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

Abstract

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. IMPORTANCERegulatory 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. Communities of surface-attached bacteria, called biofilms, account for the majority of cells on the surface of our planet. Biofilms allow bacteria to engage in metabolic cooperation, share genetic information, and afford protection from chemical and physical stresses in the environment. Moreover, organic biopolymers and ions accumulate at surfaces through a process known as conditioning (1-4); thus, the ability of a bacterial cell to adhere can confer a nutritional advantage, particularly in oligotrophic environments where nutrients are extremely scarce (5). Clearly, the capacity to transition from a free-living, unicellular lifestyle into a surface-attached ...

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

“…When an OD 660 of 0.3 was reached, cells were harvested by centrifugation at 21,000 g for 1 min at room temperature, yielding 0.5 OD*ml of cells. Samples were then resuspended in lysis buffer (25 mM Tris-HCl (pH 7.6), 125 mM NaCl, 10 mM CaCl 2 , 10 lg•mL 21 DNase I, 1% (w/ v) SDS, 0.1% (v/v) Triton X and EDTA-free protease inhibitor tablet) and western blotted as described previously (Eaton et al, 2016). A 1:1,500 dilution of monoclonal antimouse anti-HA antisera and a 1:4000 dilution of monoclonal goat anti-mouse antibodies conjugated to horseradish peroxidase (HRP) were used (Thermo Scientific).…”

The Two Chemotaxis Clusters in Caulobacter crescentus Play Different Roles in Chemotaxis and Biofilm Regulation

“…Environmental factors, such as blue light via the LovK-LovR system and nutrient availability, add to the control of hfiA expression (47,50). Finally, HfiA is also regulated posttranscriptionally, as the chaperone DnaK affects HfiA levels, probably ensuring its stabilization in the cell (51). Overall, this multilayered control ensures that holdfast production and subsequent irreversible adhesion are tightly controlled at different levels.…”

The Protein Quality Control Network in Caulobacter crescentus

“…The importance of maintaining σ 32 sequestration is reflected during depletion of DnaKJ/E in otherwise optimal conditions, where inappropriate HSP induction leads to a block in DNA replication through degradation of the replication initiator DnaA by the protease Lon (Jonas et al, 2013). DnaKJ/E also plays a role in Caulobacter development by interacting with the holdfast inhibitor HfiA (Eaton et al, 2016). DnaKJ/E activity keeps HfiA stabilized in a folded form, and this interaction operates in a regulatory circuit where increased levels of DnaK reduce the likelihood of developing surface attachment (Eaton et al, 2016), potentially promoting dispersal away from environments with inherent proteotoxic attributes.…”

“…DnaKJ/E also plays a role in Caulobacter development by interacting with the holdfast inhibitor HfiA (Eaton et al, 2016). DnaKJ/E activity keeps HfiA stabilized in a folded form, and this interaction operates in a regulatory circuit where increased levels of DnaK reduce the likelihood of developing surface attachment (Eaton et al, 2016), potentially promoting dispersal away from environments with inherent proteotoxic attributes.…”