«l 'x I I \lhl‘WHfi ‘. \ MIN ‘ 7 _ _—‘ ——_—:——_ — — I. .1;— _ i i _. , ’— —_—"—‘—— — Z __’—— — ’— —’—— ,___'——— ‘ ’— ”4 _,—_._ I -i_.'_‘ \ Ian. CDU'IU'I RUTRiTiONAL REQUEREMENTS ANC’? TRANSDUCJEB!‘ "r“! u; E o o rm? (:1? guawpmmg; .-:-. ffifiiJiRifiM “.31 ~— I m Thesis fur ‘i‘he Jame-a cf M s {‘("31‘ ¢::5£‘2?'3 h i Allj}: L} ‘4;“J.E:;:‘}E9§ .l‘. a; a wan-.0 Ci Mme {masts LIBRARY Michigan 5, tatc University ABSTRACT NUTRITIONAL REQUIREMENTS AND TRANSDUCIBILITY OF SALMONELLA PULLORUM by Bruce Kline Forty-five strains of Salmonella pullorum were assayed for their amino acid, vitamin, and (a) purine requirements. They were found to require predominantly only cysteine and leucine for growth on synthetic minimal agar. Eleven per cent of the strains were found to require an additional supplement of niacin or thiamine. Two strains were found which could apparently utilize either thiamine or leucine to satisfy the same growth requirement. A significant number of the strains were found to have an apparent increased back mutation frequency to leucine independence in the presence of thiamine and panto- thenate. A study of potential sulfur sources showed that 83% of the strains reSponded to either sulfite, sulfide, or cysteine as a sulfur source and 13% reSponded only to either sulfide or cysteine as a sulfur source. Significantly, cysteine sulfinic acid, an organic equivalent of inorganic sulfite ion, was not utilized. A genetic attempt was made to roughly correlate the larger of the two groups to either the cysA mutant, the sulfate- thiosulfate permeaseless mutant of Salmonella typhimurium; or its nutritional counterpart, the gygB mutant. By interSpecies transduction of §. pullorum and S. typhimurium it was possible to map the larger group of S. pullorum in the region of the S. typhimurium cysA locus. NUTRITIONAL REQUIREMENTS AND TRANSDUCIBILITY OF SALMONELLA PULLORUM By Bruce C. Kline 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 1966 This thesis is dedicated to all my family, but eSpecially to Mary Ann and Bruce Jr. 11 ACKNOWLEDGEMENTS I wish to express my deepest appreciation to Dr. D. E. Schoenhard for his very warm and knowledgeable guidance through-=- out the course of this study. During this study,1 was supported in part by a departw mental research assistantship and my wife, Mary Ann. 111 RESULT So 0 O O O O O O 0 O 0 O 0 0 O O O O 0 O 0 TABLE OF CONTENTS INTRODUCTION 0 0 O O 0 O O O 0 0 0 o 0 0 O O O O O 0 LIT ERAI‘URE REVIEW 0 O O O O 0 0 O O 0 0 0 O O O O 0 0 Nutritional Requirements . . . . . . . . . . . Transduction . . . . . . . . . . . . MATERIALS AND METHODSO 0 O o 0 O 0 0 O 0 O 0 0 O 0 0 Bacterial Strains. . . . . . . . . . . . . . . Phage. o O O O 0 O O o 0 0 O O O O o O O 0 O 0 Media. 0 0 O 0 O 0 0 O 0 0 O 0 0 O 0 O O 0 o O E medium 0 0 O o O o O O 0 0 O O 0 0 O 0 Vitamin, purine and amino acid supple- mentationo o o o o o o o o o o o. o o o M‘9 brOtho o o o o o o o o o o o o o o 0 13-2 bllffer o o o o o o o o o o o o o o o Transduction-indicator agar. . . . . . . Tryptone agar. . . . . . . . . . . . . . Nutritional Requirements . . . . . . . . . . . Generalo O O O O O 0 0 o 0 O o O O 0 0 O SpeCific O o 0 o 0 o O o o O 0 O 0 O 0 0 Preparation of transducing lysates . . . . . . Transduction . . . . . . . . . . . . . . . . . O 0 Amino Acid and Vitamin Requirements. . . . . . Transdu01bility. O O o o o .0. o. 0 O 0 O I o 0 0 Growth ReSponses of S. pullo mm Strains to Potential Sulfur Sources ... . . . . . . . . Attempts to Transduce s. pullorum Strains with Phage Lysates of S. typhimurium Deletion Mutant, CI! SA’ZO o o o o o o o o o o o 0 Growth Response of Strain 5A to Thiamine or Leucine at 25 C. . . . . . . . . . . . . . . DISCUSSION 0 O O O O O O 0 O O O O O 0 O O 0 0 O O 0 SUMMARY 0 O I O 0 O O 0 O 0 O O 0 0 0 O O 0 O O 0 O BIBLIOGRAPHY O O O 0 O O O O 0 0 0 0 O O 0 0 O 0 O 0 iv 20 21 28 32 33 Table 1. LIST OF TABLES Page Growth responses of S. typhimurium cys mutants to various potential sulfur sources . . . 16 Growth at 37 C of S. pullorum strains related to amino acids, vitamins, and a purine . . . . . . . 22 Growth responses of S. pull.om strains a.nd S. typhimurium mutants to various potential SUlfursourceSoooooooooooooooooo26 Summary of results of deletion mapping of cys marker in §. pullorum 35. . . . . . . . . . . . . 