Abstract

This project is directed at the genetic characterization of flagellar phase variation in Salmonella and the regulation of this process. Phase variation in Salmonella is the alternative expression of flagellar antigens H1 and H2. A detailed characterization of the Hin recombinase DNA binding domain will be carried out. The amino acids in Hin which make sequence-specific contacts to the hix DNA recombination sites will be determined. Altered substrate specificity mutants in Hin will be isolated in the DNA major groove recognition region of Hin. In vitro DNA binding studies will be done using purified Hin and synthetic DNA oligonucleotides which contain the base analog 3-deazaadenine or methylphosphonate derivatives. These experiments will determine whether the minor groove binding region of Hin is recognizing the phosphate backbone of the DNA in the minor groove or is making sequence-specific contacts to the bases in the minor groove. The biology of flagellar phase variation will be characterized. Regulation of hin expression will be characterized in detail. Fusions of the lac operon to genes whose expression is under the control of the phase variation regulatory network will be isolated and used to isolate linked and unlinked mutations which either inhibit Hin-mediated recombination or affect the regulation of this process. These mutants will be characterized to determine the cell functions other than Hin which are involved in Hin-mediated site-specific inversion of the Salmonella chromosome. S. typhimurium fis mutants will be isolated and characterized. Its effect on site-specific recombination and transcriptional regulation of the flagellin genes will be determined. The Salmonella genome will be surveyed for operons whose transcriptional regulation is strongly influenced by the presence or absence of fis mutations. This work will result in a better understanding of how DNA structural alterations influence gene expression; how proteins recognize specific DNA sequences in the minor groove of the DNA.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM043149-04
Application #
3302113
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1990-07-01
Project End
1995-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
4
Fiscal Year
1993
Total Cost
Indirect Cost

  Institution

Name
University of Washington
Department
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195

  Publications

Chadsey, M S; Hughes, K T (2001) A multipartite interaction between Salmonella transcription factor sigma28 and its anti-sigma factor FlgM: implications for sigma28 holoenzyme destabilization through stepwise binding. J Mol Biol 306:915-29
Karlinsey, J E; Lonner, J; Brown, K L et al. (2000) Translation/secretion coupling by type III secretion systems. Cell 102:487-97
Chadsey, M S; Karlinsey, J E; Hughes, K T (1998) The flagellar anti-sigma factor FlgM actively dissociates Salmonella typhimurium sigma28 RNA polymerase holoenzyme. Genes Dev 12:3123-36
Nanassy, O Z; Hughes, K T (1998) In vivo identification of intermediate stages of the DNA inversion reaction catalyzed by the Salmonella Hin recombinase. Genetics 149:1649-63
Chilcott, G S; Hughes, K T (1998) The type III secretion determinants of the flagellar anti-transcription factor, FlgM, extend from the amino-terminus into the anti-sigma28 domain. Mol Microbiol 30:1029-40
Adams, C W; Nanassy, O; Johnson, R C et al. (1997) Role of arginine-43 and arginine-69 of the Hin recombinase catalytic domain in the binding of Hin to the hix DNA recombination sites. Mol Microbiol 24:1235-47
Karlinsey, J E; Pease, A J; Winkler, M E et al. (1997) The flk gene of Salmonella typhimurium couples flagellar P- and L-ring assembly to flagellar morphogenesis. J Bacteriol 179:2389-400
Gillen, K L; Hughes, K T (1993) Transcription from two promoters and autoregulation contribute to the control of expression of the Salmonella typhimurium flagellar regulatory gene flgM. J Bacteriol 175:7006-15
Hughes, K T; Gillen, K L; Semon, M J et al. (1993) Sensing structural intermediates in bacterial flagellar assembly by export of a negative regulator. Science 262:1277-80
Hughes, K T; Gaines, P C; Karlinsey, J E et al. (1992) Sequence-specific interaction of the Salmonella Hin recombinase in both major and minor grooves of DNA. EMBO J 11:2695-705

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