SUMMARY

            MutL protein is part of the mismatch repair system (MMRS) which rectifies DNA replication errors and prevents recombination between related sequences. Failure of the MMRS has been associated with pathogenicity of P. aeruginosa and other bacterial pathogens. To further investigate this association in Pseudomonas, we have cloned by PCR the mutL gene of P. aeruginosa using primers designed from de mutL homologous sequence found in the genome sequence of P. aeruginosa. The PCR product was cloned in the E. coli pET15b plasmid (Novagen) which allows the expression of a histidine (6)-Tag-MutL fusion protein. The expression of the cloned mutL gene produced a protein of approximately 70 kDa, as expected from the corresponding DNA sequence. In order to characterize the role of this gene and its corresponding protein product in the MMRS of P. aeruginosa, disrupted mutL mutant strains were generated. Insertional mutL mutants, containing a kanamycin resistance gene cassette, were constructed by homologous recombination and sucrose-mediated counterselection, using the pKNG101 plasmid. Mutants defective in mutL expression showed more than 100-fold increase in the mutation frequency compared to the wild type strain. Mutant mutL strains were complemented with the PCR cloned gene, confirming the functionality of the mutL cloned gene and the correct disruption of this gene in the mutL mutant strains. At present we are evaluating the general phenotype of the mutant strains and the in vivo substrate specificity of the MMRS of P. aeruginosa.