Project Summary Our long-term objectives are to understand genetic and phenotypic properties that allow the abundant human gut Bacteroidales species to survive in the host over its lifetime. In this proposal, we will build upon our work studying antibacterial toxins produced by the gut Bacteroidales that they use to antagonize other members of ecosystem. We will study important protective responses mounted by these bacteria upon exposure to antibacterial toxins and other stressors they encounter in the gut microbiota. We found that the genes of an unusual ECF-type sigma factor/antisigma factor pair, named EcfO-Reo, are induced upon exposure of Phocaeicola vulgatus to toxins and other stress-inducing compounds. The ensuing pleiotropic response protects these bacteria from various stressors that perturb the outer membrane. efcO-reo and the genes of its regulon are among the most highly expressed in vivo. The genes of this regulon include those encoding three families of proteins with unknown functions: the NigD proteins, a family of outer membrane porins, and proteins involved in the synthesis of long LPS. Our preliminary data suggest that these genes are regulated in different manners and provide distinct mechanisms of protection. In Aim 1, we will use a transcriptional reporter and phenotypic analyses to identify microbial, host and abiotic stressors that induce expression of the ecfO-reo operon and determine if induction of the response protects the bacteria from those stressors. In Aim 2, we will determine the molecular basis of long LPS (O-antigen) synthesis and the protection it confers to diverse stressors. In Aim 3, we will use genetic and phenotypic analyses combined with unbiased approaches to identify the protective functions of the NigD family of proteins. These proteins are are released in outer membrane vesicles when the EcfO regulon is induced by outer membrane perturbations. In Aim 4, we will study a family of outer membrane proteins that are highly upregulated when the EcfO-Reo operon is induced. We will use a combination of techniques to identify the unique mechanism of regulation and the contribution of these OMPs to the EcfO-Reo- induced protective response. For all aims, we will study the contribution of the different arms of the protective response to the fitness of the bacteria in the gnotobiotic mouse gut. The knowledge gained from this proposal will greatly increase our understanding of the molecular mechanisms that this order of bacteria use to survive in the human gut microbiota when exposed to various stressors of host, microbial and xenobiotic origin.