Identification of the DNA-binding site of the Rgg-like regulator LasX within the lactocin S promoter region

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Abstract
LasX regulates the transcription of the divergent operons lasXY and lasA–W, which specify the production of lactocin S in Lactobacillus sakei L45. Using histidine-tagged LasX, and a DNA fragment containing the complete intergenic lasA–lasX region, electrophoresis mobility-shift (EMSA) analyses were employed to demonstrate that LasX binds to the lasA–lasX intergenic DNA. Two direct heptanucleotide motifs directly upstream of P lasA–W , and a third imperfect copy of this motif, overlapping the −10 element of P lasA–W , were identified as possible LasX-binding sites. To assess the role of the direct repeats in the binding of LasX to the intergenic lasA–lasX region, binding experiments were performed using DNA probes with different combinations of the repeats, and with arbitrarily chosen repeat substitutions. The result of these experiments demonstrated that only the middle repeat was required for the binding of LasX to the las-promoter region. This observation correlated with the results of subsequent reporter-gene analyses, thereby weakening the hypothesis of the involvement of the direct repeats in LasX-mediated transcription regulation. By analysing the ability of LasX to bind successively shortened derivatives of the original intergenic fragment, a tentative 19 bp minimum LasX-binding site was identified.