27 INTRODUCTION Salmonella pullorum, the causative agent of bacillary white diarrhea in chickens, is a natural auxotrophic organism. A determination of the nutritional requirements of this organism would be of advantage in establishing a genetic syse tem to analyse the virulent state of this organism. At present the molecular basis of virulence of this pathogen is not completely understood. An endotoxin has been detected and partially characterized by Dooley gt,§l (1958). Nonetheless, it is very difficult experimentally, if not impossible,to work backward from the presence of endotoxin to a complete molecular model of virulence. However, if one could establish that a region or certain regions of the chromosome control the heredity of virulence, then one would have taken the first step toward the intended goal; i.e., a basis would be established2finrbelieving one or several celn lular constituents were responsible for virulence. Moreover, it might even fortuitously happen that the virulence deter~ minant(s) would map in the same location as a known marker or markers. To begin a genetic analysis one must first satisfy cer- tain prerequisites, i.e.,the bacteria must be genetically distinct and a competent transfer system must exist. The objects of this work are to determine what naturally occurring genetic distinctions can be inferred by examining the l 2 nutritional requirements of auxotrOphio strains of S. pullcrum and which strains are competent recipients in transductional work. The biochemical characterization of the strains, which represent samplings from 10 different states of the United States, also has a noteworthy measure of evolu- tionary and epidemiological significance. LITERATURE REVIEW Nutritional Requirements Most Species of Salmonellae grow well in a simple medium consisting of glucose9 inorganic nitroger,and mineral salts (Lederberg, 1947). However, naturally occurring auxotrophic organisms of this genus appear and may be detected on ordinary nutrient agar as dwarf colonies or slow growers (Stokes and Bayne, 1957 and 1958a). With respect to those species which are usually isola~ ted from nature as natural auxotrOphs, the nutritional requirement may be the same for all membersof the species. An example of this is the omnipresent requirement for trypto- phan in Salmonella typhosa (Fildes 2E.Ela 1933). Conversely, although thecuma are sparse, there may not be a general requirement for a particular nutrient in other naturally occurring auxotrophic species of Salmonella (Stokes and Bayne, 1958b). The basis for natural auxotrOphy is assumed by this author to be mutational in character. The precise way or ways in which a natural alteration of the genetic information encoded in DNA is altered is presently unknown. Possibly, an unstable tautomeric shift in the position of the hydrogen atoms on the purine and pyrimidine bases of the replicating DNA could cause, for example, a pairing of adenine with cytosine subsequently changing a codon's meaning (Watson ,3 4 and Crick, 1953). Further, it has been observed in some strains of Salmonella typhimurium, for example Lm-7, that natural.mutants consistently occur at a rate.greater than that in other strains of this Species. Demerec et_al (1957) pos- tulated the existence of a mutator gene to explain this phenomenon. Kirchner (1960) noted the ability of histidine requiring Lm-7 strains, which possessed the postulated mutator gene, to revert to histidine prototrophy when treated with the base analogues 5-bromodeoxyuridine or 2~amin0purine in 95% of the mutants tested. Apparently, the mutator gene facilitates an event leading to a transition. The fact that a Species, such as s. t hosa, is so stable in its requirement for trypto- phan indicates that a naturally occurring deletion-type muta- tional event may have occurred. Thus, a.third possible way of incurring a natural mutation may exist. Salmonella gallinarum, and its variant in the older literature, g. ullorum, are natural auxotrOphs. Today, both species are classified as §. gallinarum in the seventh edition of Bergey's Manual of Determinative Bacteriology. However, in the literature to be cited in this discussion they are treated as separate species. Further, the 45.strains used in this investigation were originally classified as s. pullorum on the basis of the distinguishing biochemical reactions reported in the older literature and the gross pathology of the host in response to infection. Historically, difficulties were encountered in classifi- cation because cf the weak biochemical differences between the two organism. For this reason, and because of the 5 significant economic losses incurred with infection by the more virulent strains of g: pullorum, and because of the basic scientific interest in the "in vitro” phenomenon of slow growth exhibited by both of these organisms, early investiga- tional efforts were directed to a study of nutritional requirements. The results, while showing definite patterns, were more or less easily interpretable depending on which one of the three aforementioned problems was considered. Let us consider the first problem mentioned, classifica- tion, using nutritional requirements to distinguish between §. gallinarum and g. pullorum. In a total of 33 strains of §. pullorum tested by several workers, polyauxotrOphy for some combination of cysteine, leucine, proline, and aspar- tic acid is the rule; whereas an infrequent requirement is