JB Accepts, published online ahead of print on 29 August 2008

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J. Bacteriol. doi:10.1128/JB.01003-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Characterization of the Caulobacter crescentus Holdfast Polysaccharide Biosynthesis Pathway Reveals Significant Redundancy in the Initiating Glycosyltransferase and Polymerase steps

Evelyn Toh, Harry D. Kurtz Jr., and Yves V. Brun^*

Department of Biology, Indiana University, Bloomington, IN 47405-3700

^* To whom correspondence should be addressed. Email: ybrun{at}indiana.edu.

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