Positive regulation of flhDC expression by OmpR in Yersinia pseudotuberculosis

Abstract

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...

OmpR controls Yersinia enterocolitica motility by positive regulation of flhDC expression

“…Two putative OmpR binding sites (B 1 , B 2 ) are shown in bars within the flhDC promoter sequence. Two nucleotide sequences P 1 and P 2 similar to the O 1 and O 2 binding sites for OmpR identified within the Y. pseudotuberculosis flhDC promoter region (Hu et al 2009) are underlined . These four sequences (B 1 , B 2 , P 1 , P 2 ) are shown separately in alignments with the consensus OmpR binding sequence; the conserved C residues are in bold .…”

“…During our studies of OmpR binding abilities to the flhDC promoter region in Y. enterocolitica , the involvement of OmpR in the regulation of flhDC in Y. pseudotuberculosis was reported and two binding sites for OmpR in the promoter region (O 1 and O 2 ) were identified (Hu et al 2009). In silico examination of the nucleotide sequence, within the 448-bp flhDC promoter region of Y. enterocolitica , corresponding to the putative OmpR binding sites O 1 and O 2 of Y. pseudotuberculosis , led to the identification of these sequences (P 1 and P 2 ) with 66 and 50% identity to the OmpR consensus, respectively (Fig.…”

“…During these studies, the involvement of OmpR in the positive regulation of flhDC in Y. pseudotuberculosis was reported and two binding sites for OmpR in the promoter region (O 1 and O 2 ) were identified by Hu et al (2009). However, alignment of the flhDC regulatory regions of Y. enterocolitica and Y. pseudotuberculosis shows high nucleotide sequence divergence, including the P 1 and P 2 sites (corresponding to O 1 and O 2 sites of Y. pseudotuberculosis ) identified in promoter region of Y. enterocolitica .…”

Phenotypic and transcriptional analysis of the osmotic regulator OmpR in Yersinia pestis

“…OmpR is a repressor of the inv gene, which encodes the major virulence determinant invasin in Y. enterocolitica [33]. In Y. pseudotuberculosis , OmpR regulates positively the urease expression to enhance acid survival [34], whereas it controls negatively the expression of FlhD and FlhC that form a heterohexameric transcriptional activator of the flagellar genes [35]. In this work, the ompR mutation likely had not affect on the virulence of Y. pestis 201, which was a human-attenuated enzootic strain in a mouse model after subcutaneous infection (data not shown).…”

A starvation-induced regulator, RovM, acts as a switch for planktonic/biofilm state transition in Yersinia pseudotuberculosis

“…In Y. pseudotuberculosis , swimming motility is primarily controlled by the expression of the flagellar master regulator flhDC . Previously studies have shown that the YpsRI and YtbRI quorum sensing systems 23 , CsrA 24 , OmpR 25 , and RpoS 26 indirectly modulate swimming motility by controlling the expression of flhDC . The Y. pseudotuberculosis hmsHFRS operon is responsible for synthesis and transport of the exopolysaccharide β-GlcNAc, the primary dry component of the biofilm matrix 27 .…”