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

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 0 RACE method, and two consensus Clp-binding sites, site I and site II centered at À69.5 and À42.5, 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.

Expressional Regulation of the Virulence Gene eglXoA Encoding Endoglucanase, Dependent on HrpXo and Cyclic AMP Receptor-Like Protein (Clp) in Xanthomonas oryzae pv. oryza

“…It has been reported that the Clp protein directly binds to the promoter regions of the endoglucanase (engA) and polygalacturonase (pheA) genes of X. campestris, presumably enhancing transcriptional expression [26,28]. Therefore, it is reasonable to assume that ClpXo can also bind to the promoter region of eglXoA.…”

Regulation of the Type II Secretion Structural Gene xpsE in Xanthomonas campestris Pathovar campestris by the Global Transcription Regulator Clp

“…The procedures for EMSA experiments were conducted as described [9] with the exception that a 6% polyacrylamide gel was used for separation of the DNAprotein complex. The LightShift TM Chemiluminescent EMSA kit (Pierce, Rockford, IL) was used for detection.…”

“…However, the leaf symptoms caused by WTM and XC472C strains appeared 1-2 days later than the wild-type strain 8004 (data not shown). Clp regulates a large set of genes directly or indirectly [1,3,8,9,10]. Therefore, it is possible that in WTM and XC472C strains some unknown virulence genes were also downregulated to such low levels that the virulence of Xcc was affected.…”

“…GUS activity was expressed as pmol 4-MU per OD 600 per min. Data represent means ± SD of three experiments engXCA promoter [9]. The DNA sequence for Clp binding has been evaluated and found to be similar to that of the E. coli CRP-binding site (5 0 -AAATGTGA-TCTAGA-TCACATTT-3 0 ) [4].…”

“…It was suggested that the position of the center of the CRP-binding site varies from -40 to -200 from the transcript start site at different promoters [15]. However, the Clp binding site of xpsE is located far from its translation start codon compared to that of the engXCA gene, which has two Clp binding sites centered at -69.5 and -42.5 relative to transcription initiation site [9]. In order to authenticate this putative Clp binding site, EMSA was performed using purified Clp with a 500-bp DNA fragment (probe A) containing the putative binding sequence from the xpsE promoter (Fig.…”