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.
