HfaE Is a Component of the Holdfast Anchor Complex That Tethers the Holdfast Adhesin to the Cell Envelope.

Bacteria use adhesins to colonize different surfaces and form biofilms. The species of the order use a polar adhesin called holdfast, composed of polysaccharides, proteins, and DNA, to irreversibly adhere to surfaces. In Caulobacter crescentus, a freshwater species, the holdfast is anchored at the cell pole via the oldast nchor (Hfa) proteins HfaA, HfaB, and HfaD. HfaA and HfaD colocalize with holdfast and are thought to form amyloid-like fibers that anchor holdfast to the cell envelope. HfaB, a lipoprotein, is required for the 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 being properly attached to the cell and shed into the medium. This phenotype is greater in a Δ mutant than in a Δ Δ double 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 species, with no predicted function. In planktonic culture, mutants produce holdfasts and rosettes similar to those produced by the wild type. However, holdfasts from mutants bind to the surface but are unable to anchor cells, similarly to other anchor mutants. We showed that fluorescently tagged HfaE colocalizes with holdfast and that 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. For surface attachment and biofilm formation, bacteria produce adhesins that are composed of polysaccharides, proteins, and DNA. Species of the 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 members of the with drastically different environments. We show that HfaE plays an important role in adhesion and biofilm formation in the . Our results provide insights into bacterial adhesins and how they interact with the cell envelope and surfaces.