found for the B vitamhnsw-thiamine, niacin, pantothenate, and riboflavin; and no requirement for other vitamins or nucleic acid precursors is found (Johnson and Rettger, 1943; Lederberg, l9h7; Kuwahara 32 El, 1958; Stokes and Bayne, 1958b). In contrast, in a total of 46 strains of g. ‘ gallinarum tested, there appeared an infrequent require- ment for amino acids but a very frequent requirement for thiamine (Johnson and Bettger, l9h3; Lederberg, l9h7; and Stokes and Bayne, 1958b). In contrast to this last state- ment, of 11 additional strains of g. gallinarum tested for a thiamine requirement by Kuwahara 83.2; (1958) only one strain required thiamine. Also, Stokes and Bayne (1958b) found in the 12 strains of §. gallinarwn which they determined as only requiring thiamine that a supplementary mixture of 6 cysteine, leucine, and aSpartic acid markedly stimulated growth in 10 of the strains. Moreover, Schoenhard and Stafseth (1953), as well as Gilfillian gt 31(1955), have shown that a mixture of vitamins stimulates the growth rate of a variety of g. pullorum strains. Thus.there seems to be an apparent overlap in the nutritional requirements of these species. To what extent this overlap is real or simply due to the test procedure is difficult to assess. In view of the facts, however, that s. pullorum does grow very slowly on synthetic media, it does mutate quite frequently in at least the leucine locus (D. E. Schoenhard, personal communica- tion), and that Lederberg (1950) has reported the presence of detectable amounts of sulfide ions in amino acid prepara- tions, it seemed very desirable to find an assay procedure which would take these facts into account. This desire was intensified since it was suSpected that many of the strains to be tested in this work would be cysteine or leucine auxotrOphs. The addition of Just the suSpected requirements, utilizing the soft agar auxanographic method of Beijerinck as reported by Lederberg (1950), admirably resolved the complications of interpretation in the classical ommission technique performed in synthetic broth. The results show that all #5 §. pullorum strains tested required at least cysteine and leucine; the leucine require- ment has a high reversion frequency with both large and small colonial forms arising within the same strain; and that 7 thiamine and pantothenate may affect the requirement for leucine in some,tnn:not all,s¢rains. Having once established the nutritional requirements of the strains under considera- tion, the next step is to determine which of the strains are amenable to a transductional type of analysis. Transduction Transduction, the bacterial virus mediated transfer of bacterial genetic material from a donor organism to a recipient organisms, was first observed between auxotrophic mutants of s. typhimurium (Zinder and Lederberg, 1952). It was subsequently shown that 3 types of transduction are possible, vis., generalized, specialized and abortive transduction. In generalized transduction any marker may be transferred from the donor to the recipient cell (Zinder and Lederberg, 1952). In contrast, in specialized transduction only a given region of the donor chromosome is transferred (Morseg l954). This region is contiguous to the prophage region on the host chromosome. In abortive transduction any marker may be transferred, but the donor marker is not inte» grated into the genome of the reel ient. It is transferred unilinearly within a clone (Ozeki9 1956, 1959). A successful generalized transduction process is a function of several variables. Given that a particular recipient is permissive, it is a preliminary necessity that the recipient contains the prcper surface receptor for donor virus attachment. g. tythimurium and g. Hgllgggm share the common 0 antigen,IU2. This antigen has been shown to be the phage receptor site of g. typhimurium for phage PDTZE. 8 Fortuitously, §. pullorum phage P38 will transduce s. typhimurium recipient cells (Snyder and Schoenhard, 1960). This observation was employed as the basis for using a proto- trophic §. typhimurium strain, LTZ, as the donor in establish- ing the transducibility of §. pullorum auxotrOphs. The results of such transductions show that most §o pullorum are transducible to cysteine prototrOphy by §. pullorum phage P38 grown on wild type g. typhimurium. MATERIALS AND METHODS Bacterial Strains *4 S SD S. pullorum: 45 strains, isted in table 2, were employed. They are from the collection of D. E. Schoenhard. They were originally isolated from a variety of sources in 10 different states of this country. S. typhimurium: strain LTmZ. a prototrcph; strain DT-Z mutants: gygA-ZO. a cryptic mutant for sulfate and thic- sulfate ions. which is deleted in all of the cistrons-a, b, and c9 of the A locus (Dreyfuss, 1963); gy§J~5389 a deleu tion mutant of the cysteine pathway which cannot reduce sulfite to sulfide,were