High-throughput Sequencing Reveals Suppressors of Vibrio cholerae rpoE Mutations: One Fewer Porin is Enough
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CitationDavis, Brigid M., and Matthew K. Waldor. 2009. High-throughput sequencing reveals suppressors of Vibrio cholerae rpoE mutations: One fewer porin is enough. Nucleic Acids Research 37(17): 5757-5767.
AbstractAnalyses of suppressor mutations have been extremely valuable in understanding gene function. However, techniques for mapping suppressor mutations are not available for most bacterial species. Here, we used high-throughput sequencing technology to identify spontaneously arising suppressor mutations that enabled disruption of rpoE (which encodes σ\(^E\)) in Vibrio cholerae, the agent of cholera. The alternative sigma factor σ\(^E\), which is activated by envelope stress, promotes expression of factors that help preserve and/or restore cell envelope integrity. In Escherichia coli, rpoE is an essential gene that can only be disrupted in the presence of additional suppressor mutations. Among a panel of independent V. cholerae rpoE mutants, more than 75% contain suppressor mutations that reduce production of OmpU, V. cholerae’s principal outer membrane porin. OmpU appears to be a key determinant of V. cholerae’s requirement for and production of σ\(^E\). Such dependence upon a single factor contrasts markedly with regulation of σ\(^E\) in E. coli, in which numerous factors contribute to its activation and none is dominant. We also identified a suppressor mutation that differs from all previously described suppressors in that it elevates, rather than reduces, σ\(^E\)’s activity. Finally, analyses of a panel of rpoE mutants shed light on the mechanisms by which suppressor mutations may arise in V. cholerae.
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