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.
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