Type 1 pili expressed by Escherichia coli and other enteric bacteria are responsible for mediating mannose-specific adhesion to eucaryotic cells. These organelles have a critical role in the ability of the bacteria to colonize a human or animal host. type 1 pili expression is subject to reversible ON or OFF phase variation. this variation is executed by the site-specific inversion of a 314 base pair DNA segment containing the promoter for pilA, the gene encoding the type 1 pilin monomer. The pilA promoter-containing DNA segment inversion rate is normally 1 X 10- 4/cell/generation. This inversion rate is increased over 100-fold in E. coli mutants lacking the histone-like protein H-NS. In other words the H- NS protein effectively inhibits this site-specific DNA inversion event. Experiments using both biochemical and genetic approaches are described in this proposal to establish the function of H-NS in pilA promoter inversion. Either H-NS regulates the expression of gene products involved in promoting pilA inversion or this protein affects inversion due to direct interaction with the invertible DNA substrate. H-NS binding to the invertible DNA substrate will be assessed. the transcription level of genes encoding proteins that mediate pilA promoter inversion will be compared in strains that possess and lack H-NS. Two second-site mutations have been identified that suppress the rapid promoter inversion observed in mutants lacking H- NS. The location and nature of these lesions will be analyzed to determine what other gene products or DNA sequences interact with H-NS in affecting DNA inversion. Also, essentially random mutations will be introduced into the cloned gene encoding H-NS. Resulting mutants will be analyzed to determine the role of potential functional domains in H-NS activity. In short, three independent lines of investigating H-NS activity in pilA promoter inversion are proposed; a) determining the specific role of this protein in DNA inversion, b) investigating the interaction of H-NS with other gene products or DNA sequences that affect inversion, and c) analyzing domains possessed by this histone-like protein that function in the inversion process.
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