Elucidation of a pathway for holdfast polysaccharide biosynthesis in Caulobacter crescentus

Bacterial adhesion plays a crucial role in establishing a community of microorganisms on surfaces. The strategies employed by bacteria for mediating attachment to surfaces vary, although adhesins and secondary contributions from the flagellum and pili are essential for effective adherence. The aquatic bacterium Caulobacter crescentus produces an exopolysaccharide adhesin called the holdfast to facilitate permanent attachment to surfaces. Holdfast biosynthesis is controlled by the holdfast synthesis (hfs) genes, and deletion mutants of some hfs genes exhibit severe defects in holdfast formation. Surprisingly, I discovered unprecedented redundancies in the initiating (HfsE) and polymerizing steps (HfsC) in the polysaccharide biosynthesis pathway. hfsE has two paralogs (pssY and pssZ), that can substitute for hfsE in holdfast synthesis, while hfsI serves as the hfsC paralog. Interestingly, Delta hfsF exhibits nearly wild-type levels of adhesion and holdfast synthesis, even though HfsF is predicted to translocate the holdfast polysaccharide repeat unit from the cytoplasmic membrane to the periplasm. A genetic screen for non-binding DeltahfsF transposon mutants revealed the potential involvement of hfaD, a holdfast anchor gene. Phenotypic analysis of DeltahfsFDeltahfaD confirmed that holdfast synthesis was abolished in this double deletion mutant, demonstrating for the first time a link between the hfa and hfs genes in regulating holdfast synthesis. To gain insights into the nature of the interaction between HfaD and HfsF, restored binding suppressors of DeltahfaDDeltahfsF were characterized. One suppressor mutation mapped to capD, and the other mapped to rfbB. Both CapD and RfbB are epimerase-like enzymes that catalyze the interconversion of different sugar precursors. Loss of function of either CapD or RfbB singly in DeltahfaDDeltahfsF restores holdfast formation. Therefore, I hypothesize that loss of either of these enzymes can cause an accumulation of different sugar precursors within the cell that ultimately serve as signals to jumpstart the manufacture of the holdfast polysaccharide under certain conditions. Defining the functions of the holdfast synthesis (Hfs) proteins may advance our understanding of the mechanisms responsible for the biosynthesis and export of the holdfast, and provide insights to what governs bacterial adhesion to surfaces. More globally, holdfast synthesis can be used as a model to study bacterial adhesion to surfaces.