VINCENT Paula Andrea
congresos y reuniones científicas
OVEREXPRESSION OF YDIE, AN ORF OF UNKNOWN FUNCTION, CONFERS HIGH RESISTANCE TO MICROCIN J25
SOCíAS SERGIO B; VINCENT PAULA A.; SALOMóN RAúL A.
Mar del Plata
Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología molecular.; 2007
Sociedad Argentina de Investigaciones en Bioquímica y Biología molecular.
Previously, we observed an intrinsic, chromosomally-encoded resistance, in several E. coli strains to the peptide antibiotic MccJ25. To identify genes responsible for this phenotype, a genomic library in MudII4042 was constructed by thermoinduction of MC4100 Mucts ÄyojI (pEG109). Strain AB1133 (sensitive to MccJ25) was infected with the resulting lysate and muductants were selected on medium containing chloramphenicol and MccJ25. Plasmid DNA was extracted from one of several MccJ25-resistant clones, digested with HindIII and cloned into pACYC177. This plasmid conferred resistance to MccJ25 when transformed into a sensitive strain and was named pACAX66. The fragment carried by pACAX66 was sequenced and located on the 38-min region of the E. coli chromosome. Additional subclones of this plasmid in the vector pACYC177 were constructed. One of them, containing a 655 bp fragment, was able to confer resistance to MccJ25. The insert should encompass only a single chromosomal ORF, ydiE. Interestingly, several experiments showed that YdiE could not export MccJ25 and conferred resistance even in a TolC background. This suggests that ydiE neither acts as a MccJ25 pump nor activates one. Whatever the mechanism, this represents a new form of microcin resistance. This is currently under study in our laboratory.