Chemosensory regulation of developmental gene expression in Myxococcus xanthus

Abstract

The delta-proteobacterium Myxococcus xanthus coordinates its motility during aggregation and fruiting body formation. While searching for chemotaxis genes in M. xanthus, we identified a third chemotaxis-like gene cluster, the che3 cluster, encoding homologs to two methyl-accepting chemotaxis proteins (MCPs), a CheW, a hybrid CheA, a CheB, a CheR, but no CheY. Mutations in mcp3A, mcp3B, and cheA3 did not show obvious defects in motility or chemotaxis but did affect the timing of entry into development. Mutations in these genes caused early aggregation of starving cells, even at low cell densities. Furthermore, these mutants showed pronounced overexpression of the developmentally regulated Tn5lac fusions Omega4403, Omega4411, and Omega4521 as well as overexpression of mbhA and tps, markers for peripheral rods and aggregating cells, respectively. Divergently transcribed from the che3 promoter region is another gene, crdA (chemosensory regulator of development), predicted to encode a transcriptional activator of sigma(54)-dependent promoters. To test the hypothesis that CrdA functions as the cognate response regulator for the histidine kinase CheA3, CrdA and CheA3 were assayed and found to interact strongly in the yeast two-hybrid system. Mutant analysis showed that crdA cells were delayed in development (12-24 h) and delayed in MbhA production relative to the wild type. An mcp3BcrdA double mutant displayed the crdA phenotype, indicating that crdA is epistatic to mcp3B. We conclude that the Che3 chemotaxis-like system functions to control developmental gene expression by regulating a sigma(54) transcriptional activator, CrdA.

Figure 1

Genetic organization of the che3 cluster. crdA (yellow) encodes a σ⁵⁴-dependent transcriptional activator. crdB (red) encodes a hybrid lipoprotein receptor/peptidoglycan binding protein. Green arrows represent ORFs encoding proteins with predicted transmembrane domains (crdC, mcp3A, and mcp3B). ORFs encoding cytoplasmic chemotaxis protein homologs (cheW3, cheA3, cheB3, and cheR3) are blue. The white arrows encode a nifS homolog, an ORF with no similarity to any sequence in the database, and a hypothetical protein (left to right, respectively). The transcription start site (black arrow) was identified by using primer extension, and a putative terminator was identified by using GCG StemLoop. RT-PCR confirmed that crdB-cheA3 comprise a single transcript. crdC, mcp3A, mcp3B, and cheA3 appear to be translationally coupled.

Figure 2

Effect of the che3 mutations on development. The assays were performed as described in Materials and Methods. (a) Effect of the mcp3B and crdA mutations on the timing of aggregation. Cells were plated at an initial concentration of 2 × 10⁹ cells per ml. Each black dot is an aggregate or fruiting body containing ≈10⁵ to 10⁶ cells. Note that aggregates remain translucent before sporulation [wild type (WT) at 24 h and mcp3B at 12 h]. (b) Effect of the mcp3B mutation on density-dependent aggregation. The plates were photographed at 24 h. Wild-type aggregates remain translucent (Upper Right), indicating that refractile spores have not yet formed. (c) Effect of the mcp3B mutation on cells growing on rich medium. The colonies were ≈2 cm in diameter when photographed. Right shows higher magnification (×5) of the mcp3B mutant. Small semitranslucent mounds (circle) were observed after 3 days in the outer edge of the growing mcp3B colony; none were observed in the wild-type colony. The corresponding area for both colonies was harvested and analyzed for heat- and sonication-resistant spores.

Figure 3

Effect of mcp mutations on developmentally regulated genes. Assays were performed as described in Materials and Methods. (a) Effect of the mcp3A mutation on lacZ expression from Tn5lac developmental reporters Ω4403, Ω4411, and Ω4521. Shown are the expression patterns in the DZ2 (wild type) background for Ω4403, Ω4411, and Ω4521 strains (open circles) and the Δmcp3AΩ4403, Δmcp3AΩ4411, and Δmcp3AΩ4521 strains (filled circles). (b) Effect of the mcp3B mutation on the expression of Protein S. Shown is a dot-blot assay for tps mRNA (Upper) and immunoblot assay for Protein S production (Lower). (c) Effect of the mcp3B and crdA mutations on the expression of myxobacterial hemagglutinin. Shown is an immunoblot assay performed with anti-MbhA antibody diluted 1:5,000.

Figure 4

Yeast-two-hybrid assay for CheA3 and CrdA interaction. The assay was performed as described in Materials and Methods. The full-length cheA3 gene and a truncated version (cheA3s) were subcloned into both the activation domain pGAD and the binding domain pGBDU plasmids. Yeast cells (PJ69-4A) were transformed with the plasmids indicated and assayed for growth in the absence of histidine and/or adenine. Only those transformants that contained both cheA3 and crdA were capable of growing (++) in the absence of both adenine and histidine. nd, not determined.

Figure 5

Model for chemosensory regulation of gene expression. The model is described in Discussion. Proteins predicted to be integral membrane proteins are green, cytoplasmic proteins are blue, and the DNA binding response regulator (CrdA) is yellow. The cross hatch represents the peptidoglycan layer. The light blue arrow indicates a putative target gene whose expression is controlled by the Che3 system. CrdB (red) is depicted as bridging the peptidoglycan layer because the C terminus shows strong similarity to a domain within OprF known to interact with peptidoglycan (46); the N terminus of CrdB shows great similarity to a lipoprotein receptor. CrdC is depicted as an inner membrane protein because it has a putative transmembrane domain in its C terminus; CrdC is shown as part of the complex with the receptors because the genes for crdC, mcp3A, mcp3B, and cheA3 are translationally coupled. A signal, possibly a lipoprotein, may induce a conformational change in CrdB, which is transmitted to the CrdC–chemoreceptor complex. Subsequently, the receptor complex would alter CheA3 autophosphorylation levels in a manner similar to that observed during chemotaxis signal transduction (13). The overall level of CrdA–P would regulate expression of target genes involved in development in M. xanthus.