used. gygAm2O and gygfim538 are separated in their chromoscmal location by approximately eleven minutes in experiments which determine time of entry during canugation (Sanderson and Demerec9 1965). The involve» ment of the gng mutant in cysteine synthesis may be ascertained from the schematic pathway shown on page 15. These mutants are originally from the collection of M. Demerec and were kindly supplied by D. E. Schoenhard. Phage Phage P38, a generalized transducing phage of S. ullorum, was employed in this study. It has been described by Vaughan (1962). S. typhimurium phage P22 and Zinder°s nonlysogenizing variant, Emu, were also employed in this study. They have been described previously by Zinder and ' 9 lO Lederberg (1952), Zinder (1958), and Adye (1962). Media The basal medium employed in this study was the minimal E medium of Vogel and Bonner (1956). Mg804°7H20 0.2 g Citric acid°H20 2.0 g KZHPOu (anhydrous) 10.0 g NaHNHuPOquHEO 3.5 g Glucose 5.0 g Distilled water 1000 ml Minimal E salts at single strength (ElX) is equivalent to the minimal E medium minus the carbohydrate. inimal E agar (E) was made by mixing a double strength, sterile, E salts solution at 45 C with an equal volume of sterilized, double strength (3% w/v) agar. The agar was supplemented with 0.5 g Na citrate before autoclaving. An aliquot of autoclaved 40%;glucose solution ( w/v) was added aseptically to make the final concentration of glucose 0.5% (w/V). E soft agar was prepared the same as E agar, except the final concentration of agar was 0.7% (w/v). Supplementation of E agar with amino acids was at a final concentration of 20 ug/ml of each amino acid. All organic supplements, except glucose, were sterilized by filtration through a millipore membrane, pore size, 0.u5 u. The final concentration of vitamins added wass Thiamine°HC1 ling/ml Niacin 1 pg/ml Ca pantothenate 2 pg/ml Xanthine was also added to a final concentration of 5 pg/ml. To indicate what supplementation was accomplished. the following abbreviations and their significance are employed in this communicatione Ec, El, and Ecl indicate E agar with cysteine.9 leucine, and cysteine and leucine respectively. 8 agar is E agar supplemented with thiamine°HCl, niacin, calcium pantothenate9 and Kanthine to the final concentran tions previouSly mentioned. The interpretation of the symm bols Sc, 31, and Sol is analogous to Be. etc. Mn9 broth was employed in phage propagation. It was prepared according to Vaughn (1962). 2X M~9 Salts ~~ Each salt is added singly in the order listed until dissolved. KHZPOQ 6.0 g NaZHPop 12.0 g NHLPCI 200 8 Distilled water 1000 ml Casamino acids (C.A.) an 2X Difco C.A. (lot 0230m0l)30.0 g Distilled water 1000 ml Norite A charcoal 2.0 teaSpoons (activated) This solution was mixed thoroughly and stored overnight at H C; filtered; and stored over chloroform until used. 12 M~9 Broth 2X M39 salts 2X Casamino acids 1 M MgSOuv7HZO 25% NaCl 20% Glucose The T2 buffer of Hershey a (I) a KHZPO4 NaCl K280“ MgSOu (001 M) CaCl2 (0001 M) Gelatin (1%) istilled water Final pH, about 700 H. indicated, was prepared as follows: 500,0 50000 205 200 500 1d Chase (19 rs 100 105 b.0 5o0 10 10 H \O ‘Q \0 ml ml ml ml ml 52), employed Autoclave at 10 lbso pressure for ten minuteso The indicator agars used for the detection of cysteine positive transductants were 31 or El agar supplemented with 1°25% (v/V) reconstituted Difco nutrient brotho The Si enriched agar (Slem) was used for §o Egllprum recipientso Slem or E enriched agar (Elem) was used for go typhlmuriqm_ recipients. According to Demerec and Hartman U956)9 enriohm ment with nutrient broth increases ‘ _,. ,, $.33 the efficiency of transn 13 The complex media employed in this work were nutrient agar (NA), penassay agar (PA)~‘both purchased from the Difoo Co.9 Detroitfi Michiganwwand try tone agaro The trypm tone agar was prepared as follows: Tryptone9 Difco 10.0 g NaCl 800 g Glucose 800 g CaC12 (1N) 0.1 ml Agar hard: 1000 g SOft: 700 g Distilled water l000 ml AdJust pH to 700 and autoslave at 121 C for 15 minutesg Mutritional Requirements A0 General The euxanographie method of Beijerniek9 as described by Lederberg (1950)9 was employed in determining the nutrim tional requirements of the #5 strains of §. pullorum. Prior work by Robinson and Schoenhard (unpublished results) an a few of these strains had indicated a frequent require» ment for eyeteine and leucineo Earlier work by Sohoenhard and Stafseth (1953) and Gilfillian 23 gl (1955) had also indicated that a mixture ofvitaminsenfi.xanthine was necessary for a rapid growth response in some of these same strains. Using these findings9 the following test procedure was devised to pinpoint the requirements of the strains for cysteine, leueine9 thiamine°HCl, niacin9 Ca pantothenate a, and xanthineo The bacterial str lo (1“ m s. .o be tested was grown 94 heurs in 10 ml of