Davis, Brigid M.Waldor, Matthew2011-11-012009Davis, 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): 5757-5767.0305-1048http://nrs.harvard.edu/urn-3:HUL.InstRepos:5336036Analyses 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.en-USJournal Article2011-11-0110.1093/nar/gkp568