DifA, a methyl-accepting chemoreceptor protein-like sensory protein, uses a novel signaling mechanism to regulate exopolysaccharide production in Myxococcus xanthus

Abstract

DifA is a methyl-accepting chemotaxis protein (MCP)-like sensory transducer that regulates exopolysaccharide (EPS) production in Myxococcus xanthus. Here mutational analysis and molecular biology were used to probe the signaling mechanisms of DifA in EPS regulation. We first identified the start codon of DifA experimentally; this identification extended the N terminus of DifA for 45 amino acids (aa) from the previous bioinformatics prediction. This extension helped to address the outstanding question of how DifA receives input signals from type 4 pili without a prominent periplasmic domain. The results suggest that DifA uses its N-terminus extension to sense an upstream signal in EPS regulation. We suggest that the perception of the input signal by DifA is mediated by protein-protein interactions with upstream components. Subsequent signal transmission likely involves transmembrane signaling instead of direct intramolecular interactions between the input and the output modules in the cytoplasm. The basic functional unit of DifA for signal transduction is likely dimeric as mutational alteration of the predicted dimeric interface of DifA significantly affected EPS production. Deletions of 14-aa segments in the C terminus suggest that the newly defined flexible bundle subdomain in MCPs is likely critical for DifA function because shortening of this bundle can lead to constitutively active mutations.

FIG. 1.

Identification of DifA start codon. (A) The upstream and 5′ coding sequence of difA with deduced amino acids. The oligonucleotide used for primer extension (see Fig. S1 in the supplemental material) is complementary to the underlined DNA sequence centered around the codon for D77. The two transcription initiation sites identified by primer extension are marked by two forward arrows, respectively. The two potential GTG start codons for DifA at −138 and +1 are shaded in black, with their consensus ribosomal binding sites boxed with solid lines. The ATG proposed by bioinformatics as the start codon for DifA is shaded in gray. The amino acids in bold are the two predicted transmembrane helices TM1 and TM2. The charged residues preceding TM1 are in bold and underlined. Codons 34 (CGC) and 138 (CTG), boxed by dashed lines, were changed to CGG and CTC as silent mutations to introduce two restriction sites in pWB230 (Table 2). (B and C) Immunoblot analysis (B) and EPS production (C) of strains with mutations of the potential start codons. For immunoblotting, whole-cell lysates from 5 × 10⁸ cells for each strain were separated by SDS-PAGE and probed with anti-DifA antibodies. For EPS analysis, 5 μl of cells at approximately 5 × 10⁹ cells/ml was spotted onto CTT plates containing calcofluor white at 50 μg per ml; the plates were photographed under UV illumination after 5 days of incubation at 32°C. The diameter of the plates shown is ∼9 cm. Strains: difA⁺, YZ619; ΔdifA, YZ601; −138GTC, YZ709; +1GTC, YZ710; +1ATG, YZ711; and M49A, YZ606.

FIG. 2.

Deletions in the N-terminus extension of DifA affect EPS production. Shown are immunoblot analysis (A) and EPS production (B) of mutants with deletions in the predicted cytoplasmic N terminus of DifA. Experiments were performed as described for Fig. 1B and C. Strains: difA⁺, YZ619; ΔdifA, YZ601; Δ2-40, YZ776; Δ10-35, YZ1710; Δ2-8, YZ1711; Δ9-18, YZ1712; Δ19-28, YZ1713; and Δ29-38, YZ1714.

FIG. 3.

Point mutations in the N terminus of DifA influence EPS production. (A) DifA protein levels by immunoblot analysis. Four micrograms of whole-cell lysates from each strain was analyzed by SDS-PAGE and probed with polyclonal anti-DifA antibodies. (B) DifA membrane localization. The membrane (M) and the soluble (S) fractions normalized to whole-cell lysates were analyzed by immunoblotting as in panel A. (C) EPS production of the point mutants was examined as described for Fig. 1C. Strains: difA⁺, YZ619; ΔdifA, YZ601; L38P, YZ1822; G45A, YZ1820; Y46C, YZ1821; and P96L, YZ1823.

FIG. 4.

Deletions of DifA indels impact EPS production. Shown are immunoblot analysis (A) and EPS production (B) of indicated deletions of indel segments. See Fig. 1 for experimental details. Strains: difA⁺, YZ619; ΔdifA, YZ601; ΔI, YZ777; ΔIV, YZ778; Δ(I+IV), YZ779; ΔII, YZ780; ΔIII, YZ781; and Δ(II+III), YZ782.

FIG. 5.

Mutations of certain E/Q residues impact EPS production. Immunoblot analysis (A) and EPS production (B) of indicated glutamate (E) or glutamine (Q) substitution mutants. See the legend to Fig. 1 for experimental details. Strains: difA⁺, YZ619; ΔdifA, YZ601; E183A, YZ745; Q394N, YZ745; E400D, YZ714; E400Q, YZ751; E407A, YZ748; E407Q, YZ738; and Q429E, YZ763.

FIG. 6.

(A) Predicted topology of DifA and the effect of selected mutations on EPS production. The N-terminal extension (NTE) is cytoplasmic. It is followed by two transmembrane helices, TM1 and TM2, which are separated by nine residues in the periplasm. The HAMP domain connects TM2 to the C-terminal signaling domain, which forms a coiled-coil hairpin with three subdomains: the signaling subdomain, flexible bundle subdomain (FBS1 and FBS2), and the methylation helices (MH1 and MH2). Indels I and IV are in the MHs, whereas II and III are in the FBSs. Also indicated in the drawing are the positions of selected mutations and their effects on EPS production. Only mutations with minimum impact on DifA protein level are shown, and those in green led to increased EPS production, while those in red resulted in reduced or undetectable EPS levels. (B) The modeled structure of a DifA_C dimer and the position of seven mutated E/Q residues in Fig. 5. The DifA_C dimer is shown on the left. The two chains of the dimer are labeled blue (chain A) and red (chain B), respectively. The positions of the mutated E/Q residues in chain A are shown as white, green, or yellow spheres, corresponding to their mutations with either no phenotype, reduced EPS level only, or both increased and decreased EPS levels, respectively (see text). For a clearer illustration of the dimer interface, the panels on the right show the zoomed-in view of residues that were mutated. The same E/Q residues in chain B of the dimer are located in similar positions to those in chain A, although they are not in perfect symmetry.