Identification of genes required for synthesis of the adhesive holdfast in Caulobacter crescentus

Adhesion to both abiotic and biotic surfaces by the gram-negative prothescate bacterium Caulobacter crescentus is mediated by a polar organelle called the "holdfast," which enables the bacterium to form stable monolayer biofilms. The holdfast a complex polysaccharide composed in part of N-acetylglucasamine, localizes to the tip of the stalk a thin cylindrical extension of the cell wall and membranes). We report here the isolation of adhesion mutants with transposon insertions in an uncharacterized gene cluster involved in holdfast biogenesis (hfs) as well as in previously identified polar development genes (podJ and pleC), and the holdfast attachment genes (hfa). Clean deletions of three of the four genes in the his gene cluster (hfsDAB) resulted in a severe holdfast biogenesis phenotype. These mutants do not hind to surfaces or to a fluorescently labeled lectin, specific for N-acetylglucosamine. Transmission electron microscopy indicated that the hfsDAB mutants fait to synthesize a holdfast at the stalk tip. The predicted hfs gene products have significant sequence similarity to proteins necessary far exopolysaccharide export in gram-native bacteria. HfsA has sequence similarity to GumC from Xanthomonas campestris, which is involved in exopolysaccharide export in the periplasm. HfsD has sequence similarity to Wza from Escherichia cot, an outer membrane protein involved in secretion of polysaccharide through the cuter membrane. HfsB is a novel protein involved in holdfast biogenesis. These data suggest that the hfs genes play an important role in holdfast exhort.