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.

FIG. 1.

Morphology of developmental mutants compared to that of wild-type strain CB15. The cellular morphology of representative populations of C. crescentus (A) CB15, (B) YB2861, and (C) YB2863 was image captured with a Nikon E800 light microscope equipped with a ×100 Apo oil objective, Princeton Instruments cooled CCD camera, and Metamorph imaging software, v.4.5.

FIG. 2.

Location of transposon insertions in holdfast-deficient mutants. Genes are delineated by boxes with corresponding gene name and GenBank accession number. Arrows indicate the direction of transcription for each gene relative to one another. Wedges indicate the mapped locations of mini-Tn5lacZ2 insertions.

FIG. 3.

Glass coverslip binding assay of holdfast-shedding transposon mutants. Phase-contrast (left panels) and fluorescence (right panels) images of representative areas of glass slides submerged in cultures of various strains and assayed for lectin binding with FITC-WGA are shown for the following strains: CB15 (A and B), NA1000 (C and D), YB2779 (E and F), YB2782 (G and H), YB2780 (I and J), YB2783 (K and L), YB2781 (M and N), and YB2784 (O and P).

FIG. 4.

Analysis of the holdfasts of hfs deletion mutants as compared to CB15 and NA1000 by TEM. Electron micrographs of representative members of stalked cell population (positive stained with 7.5% uranyl magnesium acetate) at ×50,000 magnification. (A) CB15. (B) NA1000. (C) CB15 ΔhfsA. (D) CB15 ΔhfsB. (E) CB15 ΔhfsC. (F) CB15 ΔhfsD. Bars, 100 nm.

FIG. 5.

Detection of N-acetylglucosamine in the holdfast of hfs deletion mutants. FITC-WGA was used to label the holdfasts of CB15 (A), NA1000 (B), CB15 ΔhfsA (C), CB15 ΔhfsB (D), CB15 ΔhfsC (E), and CB15 ΔhfsD (F).