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Population Heterogeneity of Salmonella enterica Serotype Typhimurium Resulting from Phase Variation of the lpf Operon In Vitro and In Vivo. Robert A. Kingsley, 2002.The lpf fimbrial operon oscillates between phase ON and phase OFF expression states, thereby generating heterogeneity within S . enterica serotype Typhimurium populations with regard to expression of long polar fimbrial antigens . To determine whether the proportion of lpf phase variants changes with growth conditions, the lpf phase ON content of cultures was determined after in vitro and in vivo passage . After passage in Luria-Bertani (LB) broth for 120 generations, 96% of cells in a serotype Typhimurium culture carried the lpf operon in the phase ON expression state, regardless of the phase ON/OFF ratio in the inoculum . In contrast, a culture passaged on LB agar plates for 500 generations contained approximately 2% lpf phase ON cells . Differences in the lpf phase ON content of cultures passaged in broth and on plates were not caused by an outgrowth of lpf phase ON or lpf phase OFF cells, since deletion of lpf biosynthesis genes did not alter the phase ON/OFF ratio attained after passage . Instead, growth in LB broth resulted in a eightfold increase in the phase OFF-to-ON transition frequency and a decrease of the lpf phase ON-to-OFF transition frequency by a factor of 150 compared to growth on LB agar plates . After infection of naïve CBA/J mice with an lpf phase ON culture of serotype Typhimurium, the proportion of lpf phase ON cells continuously decreased over time, regardless of whether the strain carried intact fimbrial biosynthesis genes . These data suggest that elaboration of fimbriae does not have a major influence on the population heterogeneity produced by phase variation of the lpf operon in naïve mice . Chaperone-Subunit-Usher Interactions Required for Donor Strand Exchange during Bacterial Pilus Assembly. Michelle M. Barnhart, 2003.The assembly of type 1 pili on the surface of uropathogenic Escherichia coli proceeds via the chaperone-usher pathway . Chaperone-subunit complexes interact with one another via a process termed donor strand complementation whereby the G1ß strand of the chaperone completes the immunoglobulin (Ig) fold of the pilus subunit . Chaperone-subunit complexes are targeted to the usher, which forms a channel across the outer membrane through which pilus subunits are translocated and assembled into pili via a mechanism known as donor strand exchange . This is a mechanism whereby chaperone uncapping from a subunit is coupled with the simultaneous assembly of the subunit into the pilus fiber . Thus, in the pilus fiber, the N-terminal extension of every subunit completes the Ig fold of its neighboring subunit by occupying the same site previously occupied by the chaperone . Here, we investigated details of the donor strand exchange assembly mechanism . We discovered that the information necessary for targeting the FimC-FimH complex to the usher resides mainly in the FimH protein . This interaction is an initiating event in pilus biogenesis . We discovered that the ability of an incoming subunit (in a chaperone-subunit complex) to participate in donor strand exchange with the growing pilus depended on a previously unrecognized function of the chaperone . Furthermore, the donor strand exchange assembly mechanism between subunits was found to be necessary for subunit translocation across the outer membrane usher .
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