Identification of Genes Required for Synthesis of the Adhesive Holdfast in Caulobacter crescentus

Abstract

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-acetylglucosamine, 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 hfs gene cluster (hfsDAB) resulted in a severe holdfast biogenesis phenotype. These mutants do not bind to surfaces or to a fluorescently labeled lectin, specific for N-acetylglucosamine. Transmission electron microscopy indicated that the hfsDAB mutants fail to synthesize a holdfast at the stalk tip. The predicted hfs gene products have significant sequence similarity to proteins necessary for exopolysaccharide export in gram-negative 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 coli, an outer membrane protein involved in secretion of polysaccharide through the outer membrane. HfsB is a novel protein involved in holdfast biogenesis. These data suggest that the hfs genes play an important role in holdfast export.

Pivotal Roles of the Outer Membrane Polysaccharide Export and Polysaccharide Copolymerase Protein Families in Export of Extracellular Polysaccharides in Gram-Negative Bacteria

“…There is limited biochemical information concerning polysaccharide export in most of these bacteria. However, the OPX proteins are involved in the assembly of products, including traditional EPS such as xanthan gum (114) and succinoglycan (93), as well as polymers with specialized functions, such as the holdfast adhesive that allows the stalks of Caulobacter crescentus to stick to surfaces (104). Group C proteins are among the smallest identified OPX protein (Fig.…”

Signals, Regulatory Networks, and Materials That Build and Break Bacterial Biofilms

“…The flagellum of cells destined for attachment is removed by a protease (3,157). In its place, a protrusion called a holdfast composed of oligomers of N-acetylglucosamine appears (150,296). The holdfast has a strong adhesive polysaccharide that ensures tight adhesion to the surface (197,326).…”

Getting in the Loop: Regulation of Development inCaulobacter crescentus

“…That factor was found to be the protein PodJ. PodJ was identified in multiple developmental screens, and mutants have a phenotype similar to that of pleC mutants (43,85,229,256). Both mutant strains lack pili and a holdfast and have difficulty ejecting the flagellum.…”

“…These paralyzed flagella are also not shed during the swarmer 3 stalked cell transition. These mutants also do not produce pili (as measured by pilus-tropic phage sensitivity), holdfast, or stalks (229,265). Intensive mapping techniques finally led to the identification of the pleC gene, encoding a histidine kinase (265).…”

“…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).…”