Abel Ferrandez

Cluster II che Genes from Pseudomonas aeruginosa Are Required for an Optimal Chemotactic Response

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.

Chemotaxis in Pseudomonads

All Pseudomonas species are motile by one or more polar flagella and are highly chemotactic. Chemotaxis and motility have been implicated in virulence in Pseudomonas aeruginosa 21, and are important for plant root associations in Pseudomonas fluorescens 17. The chemotaxis machinery has not been studied in detail in any Pseudomonas species and the range of attractants and environmental conditions to which Pseudomonads can respond behaviorally remains largely unexplored. However, the availability of four Pseudomonas genome sequences has allowed the identification of numerous potential chemotaxis genes. Experiments in P. aeruginosa and Pseudomonas putida indicate that the general chemotaxis machinery present in these organisms is similar to that of the well-studied enteric bacteria Escherichia coli and Salmonella. The vast array of chemotaxis and receptor genes present in the Pseudomonas genomes suggests that chemotaxis may be more complex and sensory transduction may be more versatile in the Pseudomonads than in enteric bacteria. This chapter will focus primarily on the information gleaned from the complete genome sequences of P. aeruginosa PAOl74, P. putida KT244050, Pseudomonas syringae DC3000 (ref. 8a) and the unfinished P. fluorescens PFOl sequences (http://www.jgi.doe.gov/JGI_microbial/html/index.html), correlating available functional data whenever possible.