Clp upregulates transcription of engA gene encoding a virulence factor in Xanthomonas campestris by direct binding to the upstream tandem Clp sites

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Abstract
In Xanthomonas campestris, the causative agent of black rot in crucifers, the endoglucanase level is greatly decreased in the mutant deficient in Clp, a homologue of cyclic AMP receptor protein (CRP). It is established that Clp has the same DNA binding specificity as CRP at positions 5, 6, and 7 (GTG motif) of the DNA half site. In this study, the engA transcription initiation site was determined by the 5′ RACE method, and two consensus Clp‐binding sites, site I and site II centered at −69.5 and −42.5, respectively, were located. Transcriptional fusion assays indicated that Clp greatly activates engA transcription. Site‐directed mutagenesis indicated that position 5 of GTG motif in site II is essential for both DNA‐protein complex formation in electrophoretic mobility shift assays and engA transcription in vivo. In addition, mutation at position 5 of site I drastically reduces the promoter activity, indicating that binding of Clp to site I exerts a synergistic effect on the transcription activation by site II. engA appears to be the first X. campestris gene known to be activated by Clp via a direct binding to the promoter.