penassay broth supplemented with 14 cysteine and leucine, 20 pg/ml of each. The cells were centrifuged; washed 2 times in equal volumes of ElX salt) solution;and,finally.resuspended in an equal volume of minimal E broth. Next the minimal E broth suspension was incubated for 3 hours at 37 C in a water bath to starve the cells. To test the organisms, 0.3 ml of the starved cells were added to 3.0 ml of E soft agar (final concentration about 1.06 to 108/ml) in each of 8 test tubes. Then duplicate agar plates of E, Ec, El. and Eel were overlayed with the soft agarwbacteria mixture. Each plate of a set, consisting of E, E3, El, and Ecl.vun3then Spotted with 4 disks, each disk containing 1 of the following 4 compounds in the amounts indicated: thiamine°HCl, 0.0025 pg; Ca pantothenate9 0.005 pg; niacin, 0.0125 pg, and xanthine, 0.0125 pg. The remaining set of plates was used to test the various combinations of cysteine and leucine. Although most positive results appeared within 15 to 24 hours at 37 C on the plates with adequate supplementation. incubation was prolonged to observe for back mutants or delayed growth responses. If no growth or poor growth resulted on the most highly supplemented plate, additional crystal tests with amino acids and/or vitamins were performed to determine the exact requirements. All tests were performed at least twice. B. Specificm~Cysteine Pathway The nutritional classification of cysteine requiring mutants of s. typhimurium was originally performed by Clowes (1958). Since then it has been confirmed and extended by Dreyfuss and Monty (1963). The working assumption is made 15 that cysteine Synthesis in §. typhimurium and §. pullorum is the same. To substantiate a nutritional finding in g. ‘ullorum, deletion mapping utilizing phage propagated on known deletion mutants of S. typhimurium was performed. Cysteine is synthesized from sulfide (reduced enzymati- cally from sulfate) and serine by the action of the enzyme serine transulfhydrase (Schlossman and Lynen, 1957). The pathway of sulfate reduction in S. typhimurium has been determined by Dreyfuss and Monty (1963) and is as follows: Ba, Bb, Ba, Bb, BC, = D C Bo, H = G, I, J = E 804 ----9 AFB ----9 PAPS --------- 9 803 --------- ) S --§ CYS- Ba, Bb, Bo \// 8203 (APS = adenosine-5'phOSphosulfate; PAPS = 3'-phOSphoadenine- 5'phosphosulfate.) D, C, H, J, I, B, and E represent the corresponding mutant cysteine genes controlling the indicated step. Dreyfuss and Monty (1963) established the relationships between the sulfur sources utilized and the mutant gene as indicated in Table 1. Therefore, to determine the nature of the cysteine deficiency, soft agar auxanography was performed with cysteine sulfinic acid (CSA), sulfite, and thiosulfate, 50 ug each' per disk. Special sulfur-free E medium was used in which 0.2 g of MgClZ-SHZO was substituted for the MgSOu and Nobel (Difco) agar.was substituted for regular agar. Sulfide utilization was tested in screw cap test tubes, utilizing sulfur free El broth and a filter-sterilized solution of Nags, final concentration of 8:: about 4 x 10.“ M. 16 T.E1e 1. Growth responses of S. typhimurium #agsmutants to various potential sulfur sources Sulfur Mutant Classification DLJU.I‘C e C,D,H J.I,G Ba, Bo Bb Ea,Eb Aa,Ab,Ao SO4 - m _ a - a CSA or so; + n Slow - a + S ...L a? m m m u 203 + S = r + + + "e + Cysteine + +, + +. + + 21% Drevfuss and Monty, 1963. Preparation of Transduclng Lysates The phage were cloned three times on donor cellss. using the tryptone soft agar technique of Adam's (1959), prior to their usage in the production of transducing phage. Onemtenth ml of an overnight M~9 broth culture of bacteria was transferred to 10 ml of fresh Mw9 broth. The culture was aerated for three hours at 3? C. Phage partichmswere added to a final concentration of about 106/ml. The culture was then incubated at 37 C until cellular debris accumulating on the walls of the tube indicated extensive lysis had occurred. One ml of chloroform was then added and the incuba-= tion with aeration was continued for 3 to 5 minutes. Next the culture was centrifuged at 6,”)00xg for 10 minutes to remove the cellular debris. The phage in the supernatant were assayed on the homologous cells. Phage stocks were 1? stored over chloroform at 4 C. Transduction Ten ml of an aerated, overnight PA culture were eentrim {aged at 6Jw03(g for 10 minutes and resuspended in 1 ml of T2 buffer. All transducing phage lysates were diluted to 5 X 109/m1 and irradiated while shaken, with a UV light for 50 seconds at 4J.cm. The lamp was a G. E. Germicidal, G3OT8. Approximately 99.99% of the phage were not able to form plaques after the irradiation. The transduction was performed by adding 0.1 ml of the cell suspension to 0.9 ml of pre- warmed (37 C), irradiated phage (m.o.i. m 5). The mixture was incubated at 37 C for 10 minutes before plating 0.2 ml aliquots in