OmpR has been demonstrated to negatively regulate the expression of the flagellar master operon flhDC in a wide variety of bacterial species. Here we report the positive regulation of flhDC expression by OmpR in Yersinia pseudotuberculosis. A s 70 -dependent promoter was identified by primer extension analysis and an active region with two conserved OmpR-binding sites around the flhDC promoter was confirmed. To confirm the regulation of flhDC expression by OmpR, flhDC as well as the downstream flagellar genes fliA, flgD, flgA, flgM, fliC and flaA were fused to lacZ, and decreased expression of all these genes in an ompR mutant (DompR) was detected. Furthermore, DompR was defective in bacterial motility and flagella synthesis. This defect was due to the low level of expression of flhDC in DompR since overproduction of FlhDC in DompR restored bacterial motility. The importance of two conserved OmpR-binding sites around the flhDC promoter region in the regulation of flhDC expression by OmpR was demonstrated by the fact that mutation of either one or both sites significantly decreased the promoter activity in the wild-type but not in DompR. The binding of OmpR to these two sites was also demonstrated by DNA mobility shift assay. The possible mechanism underlying this positive regulation in Y. pseudotuberculosis is discussed. To our knowledge, this is the first report to demonstrate that OmpR positively regulates flhDC expression.
INTRODUCTIONFlagella, the unique structure for motility, represent an important advantage for bacteria in moving towards favourable conditions or in avoiding detrimental environments. In pathogenic bacteria, flagella are usually considered as virulence factors (Josenhans & Suerbaum, 2002), and they play crucial roles in adhesion, colonization and biofilm formation of many bacteria such as Pseudomonas aeruginosa (Toutain et al., 2007), Escherichia coli (Pratt & Kolter, 1998), Vibrio cholerae (Gardel & Mekalanos, 1996) and Salmonella typhimurium (Ciacci-Woolwine et al., 1998). Flagellar gene regulation and assembly have been extensively characterized in E. coli and S. enterica serovar Typhimurium (Aldridge & Hughes, 2002). Over 60 genes, which are arranged into three classes (class I, class II and class III), are coordinately regulated and expressed in a transcriptional hierarchy (Chilcott & Hughes, 2000;Kanehisa et al., 2004;Kutsukake et al., 1990). Transcription of the downstream flagellar genes is dependent on a high expression level of some upstream genes. At the top of this hierarchy is the master operon composed of the genes flhD and flhC. FlhD and FlhC together form a heterohexameric transcriptional activator that binds to the class II operons (Wang et al., 2006). Due to the key role of flhDC in flagella synthesis, the regulation of its expression has been studied at both the transcriptional and post-transcriptional levels (Soutourina & Bertin, 2003). Many regulatory proteins, such as H-NS (Bertin et al., 1994;Soutourina et al., 1999), cAMP-CAP complex (catabolite gene activator protei...