A single amino acid substitution in the C terminus of OmpR alters DNA recognition and phosphorylation

Tran, Van Khanh; Oropeza, Ricardo; Kenney, Linda J.

doi:10.1006/jmbi.2000.3809

Cited by 46 publications

(49 citation statements)

References 56 publications

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“…It has been reported recently that OmpRV203 M was unable to be phosphorylated by acetyl phosphate, but it was still able to be phosphorylated by EnvZ both in vivo and in vitro (31). A similar result has been observed with several chemotaxis response-regulator CheY mutants (32).…”

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confidence: 83%

The critical role of DNA in the equilibrium between OmpR and phosphorylated OmpR mediated by EnvZ in Escherichiacoli

Qin¹

2001

Proceedings of the National Academy of Sciences

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Escherichia coli modulates its porin expression through a histidine kinase, EnvZ, and its cognate response regulator, OmpR. EnvZ is a bifunctional enzyme that possesses both OmpR kinase and phosphorylated OmpR (OmpR-P) phosphatase activities and thus controls the cellular level of OmpR-P. In an in vitro-assay system, the addition of OmpR to the reaction mixture consisting of the cytoplasmic domain of EnvZ (EnvZc) and ATP produces a barely detectable amount of OmpR-P because of the dual activities of EnvZ. Here we report that DNA fragments containing the upstream promoter regions of the porin genes (ompF and ompC) can shift the equilibrium between OmpR and OmpR-P dramatically toward OmpR-P. Among the four reactions occurring in the mixture, only the EnvZ phosphatase activity was inhibited severely by the specific DNA, in contrast to the previous report by Kenney and her associates that The autophosphorylation of EnvZc and the phosphotransfer from phosphorylated EnvZc to OmpR were not affected by DNA, whereas the autodephosphorylation of OmpR-P was inhibited slightly. We propose that the apparent inhibitory effect of DNA on the EnvZ phosphatase function is caused by sequestrating OmpR-P from the reaction as a result of OmpR-P binding to DNA.

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confidence: 83%

The critical role of DNA in the equilibrium between OmpR and phosphorylated OmpR mediated by EnvZ in Escherichiacoli

Qin¹

2001

Proceedings of the National Academy of Sciences

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“…The molecular basis for this reduced affinity is now apparent, the result of deviation from the preferred core sequence of GTXTCA (5Ј-3Ј of the template strand, where X is A or T). Our results also account for the structural basis of the reduced affinity of an OmpR2 mutant V203M for ompC (33).…”

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confidence: 56%

Amino Acids Important for DNA Recognition by the Response Regulator OmpR

Rhee

Sheng

Morgan

et al. 2008

Journal of Biological Chemistry

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Response regulators undergo regulated phosphorylation and dephosphorylation at conserved aspartic acid residues in bacterial signal transduction systems. OmpR is a winged helix-turnhelix DNA-binding protein that functions as a global regulator in bacteria and is also important in pathogenesis. A detailed mechanistic picture of how OmpR binds to DNA and activates transcription is lacking. We used NMR spectroscopy to solve the solution structure of the C-terminal domain of OmpR (OmpR C ) and to analyze the chemical shift changes that occur upon DNA binding. There is little overlap in the interaction surface with residues of PhoB that were reportedly involved in protein/protein interactions in its head-to-tail dimer. Multiple factors account for the lack of overlap. One is that the spacing between the OmpR half-sites is shorter than observed with PhoB, requiring the arrangement of the two OmpR molecules to be different from that of the PhoB dimer on DNA. A second is the demonstration herein that OmpR can bind to its high affinity site as a monomer. As a result, OmpR C appears to be capable of adopting alternative orientations depending on the precise base composition of the binding site, which also contributes to the lack of overlap. In the presence of DNA, chemical shift changes occur in OmpR in the recognition ␣-helix 3, the loop between ␤-strand 4 and ␣-helix 1, and the loop between ␤-strands 5 and 6. DNA contact residues are Val 203 (T), Arg 207 (G), and Arg 209 (phosphate backbone). Our results suggest that OmpR binds to DNA as a monomer and then forms a symmetric or asymmetric dimer, depending on the binding site. We propose that during activation OmpR binds to DNA and undergoes a conformational change that promotes phosphorylation of the N-terminal receiver domain, the receiver domains dimerize, and then the second monomer binds to DNA. The flexible linker of OmpR enables the second monomer to bind in multiple orientations (head-to-tail and head-to-head), depending on the specific DNA contacts.

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“…High sequence similarity exists among these species, and V203, which is important for OmpR binding to DNA (Tran et al, 2000), is conserved ( Supplementary Fig. S1).…”

Section: Discussionmentioning

confidence: 99%

Positive regulation of flhDC expression by OmpR in Yersinia pseudotuberculosis

Wang

Ding

et al. 2009

Microbiology

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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...

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A single amino acid substitution in the C terminus of OmpR alters DNA recognition and phosphorylation

Cited by 46 publications

References 56 publications

The critical role of DNA in the equilibrium between OmpR and phosphorylated OmpR mediated by EnvZ in Escherichiacoli

The critical role of DNA in the equilibrium between OmpR and phosphorylated OmpR mediated by EnvZ in Escherichiacoli

Amino Acids Important for DNA Recognition by the Response Regulator OmpR

Positive regulation of flhDC expression by OmpR in Yersinia pseudotuberculosis

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