6. THE JANE COFFIN CHILDS MEMORTAL FUND FOR MEDICAL RESEARCH 333 Cedar Street New Haven, Conn. APPLICATION FOR GRANT-IN-AID OF RESEARCH Applicant: L. 8. BARON, Ph.D. 28 October 1960 Chief, Department of Bacterial Immmolcgy Division of Immunology Walter Reed Army Institute of Research Washington le, DB. C. "Studies on the Genetics of Sex and Virulence in Salmonelia" Total: hoo. Permanent Equi t: Binocular Tone Contrast Microscope and Photographic Attachment Photomicrographic Exposure Meter Cailloux Micromanipulator Electropho tometer Micro-Wering Blendor Cups Ultraviolet Lamps Binocular Dissecting Microscope Travel: Consultation and attendance at scientific meetings Period of Investigation: 1961 - 1%2. $1800. 120. T19. 320. 260. 106, TOO. 7. A. Specific Aims: 1) To obtain strains of Salmonella typhose and Salmonella typhimurium having easily identifiable genetic markers and which are capable of acting as genetic donors (Ft, Hfr male bacteria). 2) To study the inheritance of Salmonella genes by strains of other enteric bacteria, especially Escherichia coli. 3) To investigate possible genetic determinants of virulence in the Salmonella group. B. Plan of Procedure and Outline of Methods: Previous studies in this laboratory have established a number of genetic homologies between Escherichie and Salmonella by means of conjugation between Hfr E. coli and FP" Salmonella. The interactions between the Salmonella chromosome and the injected Escherichia genome have been analyzed by investi- gations of stable and unstable diploid heterozygote recombinants. We hope to extend these previous studies to the analysis of recombinants obtained by conjugation between F* and Bf Salmonella and F~ Escherichia and Shigella. These recombinants will be analyzed for the inheritance of biochemical, nutritional, antigenic and phage determinants. Our primary goal is to obtein information on the genetic determinants of virulence in Salmonella species. We have already reported that diploid heterozygotes of Salmonella typhoss. chtained by conjugation with Hfr Esche- richia are significantly less virulent for mice. Progress in understand ing the nature of the genetic determinants of virulence should be greatly accele- rated if it is possible to transfer virulence from Salmonella to Escherichia. fm especially useful system should be the use of Salmonelis dmurium Her and F” Escherichia. S. t urdu is a naturel pathogen o ee and one organism injected igtravertoreatly is capable of causing death. On the other hand, as many as 10” Escherichia injected intraperitoneally can not cause death of the host. Titration of challenge suspensions of various classes of Salmonella-Kscherichia hybrids in mice would be the procedure used to localize virulence characters to short segments of the chromosome. 4A Closer analysis of chromosomal virulence determinante could then be afforded by means of phage-mediated transduction. The besic methods for obteining donor (F*, Afr) strains of bacteria have already been established by a number of workers. Using these technics we have isolated several donor strains of Salmonella typhosa. While these etraing have proved useful we feel that it is desirable to build a stock of a number of Salmonella donor strains so that a variety of potentially useful genetic markers will be available. The donor strains of Salmonella that we now have at hand show several differences from the Ft and Hfr strains of Escherichia coli usually employed. These differences include orientation of transformed genetic elements » trans- missabllity, and genetic stability. We therefore intend to study Ft and Rfr 10. strains of Salmonella with respect to these characteristics by means of microscopic electrophoresis, immunological technics and studies on conju- gation kinetics. Selearies of the principal investigator, three co-workers and one technical assistant are provided for by the Walter Reed Army Institute of Research (approximate total: $35,000. Satisfactory supplies of experimental animals (mice, guinea pigs, and rabbits) and consumable supplies are available. The Department of Bacterial Inmunclogy possesses a reasonable amount of laboratory space supplied with constant temperature incubators, centrifuges, and other routine bacteriologic research equipment. None. Baron, Louis 5S. Jan. 2, 1924 B.S. City College of New York 1947 Chemistry M.S. University of Illinois 1948 Bacteriology Ph.D. University of DTllincis L951 Bacteriology Teaching Assistant, Dept. Bact., Univ. of Ill. 1949-1952 Dept. Bact. Immmol., WRAIR, 1952-1956 Chief, Dept. Bacterial Inmimology, Division of Immmology, 1956- Fellowships: E. R. Squibb & Sons Fellowship, 1951 President's Fellowship, Soc. Am. Bact., 1957 Societies: Sigma Xi, AAAS, Soc. Am. Bact., Amer. Assoc. Immm., Amer. Acad. Microbiol., Genetics Soc. Amer., Soc. Gen. Microbiol., Soc. Expti. Biol. & Med., Biophysical Soe. Positions: Member, Genetics Study Section, USPHS, National Institutes of Health, Bethesda, Md. Meniber, Subcommittee on Maintenance of Genetic Stocks (Bacteria, Viruses) N.5.F. Special Lecturer in Microbisl Genetics, Dept. of Microbiclogy, George Wash- ington Univ. School of Medicine, Wash. D.C. Prof. J. Lederberg, Dept. of Genetics, Stenford Univ. Medical School, Palo Alto, Calif. Prof. E. Englesberg, Dept. of Biological Sciences, Univ. of Pittsburgh, Pittsburgh, Pa. Prof. V. Bryson, Institute of Microbiology, Rutgers Univ., New Brunswick, N.J. i2. Baron, L. S., Formal, S. B., and Spilman, W., "Vi Phage-Host Interaction in Salmonella Typhosa". J. Bacteriol. 