| Summary: | <jats:p>Adhesion allows microbes to colonize surfaces and is the first stage in biofilm formation. Stable attachment of the freshwater alphaproteobacterium <jats:italic>Caulobacter crescentus</jats:italic> to surfaces requires an adhesive polysaccharide called holdfast, which is synthesized at a specific cell pole, and ultimately found at the tip of cylindrical extensions of the cell envelope called stalks. Secretion and anchoring of holdfast to the cell surface are governed by proteins HfsDAB and HfaABD, respectively. The arrangement and organization of these proteins with respect to each other and the cell envelope, and the mechanism by which the holdfast is anchored on cells is unknown.</jats:p> <jats:p>In this study, we have imaged a series of <jats:italic>C. crescentus</jats:italic> mutants using electron cryotomography, revealing the architecture and arrangement of the molecular machinery involved in holdfast anchoring in cells. We found that the holdfast is anchored to cells by a defined complex made up of the HfaABD proteins, and that the HfsDAB secretion proteins are essential for proper assembly and localization of the HfaABD anchor. Sub-tomogram averaging of cell stalk tips showed that the HfaABD complex spans the outer membrane. The anchor protein HfaB is the major component of the anchor complex located on the periplasmic side of the outer membrane, while HfaA and HfaD are located on the cell surface. HfaB is the critical component of the complex, without which no HfaABD complex was observed in cells. These results allow us to propose a working model of holdfast anchoring, laying the groundwork for further structural and cell biological investigations.</jats:p> <jats:p><jats:bold>IMPORTANCE</jats:bold></jats:p> <jats:p>Adhesion and biofilm formation are fundamental processes that accompany bacterial colonization of surfaces, which are of critical importance in many infections. <jats:italic>Caulobacter crescentus</jats:italic> biofilm formation proceeds via irreversible adhesion mediated by a polar polysaccharide called holdfast. Mechanistic and structural details of how the holdfast is secreted and anchored on cells are still lacking. Here we have assigned the location and described the arrangement of the holdfast anchor complex. This work increases our knowledge of the relatively underexplored field of polysaccharide-mediated adhesion by identifying structural elements that anchor polysaccharides to the cell envelope, which is important in a variety of bacterial species.</jats:p>
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