Exploring envelope stress response toxicity and regulation in gram-negative bacteria - Critical to bacterial survival is the proper coordination and response to external stress. For example, the envelope stress response (ESR) allows bacteria to repair and defend against cell envelope damage, which is often sustained during antibiotic exposure. However, overactivation of the ESR is toxic in various microbes, suggesting that the ESR may be manipulated to kill bacteria. To exploit this vulnerability, how bacterial cells overcome this toxicity and regulate ESR overactivation needs to be understood. Preliminary work uncovered that the heat shock co-chaperone DnaJ regulates the sE-regulated ESR in Pseudomonas aeruginosa. The objective of this application is to uncover the mechanism of this regulation and characterize its extent. Although DnaJ, in complex with DnaK and GrpE, represses the heat shock response via degradation of this response’s alternative sigma factor, preliminary data suggest that DnaJ regulates the activity of the P. aeruginosa sE homolog AlgU via a different mechanism. The overarching hypothesis is that DnaJ does not regulate AlgU activity via changes in protein levels of known ESR regulators, that instead DnaJ regulates AlgU activity and the ESR via direct binding to this sigma factor, and that this role of DnaJ on the sE-dependent ESR may be conserved across gram-negative bacteria. This hypothesis will be tested via three specific aims. In Aim 1, the effect of DnaJ on gene expression and protein levels of AlgU-dependent ESR regulators will be determined via RT-qPCR and Western Blot under conditions of ESR activation. In Aim 2, DnaJ binding partners that affect the AlgU-dependent ESR will be identified. This Aim will examine which DnaJ domain is important for proper AlgU- dependent ESR activation, if DnaJ binds to AlgU, and if the effect of DnaJ on the ESR requires DnaK, a DnaJ- binding partner that is important for its functions in the heat shock response. In Aim 3, DnaJ-dependent activation of the sE-regulated ESR will be examined in two other, highly genetically tractable gram-negative bacteria, Escherichia coli and Vibrio cholerae, to examine if this mechanism is potentially conserved across Gammaproteobacteria. The outcomes of these Aims are expected to define the mechanistic effect of DnaJ on the ESR (Aims 1-2) and address the potential universality of this mechanism (Aim 3). Furthermore, this work will add to our long-term goal of understanding the mechanism(s) underlying AlgU toxicity in P. aeruginosa, which is important if therapeutics targeting the sE-dependent ESR are to be developed. These outcomes and their potential applications are expected to have a positive impact on the growing problem of multidrug- resistant infections. In addition, as DnaJ has been shown to affect multiple stress response systems in addition to the ESR, this proposal speculates that DnaJ may be a universal stress coordination hub across bacteria, emphasizing its importance in overall bacterial stress response. Finally, this work will benefit the research excellence of a minority-serving institution by sustaining the program of a PI with a strong history of training student researchers from marginalized groups.
