Molecular cloning and characterization of a chemotactic transducer gene in Pseudomonas aeruginosa.
A Pseudomonas aeruginosa mutant, defective in taxis toward L-serine but responsive to peptone, was selected by the swarm plate method after N-methyl-N'-nitrosoguanidine mutagenesis. The mutant, designated PCT1, was fully motile but failed to show chemotactic responses to glycine, L-serine, L-threonine, and L-valine. PCT1 also showed weaker responses to some other commonly occurring L-amino acids than did the wild-type strain PAO1. A chemotactic transducer gene, denoted pctA (Pseudomonas chemotactic transducer A), was cloned by phenotypic complementation of PCT1. Nucleotide sequence analysis showed that the pctA gene encodes a putative polypeptide of 629 amino acids with a calculated mass of 68,042. A hydropathy plot of the predicted polypeptide suggested that PctA may be an integral membrane protein with two potential membrane-spanning regions. The C-terminal domain of PctA showed high homology with the enteric methyl-accepting chemotaxis proteins (MCPs). The most significant amino acid sequence similarity was found in the region of MCPs referred to as the highly conserved domain. The pctA gene was inactivated by insertion of a kanamycin resistance gene cassette into the wild-type gene, resulting in the same observed deficiency in taxis toward L-amino acids as PCT1. In vivo methyl labeling experiments with L-[methyl-3H]methionine showed that this knockout mutant lacked an MCP with a molecular weight of approximately 68,000.
Department of Fermentation Technology, Hiroshima University, Japan.
A
A
Kuroda
T
T
Kumano
K
K
Taguchi
T
T
Nikata
J
J
Kato
H
H
Ohtake
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002633:Chemotaxis / Q000235:genetics*; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010461:Peptones; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000502:physiology; D016415:Sequence Alignment; D012694:Serine
D001426:Bacterial Proteins; C110124:PctA protein, Pseudomonas aeruginosa; D010461:Peptones; D012694:Serine