VINCENT Paula Andrea
congresos y reuniones científicas
Search of chromosomal genes involved in Microcin J25 production
FERNANDA POMARES, PAULA VINCENT, RICARDO FARÍAS, RAÚL SALOMÓN
Pinamar, Buenos Aires
Congreso; XLI Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología molecular; 2005
Microcin J25 (MccJ25) is a plasmid-encoded, 21-amino-acid, antibacterial peptide produced by Escherichia coli. We have developed a two-plasmid system for identifying putative chromosomal genes involved in microcin synthesis or regulation. One of the plasmid bears the structural gene, mcjA, and two genes mcjB and mcjC, required for microcin maturation. This construction is lethal for cells since it lacks the immunity gene. However, it is viable in the presence of the other plasmid, a temperature-sensitive replicon that carries the MccJ25 immunity gene mcjD cloned. We transformed an E. coli host with both plasmids. Double transformants were viable at 30ºC and produced MccJ25. When shifted to 42ºC, the thermosensitive immunity plasmid was lost and, under this condition, the only cells expected to survive were those having a chromosomal or a plasmid mutation lowering or abolishing microcin production. Several candidate clones were isolated. After discarding plasmid mutants, one chromosomal mutant was selected for further study. The mutation abolished microcin production. It was mapped to the lrp gene, encoding an E. coli global transcriptional regulator. Most likely, the lrp gene product acts as a negative regulator at the transcription level. Currently, we are analyzing other mutants obtained by using this two-plasmid system, in the hope of identifying genes whose mutation perturb MccJ25 maturation.