Characterization of the Caulobacter crescentus Holdfast Polysaccharide 1 Biosynthesis Pathway

24 Caulobacter crescentus cells adhere to surfaces using an extremely strong polar 25 adhesin called the holdfast. The polysaccharide component of the holdfast is 26 comprised in part of oligomers of N-acetylglucosamine. The genes involved in the 27 export of the holdfast polysaccharide and anchoring of the holdfast to the cell were 28 previously discovered. In this study, we identify a cluster of polysaccharide biosynthesis 29 genes (hfsEFGH) directly adjacent to the holdfast polysaccharide export genes. 30 Sequence analysis indicates that these genes are involved in the biosynthesis of the 31 minimum repeat unit of the holdfast polysaccharide. HfsE is predicted to be a UDP-32 sugar lipid carrier transferase, the glycosyltransferase that catalyzes the first step in 33 polysaccharide biosynthesis. HfsF is predicted to be a flippase, HfsG is a 34 glycosyltransferase, and HfsH is similar to a polysaccharide (chitin) deacetylase. In-35 frame deletion mutants of hfsG and hfsH both result in severe deficiencies in surface 36 adhesion, and binding to the holdfast-specific lectin wheat germ agglutinin. In contrast, 37 hfsE and hfsF mutants exhibit nearly wild-type adhesion and holdfast synthesis. We 38 identify three paralogs to hfsE, two of which are redundant to hfsE for holdfast 39 synthesis. We also identify a redundant paralog to the hfsC gene encoding the putative 40 polysaccharide polymerase, and present evidence that the hfsE and hfsC paralogs 41 together with the hfs genes are absolutely required for proper holdfast synthesis. 42 43