triplicate on the indicator agar. Five times the number of cells per transductional plate were plated on control plates of the same agar to determine back mutation frequencies. Two~tenths ml of phage lysate without recipient cellsvmme also plated on the indicator agar as a control of the sterility of the phage lysate. Furthermore, lysates were teeted qualitatively for transductional potency before use9 and recipient cells were tested concurrently for trans- ducibility by phage lysates of "wild type" g. typhimurium or,if applicable, by nonallelic deletion mutants. Incubation was for 72 hr at 37 C. Representative transductants were identified on the basis of colonial morphology and somatic antigens D or B RESULTS The overall motivation of the work, to be reported in this section, is the deveIOpment of a deeper understanding of the biochemical and genetic nature of §. pullorumo Data of this nature are being gathered in conjunction with the results of other studies so that a systematic investigation of virulence may be conducted at the molecular levelo This particular work falls into 3 main categories: nutritional studies9 genetic studies, and preliminary molecular studies of one of the nutritional requirements, the cysteine deficiencyo A brief comment on the significance of the incubation eriod employed in the first of these studies is in ordero Lederberg (1950) reported that Escherichia ooli showed posim tive growth reSponses to supplementation in 6 to 10 hrs if the soft agar overlay technique was usedo A longer period of incubation has been found necessary with s, pullorum to observe positive resultso This observation is not at all surprising in View of the smaller size and slower growth rate of the lattero In most of the strains used in these tests9 positive results occurred in 15 to 2A hrso A strong response at #8 hrs was also taken as evidence of completed supplementaw tiono A weakly positive response after 48 hrs was taken as evidence of an additional leaky requirement. The responses of the go pullorum strains to the various combinations of 18 19 amino acids and vitamins are listed in Table 20 Zinder and Lederberg (1952) and Zinder (1953) reported that transduction, not transformation, occurred with s. typhimurium phage lysateso Likewise, Snyder and Schoenhard (1960) reported transduction with go pullcrum phage lysateso Furthermore, Schoenhard (1963) reported on the transduction of §o pullorum by phage lysates of go typhimurium and XLEE Kggsao Thusgit is the object of the second of these studies not to show the means of genetic transfer, but the fact of genetic transfero The results of the crosses between phage lysates prepared on prototrophic §o typhimurium and the various §o pullorum recipient strains showed that all the strains listed in Table 2, except strain ll,were transducible to cysteine prototrophyo Strains 13, 16, 26, 50, and 54 were indeterminable due to high reversion frequencies on the indicator agaro The highest transducticnal frequency observed was 505 x 10w8, representing approximately 50 colonies per plate. In View of the frequency of both the cysteine and leum cine requirements of these strains, it is of fundamental interest to ask if these mutants are all deficient in a common step in their respective synthetic pathways or in different stepso Thusgthe object of the third part of the study is definedo Preliminary characterization of a gyg mutant was accomu plished in g. typhimurium using the crystal tests described by Dreyfuss and Monty (1963)o The pattern of responses they found are listed in Table 10 The same tests were applied to 20 the S. pullorum strains. The primary result (Table 3) was that 84% of the 45 strains tested gave a qualitative reSponse indicative of either a gysA or a gysBa,c mutant. However, in these tests the growth reSponses of the strains to CSA were very slow and very weak, but the responses to sulfite, while not as strong as the responses to cysteine in comparable tests, were rapid. With S. typhimurium, cysA mutants can be distinguished from gyg Ba,c mutants by the generation time. Dreyfuss and Monty (1963) reported that gygA mutants with CSA as a sul- ful source, have a generation time equivalent to that of the gygf genotype; whereas gygBa,c mutants with CSA as the sulfur source have a generation time approximately 3 times longer. Because of the instability and toxicity of sulfite, and the extremely weak response of S. pullorum to GSA, this method of resolution is eliminated. However, a gygA deletion mutant of S. typhimurium was available to use for deletion mapping. All the strains that were transducible with phage + propagated on cys g. typhimurium, were tested for trans- ducibility with phage lysates from strain gygAmZO. Only strain 14 was found to be transducible. Strain 14 was found to be a gyng mutant by the crystal tests, and thus this transduction result