An extracellular matrix-associated zinc metalloprotease is required for dilauroyl phosphatidylethanolamine chemotactic excitation in Myxococcus xanthus

Abstract

An extracellular matrix connects bacteria that live in organized assemblages called biofilms. While the role of the matrix in the regulation of cell behavior has not been extensively examined in bacteria, we suggest that, like mammalian cells, the matrix facilitates cell-cell interactions involved with regulation of cohesion, motility, and sensory transduction. The extracellular matrix of the soil bacterium Myxococcus xanthus is essential for biofilm formation and fruiting body development. The matrix material is extruded as long, thin fibrils that mediate adhesion to surfaces, cohesion to other cells, and excitation by the chemoattractant dilauroyl phosphatidylethanolamine. We report the identification of a putative matrix-associated zinc metalloprotease called FibA (fibril protein A). Western blotting with FibA-specific monoclonal antibody 2105 suggests extensive proteolytic processing of FibA during assembly into fibrils, consistent with the autoprocessing observed with other members of the M4 metalloprotease family. Disruption of fibA had no obvious effect on the structure of the fibrils and did not inhibit cell cohesion, excitation by dioleoyl phosphatidylethanolamine, or activity of the A- or S-motility motors. However, the cells lost the ability to respond to dilauroyl phosphatidylethanolamine and to form well-spaced fruiting bodies, though substantial aggregation was observed. Chemotactic excitation of the fibA mutant was restored by incubation with purified wild-type fibrils. The results suggest that this metalloprotease is involved in sensory transduction.

FIG. 1.

Amino acid sequence alignment of M. xanthus FibA with the AhpB elastase of A. hydrophila (accession number AAF07184) and LasB elastase of P. aeruginosa (accession number AAG07111). Open circles mark the FibA putative active site residues. The putative nucleotide binding P-loop is marked with closed circles. The putative type II secretion signal sequence is underlined with a solid black bar, while the Behmlander and Dworkin (5) amino acid sequence determined by N-terminal sequencing is underlined with a gray bar.

FIG. 2.

The fibA mutant is proficient in fibril biosynthesis. Scanning electron micrographs of DK1622 wild type (a) and LS2200 fibA::pDB23 (b) are shown. Cells were incubated for 30 min in cohesion buffer prior to fixation. Bar = 1 μm.

FIG. 3.

The fibA mutant lacks all MAb 2105 reactive polypeptides. A Western blot with MAb 2105 as primary antibody is shown. Lane 1, lysate from 5 × 10⁸ whole cells of wild-type DK1622; lane 2, 5 μg of protein from purified DK1622 fibrils; lane 3, lysate from 5 × 10⁸ whole cells of LS2200 (fibA); lane 4, 5 μg of protein from purified LS2200 fibrils.

FIG. 4.

fibA produces elongated fruiting bodies. Wild-type (a) and fibA::pDB23 LS2200 (b) cells (5 × 10⁷) were starved on TPM agar for 5 days to induce fruiting body formation. Bar = 2 mm.

FIG. 5.

fibA has normal excitation to dioleoyl PE. The reversal periods of fibA::pDB23 LS2200 (circles) and the wild type (DK1622) (squares). Bars show the standard deviations of three replicates. The wild-type data are derived from that of Kearns et al. (22).

FIG. 6.

fibA is deficient in excitation to dilauroyl PE. The reversal periods of fibA::pDB23 LS2200 (circles), the wild type (DK1622) (squares), and LS2200 cells incubated with purified wild-type fibrils (triangles). Bars show the standard deviations of three replicates.