We propose to investigate the genetic regulation and pathobiologic significance of fimbrial phase variation in Escherichia coli. All strains of E. coli examined to date, including laboratory and clinical isolates, have preserved the ability to express type 1 fimbriae, which are adhesive organelles that promote bacterial adherence to mucosal surfaces. These organelles are probably important in permitting E. coli to colonize and infect humans, particularly in the genitourinary and gastrointestinal tracts. But type 1 fimbriae also mediate bacterial binding to human phagocytic cells, which also contain the mannose receptors specific for type 1 fimbriae. Thus, at invasive phases of infection, fimbriae might actually be suicide factors for the bacteria. Presumably to overcome this need for expressing and then shedding fimbriae, bacteria have evolved a system of phase variation that turns the fimbriae expression on and off. We demonstrated that phase variation is regulated genetically by an invertible element of DNA, whose oscillating orientation correlates with the on-off transcription of the fimbrial subunit gene. Also, at least two trans-active elements were discovered that are required to promote the DNA rearrangement. We propose two major aims: 1. Characterize the molecular details of phase variation by examining both the cis-active switch and the trans-active factors. A variety of molecular genetic and biochemical approaches will be attempted. 2. Determine the role of phase variation in bacterial physiology and pathobiology. To this and phase-locked mutants will be constructed. A minor aim is to continue the investigation of the biology of organelle assembly using immunoelectron microscopy. Eventually the investigators hope to develop a detailed understanding of the molecular mechanisms and cell biologic significance of E. coli phase variation.
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