HfaE is a component of the holdfast anchor complex that tethers the holdfast adhesin to the cell envelope

Abstract

Bacteria use adhesins to colonize different surfaces and form biofilms. The species of the Caulobacterales order use a polar adhesin called holdfast, composed of polysaccharides, proteins, and DNA to irreversibly adhere to surfaces. In C. crescentus, a freshwater Caulobacterales, the holdfast is anchored at the cell pole via the holdfast anchor (Hfa) proteins HfaA, HfaB, and HfaD. HfaA and HfaD co-localize with holdfast and are thought to form amyloid-like fibers that anchor holdfast to the cell envelope. HfaB, a lipoprotein, is required for translocation of HfaA and HfaD to the cell surface. Deletion of the anchor proteins leads to a severe defect in adherence resulting from holdfast not properly attached to the cell and shed into the medium. This phenotype is greater in a {Delta}hfaB than a double {Delta}hfaA hfaD mutant, suggesting that HfaB has other functions besides the translocation of HfaA and HfaD. Here, we identify an additional HfaB-dependent holdfast anchoring protein, HfaE, which is predicted to be a secreted protein. HfaE is highly conserved among Caulobacterales species with no predicted function. In planktonic culture, hfaE mutants produce holdfasts and rosettes similar to wild type. However, holdfasts from hfaE mutants bind to the surface but are unable to anchor cells, similar to other anchor mutants. We showed that fluorescently-tagged HfaE co-localizes with holdfast, and HfaE forms an SDS-resistant high molecular weight species consistent with amyloid fiber formation. We propose that HfaE is a novel holdfast anchor protein, and that HfaE functions to link holdfast material to the cell envelope. IMPORTANCEFor surface attachment and biofilm formation, bacteria produce adhesins that are composed of polysaccharides, proteins and DNA. Species in the Caulobacterales produce a specialized polar adhesin, holdfast, which is required for permanent attachment to surfaces. In this study, we evaluate the role of a newly identified holdfast anchor protein HfaE in holdfast anchoring to the cell surface in two different Caulobacterales with drastically different environments. We show that HfaE plays an important role in adhesion and biofilm formation in Caulobacterales. Our results provide insights into bacterial adhesins and how they interact with the cell envelope and surfaces.