Interaction of the C-terminal region of P. aeruginosa MutS with the DnaK chaperone

MONTI MARIELA R.; MIGUEL VIRGINIA ; ARGARAÑA CARLOS E.

Workshop; II Argentine Workshop on current topics in Pseudomonas and Burkholderia research; 2009

MutS recognizes misspaired bases and it is required to activate downstream events in the postreplicative DNA Mismatch Repair System (MRS). In a previous work, we determined that the C-terminal region of P. aeruginosa MutS is essential for MRS activity. Different evidences indicated that this region may be involved in the interaction with other factor necessary for the correct function of the MRS. To analyze if the C-terminal region of MutS (Cter) interacts with other protein, the DNA encoding this domain was fused in frame to the maltose binding protein (MBP) gene, and expressed in P. aeruginosa. The purified MBP-Cter protein consistently copurified with a 70 kDa protein, which was absent in MBP purified preparations. This protein was identified as DnaK by mass spectrometry analysis. The P. aeruginosa cells expressing the fusion MBP-Cter acquired a phenotype similar to that previously described for dnaK mutants in other bacteria: diminished viability, cell filamentation and high temperature sensitivity. Similar phenotypes were detected by overexpressing the full length MutS protein. On the contrary, overexpression of a C-terminal deletion MutS mutant did not produce phenotypic changes compared to the wild type strain. We propose that DnaK interacts with the C-terminal region of MutS and the overexpression of the repair protein produces a complete sequestration of DnaK. We are currently analyzing the interaction MutS-DnaK by biochemical assays and generating P. aeruginosa dnaK mutant strains to analyze in more detail its phenotype.