was expected. Strains 33, 43, and 45 also were found to be qygbb by the crystal tests, but they were not transducible in any of several attempts. These latter results rendered all the negative results of the S. pullorum X gyg ~20 transductions suSpect. Therefore, a very carefully controlled transductional analysis of one of 21 the strains, 35, was performed to ascertain if the negative results with gygA-ZO donor in this strain were due to the nature of the transductional manipulations or materials. The results shown in Table 4 indicate that the negative results are not due to non-transducing phage lysates, non- transducible recipients, or the conditions employed. The data recorded in Table 2 indicate that both strains 50 and 54 can be grown on Ec medium plus thiamine or leucine at 37 C. It was accidentally discovered that strain 54 grows at room temperature on E medium plus thiamine. Tests at 25 C of strain 54 in minimal broth resulted in Just visible growth (l~5 x 107 cells/ml) in 15 days; whereas a similar growth response using the same quantity'Ckz v/v) of inoculum in minimal broth plus thiamineoHCl (0.05 ug/ml) required only 6 days. In another type experiment 5-10 x 107 cellj; were spread on minimal agar and minimal agar plus thiamine to observe the nature of the responses and the frequency of back mutations. A heavy, confluent lawn of growth on mini- mal agar plus thiamine was observed in 10 days; whereas approximately 500-1000 distinct colonial shapes against a light background of growth were observed after 16 days incuba« tion of the minimal agar plates. In both of the afore- mentioned experiments the culture used for the inoculum was washed 3X with EIX salts solution prior to usage. 22 3 + ma 3 a» + an :H 3 + an ma 3 + NH 3 + HH 3 A» + 0H can u was Moms + m can 0 3 mm + m s 9p + i 3 mp + m abnoaasm am SQH.mmo sou who I doq.mao Sou who I. no nawnpm osanpsmx cam oprmSpOpzwm .naomdn .oQHscasp Spas haobapoommoa umpwcwmnmaa mxmdc : . mzampnoo madam 2060 modem osdsw suds mafia .H pom mm 08mm copsmSOHQQSm endows m HH pom H pom msHasm a cum msasmpab .mpaom onasw on sopmaon mnamnpm ashoaasm .m go 0 am pm spzonc .m magma 23 3 + an on 3 a» + Bo, mm 3 + mm 3 gp + an mm 3 mp + an 89 mm a as + an mm 3 + an :N szmas + an mm 3 + mm 3 + am 3 mp + om 3 Qp + Bo, ma 3 + ma 3 + S” .mafl an H ED ma doqfimo dog who I soufiho dog who I a .W mo magnum. 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I) Ga waawa wHo no wsflmmwa noapwawo no wpaswon mo hawaadm .d canoe DISCUSSION The results of the nutritional studies at 37 C show that of the 45 strains of S. pullorum tested all require at least cysteine and leucine. Moreover, in addition to these amino acids, 1 strain requires proline. Niacin and thiamine stimm ulate growth resulting in a larger pOpulation. This stimulaa tion is true for 4 strains and 1 strain respectively. Furthermore, in 1 strain, which neither requires nor is stimulated by thiamine, thiamine replaces leucine. In the strain stimulated by thiamine, thiamine also replaces leucine, but growth is heaviest when thiamine and leucine are combined. It also appears that thiamine and pantothenate stimulate to varying degrees the apparent 123 mm»? 133+ reversion in a large number of the strains. The majority of the strains respond to various inorganic sulfur ions corresponding to the gygA or gygBa,c type of mutants defined in s. typhimurium. Genetic evidence indicates in one of these strains that it is the qygA.mutation which is present. Recombination at the gyg locus of 40 of 41 strains of S. pullorum occurs, since they were transduced to cysteine independence by lysates of prototrOphic S. typhimurium, DTu2. The findings of a general nutritional requirement for cysteine and leucine are in general agreement with those of Lederberg (1947) and Stokes and Bayne (1958b). Johnson and Rettger (1943) did not include information on the cysteine requirement in 10 strains of s. pullorum, which they assayed. 28 29 Five of these strains required leucine. They did find, how- ever, that in 2 of 45 strains tested for vitamin requirementS, 2 strains required niacin. A comparison of the foregoing studies and my study indicate the amino acid and vitamin requirements are substantially similar. With respect to the transducibility of S. pullorum by phage lysates of S. typhimurium, the observation of Schoenhard (1963) has been confirmed and extended to include 38 more strains of S, pullorum. No studies have been reported about the utilization of various sulfur ions in cysteine requiring S, pullorum strains. The findings of this work show that the mutants fall into primarily 2 groups, those that utilize either sulfide or cysteine as a sulfur source, and those that utilize sulfite, sulfide, or cysteine as a sulfur source. In Neurospora (Horowitz, 1955) and S, typhimurium (Clowes, 1958) CSA has been found to be nutritionally equivalent