69:177-183, 1955. Formal, S. B., Baron, L. 5., and Spilman, W., "The Virulence and Immunogenicity vce Typhosa Grown in Continuous Culture". J. Bacteriol. 72:168-175, 1950. Kline, E. 8., and Baron, L. 8., "Xylose Metaboliam in Genetic Variants of Salmonella Typhosa". Arch. Biochem. Biophys. 66:126-139, 1957. Beron, L. 8., Formal, 8. B., and Washington, 0., "Somatic Antigen Addition in Salmonella by Bacterlophage”. Virology 3:417-425, 1957. Carey, W. ¥., Spilman, W., and Baron, L. 8., "Protoplast Formation by Mass Adsorption of Inactive Bacteriophage”. J. Bacteriol. 74:543-544, 1957. Carey, W. F., and Baron, L. S., “Comparative Immmologic Studies of Cell Structures Isolated from Salmonella Typhose". J. Inmmmol. 83:517-520, 1959. Baron, L. 5., Carey, W. P., Spilman, W. M., “Genetic Recombination between 988. 1959. Coli and Salmonella Typhimurium”. Proc. Natl. Acad. Sei. 45: 976- +, 1959. Englesberg, E., and Baron, L. S., "Mutation to Rhammose Resistance and Trens- duction to Rhamnose Utilization in Salmonella Typhosa". J. Bacterlol. 76: 675-686, 1959. Baron, L. 5., Spilman, W. M., Carey, W. F., "Hybridization of Salmonella Species by Meting with Escherichia Coli". Science, 130:566-567, 1959. Mendel, A. D., Baron, L. S., and Buckler, C. E., "Role of the Vi Antigen in Salmonelia Paratyphi C Infection in Mice". Proc. Soc. Exptl. Biol. Med. 100: 653-656, 1959. Muschel, L. H., Carey, W. F., and Baron, L. 8., "Formation of Bacterial Proto- plasts by Serum Components". J. Immunol. 82:38-42, 1960. Baron, L. 8., Carey, W. F., and Spilman, W. M., "Characteristics of a High Frequency of Recombination Strain of Salmonella Typhosa Compatible with Esche- richia, Selmonella, and Shigella Species". Proc. Natl. Acad. Sei. 45:1752-1757, 1959. Carey, W. F., Muschel, L. H., and Baron, L. 5., “Formation of Bacterial Proto- plasts In Vivo". J. Immunol. 8h 5183-188, 1960. Baron, L. S., Spilman, W. M., and Carey, W. F., “Diploid Heterozygous Hybrids from Matings between Escherichia Coli and Selmonelia Typhosa". J. Exptl. Med. 112:361-372, 1960. Brinton, C. C., and Baron, L. &., "Pransfer of Piliation from Escherichia Coli to Salmonella Typhosa by Genetic Recombination". Biochim. Biophys. Acta he: 296-311, 1960. Schneider, i, Formal, Ss. B.,; and Baron, L. 5.; "Experimental Genetic Recombi- nation In Vivo between Escherichia Coli and Salmonella Typhimurium". Bacteriol. Proc. 1960. Formal, S. B., Schneider, H., Bohner, H., and Baron, L. S., "Virulence of a Nutritional Mutant of Vibrio Comme”. Proc. Soe. Exptl. Biol. Med. 103:359-%41, 1960. Buckler, C. E., Hansen, P. A., and Baron, L. S., “Linked Transduction of L-Fucose and D-Arabinose Utilization in Sulmonelia". Bacteriol. Proc. 1960. Baron, L. S., and Formal, S. B., "Immmization Studies with Living Vaccine of Salmonella Typhimurium". Proc. Soc. Exptl. Biol. Med. 1043565-567, 1960. Carey, W. F., Spilman, W. M., and Baron, L. &., "Genetic Transfer between Saimonella Typhosa end Serratia Marcescens". Bacteriol. Proc. 1960. 13. Additional remarks: This is a request for some items of permanent equipment end for travel, both of which are important for the continued progress of these studies at the maximal rate. At the present moment, funds for these purposes are not available because of budgetary limitations. The phese contrast microscope, exposure meter, and micromenipulator are essential items of equipment for the study of the segregation patterns of diploid heterozygous hybrids. The electrophotometer, Waring blendor cups and ultraviolet iemps ere required for proposed studies on growth rates, kinetics of conjugation anc production of mutant stocks. While ea number of research groups are engaged in the study of genetic re- combination in bacterie, their efforts have not extended to the study of the genetic basis of virulence. Our adequate laboratory animal rooms as well as our previous experience in this phase of experimental vork presents us with en almost unique opportunity for pursuit of this approach; availability of the need- ed equipment will assure results sooner, and the travel funds will permit con- sultation with colleagues and the presentation of these results to the scientific comminity. 14. Application approved by Dean or other administrative officer of the institution. Signature Title 15. Signature of applicant