Cluster II che genes from Pseudomonas aeruginosa are required for an optimal chemotactic response

Abstract

Pseudomonas aeruginosa, a gamma-proteobacterium, is motile by means of a single polar flagellum and is chemotactic to a variety of organic compounds and phosphate. P. aeruginosa has multiple homologues of Escherichia coli chemotaxis genes that are organized into five gene clusters. Previously, it was demonstrated that genes in cluster I and cluster V are essential for chemotaxis. A third cluster (cluster II) contains a complete set of che genes, as well as two genes, mcpA and mcpB, encoding methyl-accepting chemotaxis proteins. Mutations were constructed in several of the cluster II che genes and in the mcp genes to examine their possible contributions to P. aeruginosa chemotaxis. A cheB2 mutant was partially impaired in chemotaxis in soft-agar swarm plate assays. Providing cheB2 in trans complemented this defect. Further, overexpression of CheB2 restored chemotaxis to a completely nonchemotactic, cluster I, cheB-deficient strain to near wild-type levels. An mcpA mutant was defective in chemotaxis in media that were low in magnesium. The defect could be relieved by the addition of magnesium to the swarm plate medium. An mcpB mutant was defective in chemotaxis when assayed in dilute rich soft-agar swarm medium or in minimal-medium swarm plates containing any 1 of 60 chemoattractants. The mutant phenotype could be complemented by the addition of mcpB in trans. Overexpression of either McpA or McpB in P. aeruginosa or Escherichia coli resulted in impairment of chemotaxis, and these cells had smooth-swimming phenotypes when observed under the microscope. Expression of P. aeruginosa cheA2, cheB2, or cheW2 in E. coli K-12 completely disrupted wild-type chemotaxis, while expression of cheY2 had no effect. These results indicate that che cluster II genes are expressed in P. aeruginosa and are required for an optimal chemotactic response.

FIG. 1.

Chemotaxis genes in P. aeruginosa. P. aeruginosa has five clusters of chemotaxis-like genes. Clusters I and V have been previously demonstrated to be involved in swimming motility chemotaxis (24, 37). Cluster IV is involved in twitching motility (8, 25). Mutations constructed in this study are indicated by a delta (Δ) within the arrow representing each gene. Names given to each mutant strain are indicated either above or below each mutation (Table 1 provides further information). The P. aeruginosa PAO1 genome map (center) was obtained from the Pseudomonas Genome Project website (http://www.pseudomonas.com), and the positional numbers flanking each cluster of genes are as previously described (Pseudomonas aeruginosa Community Annotation Project [http://www.pseudomonas.com]).

FIG. 2.

Cluster I che gene mutants are nonchemotactic. Shown is a dilute LB soft-agar plate with cluster I mutant strains. Each strain was motile but deficient in chemotaxis.

FIG. 3.

CheB2 is required for optimal chemotaxis by P. aeruginosa, and overexpression of CheB2 partially restores chemotaxis to a nonchemotactic P. aeruginosa cheB mutant. (A) Dilute LB soft-agar plate showing chemotactic rings formed by P. aeruginosa PAO1-Ig (wild type) harboring only vector (pEX1.8), the cheB2 mutant AFIB2lacZ harboring only vector (pEX1.8), and the cheB2 mutant complemented with cheB2 in trans (pEXB2). (B) Dilute LB soft-agar plate showing chemotactic rings formed by P. aeruginosa PAO1-Ig (wild type) harboring only vector (pEX1.8), the cheB mutant AFIB harboring only vector (pEX1.8), and the cheB mutant complemented with cheB2 in trans (pEXB2). Expression of CheB2 from pEXB2 was controlled by the P_tac promoter and was induced by addition of 100 μM IPTG to the medium.

FIG. 4.

McpA is required for a wild-type chemotactic response by P. aeruginosa, and overexpression of McpA inhibits the chemotactic response. (A) Tryptone soft-agar plate showing chemotactic rings formed by P. aeruginosa PAO1-Ig (wild type) and an mcpA mutant (AFD3216). (B) Tryptone soft-agar plate with 1,000 μM MgCl₂, comparing the chemotactic responses of wild-type cells and mcpA mutant cells. Addition of Mg²⁺ to the medium restores wild-type chemotaxis to the mcpA mutant. (C) Dilute LB soft-agar plate showing chemotactic rings formed by P. aeruginosa PAO1-Ig (wild type) harboring only vector (pEX1.8) and P. aeruginosa PAO1-Ig (wild type) overexpressing McpA (pHAH128). Expression of McpA from pHAH128 was induced by the addition of 10 μM IPTG to the medium. (D) Tryptone soft-agar plate showing chemotactic rings formed by E. coli K-12 harboring only vector (pEX1.8) and E. coli K-12 expressing McpA (pHAH128). Expression of McpA from pHAH128 was controlled by the P_tac promoter and was induced by the addition of 1,000 μM IPTG to the medium.

FIG. 5.

McpB is required for optimal chemotaxis by P. aeruginosa, and overexpression of McpB inhibits the chemotactic response. (A) Dilute LB soft-agar plate showing chemotactic rings formed by P. aeruginosa PAO1-Ig (wild type) harboring only vector (pEX1.8), an mcpB mutant (AFD4672) harboring only vector (pEX1.8), and the mcpB mutant complemented with mcpB in trans (pAF27B). Expression of McpB from pAF27B was induced by the addition of 10 μM IPTG to the medium. (B) Tryptone soft-agar plate showing chemotactic rings formed by E. coli K-12 harboring only vector (pEX1.8) and E. coli K-12 expressing McpB (pAF27B). Expression of McpB from pAF27B was induced by the addition of 100 μM IPTG to the medium.

FIG. 6.

Effect of expression of cluster II Che proteins on E. coli K-12 chemotaxis. Shown is a tryptone soft-agar plate with chemotactic rings formed by E. coli K-12 harboring only vector (pEX1.8) or expressing CheA2 (pEXA2), CheB2 (pEXB2), CheW2 (pEXW2), or CheY2 (pEXY2). No IPTG was added to the medium. CheY2 failed to inhibit E. coli chemotaxis even when expression was induced with 100 μM IPTG.

FIG. 7.

Domain architectures of 3 of the 26 P. aeruginosa MCPs. The structures were created with the SMART server (http://smart.embl-heidelberg.de/) (35, 47). PA numbers are indicated in parentheses and are according to the Pseudomonas aeruginosa Community Annotation Project (http://www.pseudomonas.com). The roles of McpA and McpB in P. aeruginosa chemotaxis are discussed in the text. CtpH is an MCP for inorganic phosphate in P. aeruginosa and has been previously described (59). CtpH represents the common structural motif of an MCP and is similar to E. coli MCPs. Domain representations are as follows: rectangle, transmembrane domain; square, PAS domain; elongated pentagon, highly conserved domain of MCP (MA, methyl accepting); small pentagon, HAMP domain (1). The scale represents amino acid positions.

FIG. 8.

Cluster II-like gene arrangements in γ-proteobacteria. (A) P. aeruginosa cluster II. PA numbers are according to the Pseudomonas aeruginosa Community Annotation Project (http://www.pseudomonas.com). (B) Cluster II-like gene arrangements in other γ-proteobacteria. P. syringae cluster II and S. oneidensis MR-1 cluster II were accessed through the TIGR Microbial Genome Database (http://www.tigr.org/tdbl). V. cholerae VCA numbers are according to the TIGR Complete Microbial Resource (http://www.tigr.org/tigr.scripts/CMR2/CMRHomePage.spl). V. cholerae cheB2 may not be functional due to a frame shift mutation (16).