Proteins associated with the Myxococcus xanthus extracellular matrix

Abstract

Fruiting body formation of Myxococcus xanthus, like biofilm formation of many other organisms, involves the production of an extracellular matrix (ECM). While the polysaccharide component has been studied, the protein component has been largely unexplored. Proteins associated with the ECM were solubilized from purified ECM by boiling with sodium dodecyl sulfate and were identified by liquid chromatography-tandem mass spectrometry of tryptic fragments. The ECM is enriched in proteins of novel function; putative functions were assigned for only 5 of the 21 proteins. Thirteen putative ECM proteins had lipoprotein secretion signals. The genes for many ECM proteins were disrupted in the wild-type (WT), fibA, and pilA backgrounds. Disruption of the MXAN4860 gene had no effect in the WT or fibA background but in the pilA background resulted in a 24-h delay in aggregation and sporulation compared to its parent. The results of this study show that the M. xanthus ECM proteome is diverse and novel.

FIG. 1.

Spore production and viability of ECM mutants. Total spores from 5 × 10⁷ cells incubated for 5 days were enumerated microscopically (solid bars). Viable spores were determined by plating spores on CYE agar (hatched bars). Error bars indicate the standard deviation. The top-left area of each panel displays the genetic background containing each mutation: WT, wild type, DK1622; pilA, ΔpilA, DK10410; fibA, ΔfibA, LS2429.

FIG. 2.

Primary structure analysis of MXAN4860 and developmental timing of MXAN4860 disruptions in wild-type and pilA strains. (A) Schematic of the MXAN4860 protein product. Vertical white bars indicate positions of 20 cysteine residues found in the N-terminal portion of the protein. Gray boxes indicate the tryptic peptides detected by mass spectrometry. The numbers indicate amino acid positions: 1, the start of the protein; 173, the start of the first peptide detected by mass spectrometry; 298, the end of the protein. (B) Developmental time course of fruiting body morphogenesis. Cells (5 × 10⁶) were spotted in 10 μl on TPM agar and photographed every 24 h. Strains with the MXAN4860 mutation develop normally unless it is coupled with a pilA mutation, in which case there is an approximately 24-h delay in development between the 48- and 72-h time points. Bar = 1 mm.

FIG. 3.

Sporulation time courses of MXAN4860 mutants compared to wild-type, pilA, and fibA strains. Cells (5 × 10⁷) of each strain were plated on TPM and harvested at 24-h intervals. Spores were quantified using a Petroff-Hausser counting chamber. (A) Wild-type (DK1622, filled squares) and MXAN4860 (open squares) strains. (B) pilA (DK10410, filled triangle) and MXAN4860 pilA (open triangle) strains. (C) fibA (LS2429, filled diamond) and MXAN4860 fibA (open diamond) strains. Error bars indicate standard deviations.

FIG. 4.

Analysis of FibA fragmentation patterns. (A) Primary structure analysis of FibA. Horizontal lines indicate the lipoprotein secretion signal (*; amino acids 1 to 20), propeptide domain (amino acids 93 to 228), catalytic domain (amino acids 244 to 517), PPC domain 1 (amino acids 542 to 626), and PPC domain 2 (647 to 734) (12). Gray boxes indicate peptides detected by mass spectrometry. (B) Western blot using anti-FibA (α FibA) polyclonal and monoclonal antibodies. Wild-type (WT) and ΔfibA whole-cell extracts were prepared from 5 × 10⁷ exponentially growing cells. ECM material containing 5 μg protein was also analyzed. Arrowheads indicate the major degradation product.