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

Abstract

Caulobacter crescentus cells adhere to surfaces by 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 the anchoring of the holdfast to the cell were previously discovered. In this study, we identified a cluster of polysaccharide biosynthesis genes (hfsEFGH) directly adjacent to the holdfast polysaccharide export genes. Sequence analysis indicated 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 hfsG and hfsH deletion mutants resulted in severe deficiencies both in surface adhesion and in binding to the holdfast-specific lectin wheat germ agglutinin. In contrast, hfsE and hfsF mutants exhibited nearly wild-type levels of adhesion and holdfast synthesis. We identified three paralogs to hfsE, two of which are redundant to hfsE for holdfast synthesis. We also identified 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.

Functional Characterization of UDP-Glucose:Undecaprenyl-Phosphate Glucose-1-Phosphate Transferases of Escherichia coli and Caulobacter crescentus

“…2), but all contain the C-terminal domain that is essential for catalytic activity in PHPT family members (32,43,52). HfsE has 375 amino acids and contains two predicted transmembrane helices (46). In contrast, PssY and PssZ are smaller proteins, of 267 and 188 amino acids, respectively, and they each contain one predicted transmembrane helix and the conserved C-terminal region.…”

“…In the Gram-negative aquatic bacterium Caulobacter crescentus, HfsE, PssY, and PssZ are proposed to initiate the synthesis of glycans required for the production of the holdfast adhesin, but these three proteins are believed to be redundant based on genetic complementation assays in a C. crescentus ⌬hfsE ⌬pssY ⌬pssZ mutant (46). During cell division, C. crescentus produces a motile swarmer cell containing a flagellum and pili at the same pole and a nonmotile cell which adheres to surfaces via a stalk tipped with the holdfast adhesin (21).…”

Getting in the Loop: Regulation of Development inCaulobacter crescentus

“…The holdfast is ultimately placed at the tip of the extended stalk, but the vast majority of cytoplasmic proteins are excluded from the stalk interior (see "Stalk Biogenesis" below). Holdfast polysaccharide synthesis requires some cytoplasmic biosynthetic proteins (250). It is currently unknown if the machinery that synthesizes the oligosaccharide components can be separated from the export and attachment machinery.…”

“…Treatment with lysozyme, which is known to degrade N-acetylglucosamine polymers, increases the elasticity of the holdfast by 90% but it does not destroy the holdfast, suggesting that there are other components of the holdfast or that some of the glucosidic linkages are resistant to lysozyme (138). Second, many mutations that abolish holdfast production are found in genes that are predicted to encode polysaccharide biosynthesis machinery, including oligosaccharide synthesis (163,250) and export (229). Third, the holdfast was observed to have physical properties of a polysaccharide gel by atomic force microscopy (138).…”

N-Acetylglucosamine-dependent biofilm formation in Pectobacterium atrosepticum is cryptic and activated by elevated c-di-GMP levels

“…The ability to modulate the production of key adhesion factors in some bacteria is a typical example of the biological importance of c-di-GMP regulation. The adhesins in question include proteinaceous factors (Hinsa et al, 2003;Yousef & Espinosa-Urgel, 2007), exopolysaccharide (EPS; Borlee et al, 2010), adhesive curli fimbriae and pili (Pesavento et al, 2008), flagella (O'Toole & Kolter, 1998) and the adhesive holdfast of Caulobacter crescentus (Toh et al, 2008). Indeed, the matrix composition of biofilms typically consists of a complex mixture of EPS, proteins and nucleic acids (Branda et al, 2005).…”