PLASMID EXPRESSION OF mutS, -L, AND/OR -H GENE IN E. coli dam CELLS RESULTS IN STRAINS THAT DISPLAY REDUCED MUTATION FREQUENCY
Martina, Mariana A.; Jacquelín, Daniela K.; Argaraña, Carlos E. y Barra José L.
Departamento de Química Biológica, CIQUIBIC (UNC ? CONICET), Facultad de Ciencias Químicas, Universidad Nacional de Córdoba.
Haya de
Abstract
Escherichia coli MutS, -L, -H, and Dam (DNA deoxyadenosine methyltransferase) proteins are the principal components of the postreplicative DNA mismatch repair system (MRS). The transitory hemimethylated state of adenines in the genome GATC sequences of E. coli provide the strand discrimination signal to direct the MRS action toward the damaged DNA strand. E. coli dam cells posses an active but non-directed MRS; therefore, assembly of MutSLH complex at a mismatched base pair can result in MutH mediated cleavage of GATC sites in both DNA strands. Double-strand breaks on a fraction of the replication errors occurring in dam cells can cause cell death, selectively eliminating these putative mutants from the population. An increased level of MutS, -L or ?H in E. coli dam cells results in a more active MRS. We show that E. coli dam cells transformed with plasmids containing either the mutS, mutL, or mutH gene display a mutation frequency 3-8 times lower than that of the parental dam strain, due to increased mismatch-stimulated cell killing. Transformed strains are also more susceptible to killing by the base analogue 2-aminopurine. However, dam and dam transformed cells have similar duplication time, proportion of live/dead cells, and morphology. dam strains of some bacterial species (Salmonella typhimurium, Haemophilus influenzae, Pasteurella multocida, and certain strains of Yersinia pseudotuberculosis) are attenuated. Therefore, dam strains are being used as live vaccines to generate immune protection against virulent wild-type bacteria in chickens, calves and mice; and are also being used to deliver viral antigens to generate immune protection against viral diseases. However, the fact that these dam strains are mutators is an undesirable characteristic. Our results suggest that their mutator activity can be greatly reduced by insertion of a plasmid containing the mutS or mutL gene. Then, transformed dam strains are potentially useful as a source of a live vaccine because of lower mutation frequency.
