Transcriptional Regulation by Clp in Xanthomonas campestris pv. campestris

Autor: Chih-Hua Chen, 陳智華
Rok vydání: 2010
Druh dokumentu: 學位論文 ; thesis
Popis: 98
The Gram-negative plant pathogenic Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers. It is capable of producing large amounts of an exopolysaccharide, xanthan gum, and secreting an array of extracellular enzymes, including proteases, pectinases, and endoglucanases, which have long been considered important virulence determinants. Production of these extracellular products is regulated by the global transcription factor Clp (cyclic AMP receptor protein-like protein, a homolog of the Escherichia coli CRP) and DSF whose biosynthesis involves RpfF protein. It is also known that Xcc encodes no adenylate cyclase required for the production of cyclic AMP, an effector that is involved in activation of CRP for binding to CRP-regulated promoters. Furthermore, it has previously been demonstrated in our laboratory that Clp 1) in vitro can bind to the regulated promoters in the absence of cyclic AMP, and 2) exerts transcriptional activation by binding to the promoter of some regulated genes (i.e., engA, pehA, and pelA1), but without direct binding to others (i.e., fliC, groESL, prt1, and manA). Despite these findings, prediction of the sequences for Clp binding (Clp-binding site, CBS) was merely based on similarity to the consensus E. coli CRP-binding site, which is 22-bp long (5’-AAATGTGATCTAGATCACATTT-3’) exhibiting perfect twofold sequence symmetry, because Clp has been shown to have the same DNA binding specificity as CRP at positions 5, 6, and 7 (GTG motif) of the DNA half site. However, close examination of the above CBSs that have been characterized revealed that some of them possess sequences of arms and a central region deviated from the CRP consensus sequence. Therefore, this study was aimed to deduce consensus CBS sequence(s) on the basis of a larger number of characterized Xcc CBSs than what was already available. To achieve this end, PromScan program was used to search in Xcc genome. Several putative CBSs were identified, among which promoters of clp, gumB, and xpsE were then confirmed to be up-regulated by Clp, via deletion mapping in conjunction with electrophoretic mobility shift assay (EMSA) and transcriptional fusion assay. The results suggested that 1) clp is auto-regulated positively by Clp via binding to a typical CBS, 2) gum promoter possesses two atypical CBSs, in which a more conserved right arm compensates for the lack of conservation in the left arm, a high GC content in the central region (6 bp) is important for binding, and binding is enhanced by the palindromic GC-rich central region, and 3) two typical CBSs are present upstream of xpsE and both could bind to Clp in EMSA; however, only the one in proximity to the xpsE transcription initiation site is required for transcription activation. The three atypical CBSs (gumB CBS I, gumB CBS II, and xpsF CBS) were combined to create a frequency matrix which was then used in PromScan to perform genome-wide search. Twenty-one possible atypical CBSs were identified and five of them were confirmed by competitive EMSA. These CBS-containing genes have previously been shown to be up-regulated by Clp in microarray assay; however, most of the CBSs confirmed here are situated at different positions from those predicted by the microarray assay. Based on these eight atypical CBSs, a consensus atypical CBS, 5’-AnAGGCGAACGCnGTCACAnAA-3’, was compiled. In the meantime, by aligning 8 known typical CBSs, a more informative consensus typical CBS (5’-TnGTGTGnnnnAnnTCnCATCG-3’) was deduced. Using a frequency matrix based on this consensus sequence for search with PromScan, 30 Xcc genes, which have been previously identified by microarray assay to be regulated by Clp, were identified to possess typical CBS. Further EMSA is needed for confirmation of these typical CBSs. In conclusion, these results suggest that both typical and atypical CBSs exist in Xcc genome and that Clp has evolved to gain the capability to bind both types of CBSs. In addition to the above results, two important findings were also made here: 1) xpsE promoter is also up-regulated by RpfF, and 2) binding of Clp in vitro is inhibited by cyclic di-GMP, as demonstrated by using the Xcc engA promoter region as the probe in EMSA.
Databáze: Networked Digital Library of Theses & Dissertations
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