in nitrous acid and in acetic acid buffer. ' “"1 35 Induction of Mutation with Acetic Acid Buffer pH 4.5 In three separate inactivation trials all plates con- taining ten to one hundred colonies were replicated for detection of arginine auxotrophy. No arginine mutants were isolated. Spontaneous Arginine Auxotrophy of S. gallinarum— pullorum 53W and Spontaneous Mutation Rate to Arginine Auxotrophy {A The spectrum of spontaneous arginine auxotrophs iso- lated from 53W is given in Table VIII, page 36. The spontaneous mutation rate to arginine auxotrophs in S, gallinarum—pullorum was determined according to the a formulacxre ln2M_where ,(' is the mutation rate, M is the number of argin§ne auxotrophs per ml, and N is the number of cells produced from a small inoculum, and was found to be 4.8 x lO’L‘L mutations/cell. Induction of Mutation with 2-Aminopurine and Mutation Rate to Arginine Auxotrophs with 2-Aminopurine The distribution of 2-aminopurine induced arginine mutants isolated from S, gallinarum-pullorum is given in Table IX. The mutation rate to arginine auxotrophy in 53W with 2-aminopurine was determined by the same technique as was the spontaneous rate and was found to be 2.6 x 10'3. 36 omHHomQ o m AVI omempSHw ocHHomm oQHQmem 0 Au mlllll opmcHoodmomHusmw qumem< opmcHooSmomHmemm UHo¢ m H Al I. oQHHHSLpHo oHsHooSmochme< ocHHHsano 3 mH AI I ocanHmpo ogHHHsmpHo ocanHnmo H m3 A msmsspsHm meanswcco oopmHomH manomphommn copmHomH XoOHQ OHHonmpoz mpGoEomHSUom do moQEsz manompoxzm oGHQmem HmQOHpHmpsz mo monasz .3mm EsmoHHSQIESLmnHHHmm aw mo mnaomuoxsm oanHwhm msoochCOQm mo mucosoanvon HmQOHpHmpSQ no GOHpanmpmHQII.HHH> mHm