VINCENT Paula Andrea
congresos y reuniones científicas
The yojI gene, encoding a MccJ25 pump, is positively regulated by the Leucine-responsive regulatory protein (Lrp)
BENJAMÍN SOCÍAS, PAULA VINCENT, RAÚL SALOMÓN.
Pinamar, Buenos Aires
Congreso; XLI Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología molecular; 2005
We observed a greater sensitivity to MccJ25 when the Leu concentration in the medium was increased. Intracellular amount of the global transcriptional regulator Lrp inversely depends on Leu concentration. An lrp null mutant, RO64, did not show the Leu effect on sensitivity and was hypersusceptible to MccJ25. We found two potential Lrp binding sites (one of these identical to the consensus TTTATTCtNaAT) just upstream of yojI, a chromosomal gene which codes for a MccJ25 pump. To prove that Lrp is a positive regulator of yojI transcription, we transformed lrp+ and lrp strains with the plasmids pUCLO5 and pTrcHyojI both carrying yojI, in the first under control of native promoter and in the second, under control of trc promoter. Lrp+ transformants displayed complete resistance to MccJ25 with both plasmids, while the Lrp strain became resistant only with pTrcHyojI. To confirm the influence of Lrp on the expression of yojI, a lacZ gene fusion was constructed and mapped by restriction analysis. The expression level of the yojI-lacZ fusion in lrp+ and lrp strains was growth-phase-dependent. It was 10-fold higher in lrp+ than in lrp strains in stationary-phase. During exponential growth a low expression level was detected in lrp+ strain indicating a stationary-phase induction of yojI. We conclude that an increased Leu concentration reduces yojI transcription through a reduction of Lrp levels. This, in turn, reduces MccJ25 export.