Auxotrophic mutant of Staphylococcus aureus generated by allelic replacement.

MARÍA SOL BARBAGELATA; LUCÍA P ALVAREZ; CECILIA QUIROGA; FERNANDA BUZZOLA; DANIEL SORDELLI

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2007

Several strategies have been developed for generating gene replacements in bacterial pathogens. The aim of this study was construct an deletion mutant of using a new plasmid (pMAD) for nontransformable bacteria. This termosensitive vector allows a quick colorimetric blue-white discrimination of bacteria that have lost plasmid, greatly facilitating clone identification during mutagenesis. The gene of RN6390 strain was amplified with specific primers to generate a fragment with a deletion of 900 bp. The PCR product was cloned into pMAD vector. The pMAD-1 recombinant plasmid obtained was used to transform the RN6390 strain by electroporation. Then, integration of the plasmid into the chromosome by a single crossover event was selected during growth at 44ºC. Subsequent growth of cointegrates at 30ºC led to a second recombination event, resulting in their resolution. White colonies (without pMAD-1) were characterized by PCR and subsequent sequentiation to confirm the deletion of the gene. Phenotypic characterization of the mutant SB374 resulted in the loss of growth onto plates lacking aromatic aminoacids suplements. The SB374 mutant had a higher lethal dose 50 than the parental strain demonstrating be attenuated . These results show that the deletion of gene obtained by a new tool for Gram-positive, reduced the virulence of S. aureus