to sulfite. Lineweaver and Monty (1961) have demonstrated with cell free preparations of S. typhimurium that CSA is desulfinated to sulfite. Therefore, one could readily imagine 3 possible situations whereby CSA is weakly or not at all utilized: 1) cells are impermeable to CSA, 2) cells lack or have an altered enzyme responsible for desulfination of CSA to yield sulfite, ammonia, and pyruvate, or 3) there is a branched pathway in which CSA is a precursor to cysteine, but it is genetically blocked. Since at the present moment the number of steps involved in sulfate reduction by S. pullorum and the number of genes controlling each step are unknown, it 30 is impossible to choose which one, if any, of the above 3 situations is correct. The genetic data gathered to resolve whether or not the S. pullorum deficiency is analogous to the gySA or gygBa,c, lesion, as defined in S: typhimurium,is at best weak. This follows from the following reasons: 1) the reciprocal crosses of S. pullorum donor and S. typhimurium recipients always gave negative results, even though the phage lysate was known to be a good transducing lysate, as Judged by its capacity to transduce other markers from S. pullorum to S. typhimurium. (Schoenhard, 1963), and 2) whereas the cysteine loci in S. typhimurium are in 5 different regions on the chromosome (Mizobuchi E; El, 1962), this situation may not prevail in S. pullorum. Thus, interspecies transductions in either direction may be hindered by nonhomology (Zinder, 1960) if the cotransduction of nonallelic outside markers occurs. To visu- alize this last statement more clearly, imagine that integra- tion may proceed by way of breakage and reunion(Fbx and Allen, 1964) according to a model analogous to the triple stranded DNA- messenger RNA complex suggested by Zubay (1962), and discussed recently by Schaeffer (1964). Thus, only stretches of donor qygf determining DNA, unlinked to nonhomologous, nonallelic DNA, are potentially capable of integration. This hypotheti- cal explanation only accounts for a reduced frequency of trans- duction. It still does not explain the unidirectional trans- duction observed in S. pullorum x S; typhimurium cst-538 crosses. To do this, it is necessary to define speculatively more a priori states of the donor and recipient genomes. 31 This is really not warranted by the data. The finding that thiamine substitutes for leucine in fulfilling one of the nutritional requirements of both strains 50 and 54 is an unexpected observation. Also related to this observation is the apparent increased frequency of 132 --> leaf back mutations in the presence of thiamine and pantothenate. These observations may not be the first of this type. Stokes and Bayne (1958b) observed that 10 S. gallinargm strains, which took seven days for appreciable growth in minimal broth supplemented with thiamine, produced full growth in 1 day if the supplementation was casein hydroly- sate or a mixture of cysteine, leucine and aspartic acid. These findings were not further pursued in any depth. SUMMARY The predominant nutritional requirements of the 45 strains of S. pullorum tested in this study were found to be cysteine and leucine. Five of the 45 strains have an addi~ tional leaky requirement for a vitamin, 4 requiring niacin and 1 requiring thiamine. Nutritional studies of the cysteine requirement indicated that there are primarily 2 groups of organisms. The largest group, comprising approximately 80% of the strains, grew on either sulfite, or sulfide, or cysteine as a sulfur source. The remaining group, comprising 13% of the strains, grew on either sulfide or cysteine as a sulfur source. InterSpecies transductional tests lent support to the inference that one strain, 35, which belongs to the larger of the above 2 groups, corresponds to the gySA type mutant of S, typhimurium. This mutant is impermeable to sulfate and thio~ sulfate ions. Other interspecies transductional tests indi_ cate that 40 of 41 strains of S. pullorum are transducible by lysates of ”wild type" S. typhimurium. The leucine deficiency was found to have an undetermined association with the vitamins thiamine and pantothenate as evidenced by an apparently increased back mutation rate to leucine prototrOphy. 32 ' BIBLIOGRAPHY Adams, M. H. 1959. Assay of phage by agar layer method, pp. 1450-451. Lg M. H. Adams, Bacteriophages. Intersciences Publishers, Inc., New York. Adye, J. 1962. Effects of nitrous acid on transduction by Salmonella phage P22. Virology l§:627-632. Clowes, B. C. 1958. Nutritional studies on cysteineless mutants of Salmonella typhimurium. J. Gen. Microbiol., 1§2140-153. 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A theory on the mechanism of messenger RNA synthesis. Proc. Natl. Acad. Sci. U. 8. 4g: 456-471. I ill I! II III I I I I l l I ’1' l l II III ' l 1' l l Ill II. 1293 03144 9824 3 lHIHill!”MINIMUM“ J l