Allosteric control of a bacterial stress response system by an anti-σ factor.

Bacterial signal transduction systems commonly use receiver (REC) domains, which regulate adaptive responses to the environment as a function of their phosphorylation state. REC domains control cell physiology through diverse mechanisms, many of which remain understudied. We have defined structural features that underlie activation of the multi-domain REC protein, PhyR, which functions as an anti-anti-σ factor and regulates transcription of genes required for stress adaptation and host-microbe interactions in Alphaproteobacteria. Though REC phosphorylation is necessary for PhyR function in vivo, we did not detect expected changes in inter-domain interactions upon phosphorylation by solution X-ray scattering. We sought to understand this result by defining additional molecular requirements for PhyR activation. We uncovered specific interactions between unphosphorylated PhyR and an intrinsically disordered region (IDR) of the anti-σ factor, NepR, by solution NMR spectroscopy. Our data support a model whereby nascent NepR(IDR)-PhyR interactions and REC phosphorylation coordinately impart the free energy to shift PhyR to an open, active conformation that binds and inhibits NepR. This mechanism ensures PhyR is activated only when NepR and an activating phosphoryl signal are present. Our study provides new structural understanding of the molecular regulatory logic underlying a conserved environmental response system.

Proper Control of Caulobacter crescentus Cell Surface Adhesion Requires the General Protein Chaperone DnaK.

UNLABELLED: Growth in a surface-attached bacterial community, or biofilm, confers a number of advantages. However, as a biofilm matures, high-density growth imposes stresses on individual cells, and it can become less advantageous for progeny to remain in the community. Thus, bacteria employ a variety of mechanisms to control attachment to and dispersal from surfaces in response to the state of the environment. The freshwater oligotroph Caulobacter crescentus can elaborate a polysaccharide-rich polar organelle, known as the holdfast, which enables permanent surface attachment. Holdfast development is strongly inhibited by the small protein HfiA; mechanisms that control HfiA levels in the cell are not well understood. We have discovered a connection between the essential general protein chaperone, DnaK, and control of C. crescentus holdfast development. C. crescentus mutants partially or completely lacking the C-terminal substrate binding "lid" domain of DnaK exhibit enhanced bulk surface attachment. Partial or complete truncation of the DnaK lid domain increases the probability that any single cell will develop a holdfast by 3- to 10-fold. These results are consistent with the observation that steady-state levels of an HfiA fusion protein are significantly diminished in strains that lack the entire lid domain of DnaK. While dispensable for growth, the lid domain of C. crescentus DnaK is required for proper chaperone function, as evidenced by observed dysregulation of HfiA and holdfast development in strains expressing lidless DnaK mutants. We conclude that DnaK is an important molecular determinant of HfiA stability and surface adhesion control. IMPORTANCE: Regulatory control of cell adhesion ensures that bacterial cells can transition between free-living and surface-attached states. We define a role for the essential protein chaperone, DnaK, in the control of Caulobacter crescentus cell adhesion. C. crescentus surface adhesion is mediated by an envelope-attached organelle known as the holdfast. Holdfast development is tightly controlled by HfiA, a small protein inhibitor that directly interacts with a WecG/TagA-family glycosyltransferase required for holdfast biosynthesis. We demonstrate that the C-terminal lid domain of DnaK is not essential for growth but is necessary for proper control of HfiA levels in the cell and for control of holdfast adhesin development.