The mismatch repair system (mutS, mutL and uvrD genes) in Pseudomonas aeruginosa: molecular characterization of naturally occurring mutants.
We have recently described the presence of a high proportion of Pseudomonas aeruginosa isolates (20%) with an increased mutation frequency (mutators) in the lungs of cystic fibrosis (CF) patients. In four out of 11 independent P. aeruginosa strains, the high mutation frequency was found to be complemented with the wild-type mutS gene from P. aeruginosa PAO1. Here, we report the cloning and sequencing of two additional P. aeruginosa mismatch repair genes and the characterization, by complementation of deficient strains, of these two putative P. aeruginosa mismatch repair genes (mutL and uvrD). We also describe the alterations in the mutS, mutL and uvrD genes responsible for the mutator phenotype of hypermutable P. aeruginosa strains isolated from CF patients. Seven out of the 11 mutator strains were found to be defective in the MMR system (four mutS, two mutL and one uvrD). In four cases (three mutS and one mutL), the genes contained frameshift mutations. The fourth mutS strain showed a 3.3 kb insertion after the 10th nucleotide of the mutS gene, and a 54 nucleotide deletion between two eight nucleotide direct repeats. This deletion, involving domain II of MutS, was found to be the main one responsible for mutS inactivation. The second mutL strain presented a K310M mutation, equivalent to K307 in Escherichia coli MutL, a residue known to be essential for its ATPase activity. Finally, the uvrD strain had three amino acid substitutions within the conserved ATP binding site of the deduced UvrD polypeptide, showing defective mismatch repair activity. Interestingly, cells carrying this mutant allele exhibited a fully active UvrABC-mediated excision repair. The results shown here indicate that the putative P. aeruginosa mutS, mutL and uvrD genes are mutator genes and that their alteration results in a mutator phenotype.
Oliver
Antonio
A
Unidad de Microbiología Molecular, Servicio de Microbiología, Hospital Ramón y Cajal, Carretera de Colmenar Km 9.1, 28034-Madrid, Spain.
Baquero
Fernando
F
Blázquez
Jesús
J
D000251:Adenosine Triphosphatases / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D020137:Base Pair Mismatch / Q000235:genetics*; D001483:Base Sequence; D003550:Cystic Fibrosis / Q000382:microbiology; D004265:DNA Helicases / Q000235:genetics*; D004260:DNA Repair; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein; D009154:Mutation; D011552:Pseudomonas Infections / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C074770:MutL protein, E coli; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli; D004265:DNA Helicases