PMID:19656291
| Citation |
Partridge, JD, Bodenmiller, DM, Humphrys, MS and Spiro, S (2009) NsrR targets in the Escherichia coli genome: new insights into DNA sequence requirements for binding and a role for NsrR in the regulation of motility. Mol. Microbiol. 73:680-94 |
|---|---|
| Abstract |
The Escherichia coli NsrR protein is a nitric oxide-sensitive repressor of transcription. The NsrR-binding site is predicted to comprise two copies of an 11 bp motif arranged as an inverted repeat with 1 bp spacing. By mutagenesis we confirmed that both 11 bp motifs are required for maximal NsrR repression of the ytfE promoter. We used chromatin immunoprecipitation and microarray analysis (ChIP-chip) to show that NsrR binds to 62 sites close to the 5' ends of genes. Analysis of the ChIP-chip data suggested that a single 11 bp motif (with the consensus sequence AANATGCATTT) can function as an NsrR-binding site in vivo. NsrR binds to sites in the promoter regions of the fliAZY, fliLMNOPQR and mqsR-ygiT transcription units, which encode proteins involved in motility and biofilm development. Reporter fusion assays confirmed that NsrR negatively regulates the fliA and fliL promoters. A mutation in the predicted 11 bp NsrR-binding site in the fliA promoter impaired repression by NsrR and prevented detectable binding in vivo. Assays on soft-agar confirmed that NsrR is a negative regulator of motility in E. coli K12 and in a uropathogenic strain; surface attachment assays revealed decreased levels of attached growth in the absence of NsrR. |
| Links |
PubMed Online version:10.1111/j.1365-2958.2009.06799.x |
| Keywords |
Base Sequence; Binding Sites; Chromatin Immunoprecipitation; DNA Mutational Analysis; DNA, Bacterial/genetics; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Mutagenesis; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Repressor Proteins/genetics; Repressor Proteins/metabolism; Transcription Factors/genetics; Transcription Factors/metabolism |
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ChIP-chip was done as described in Efromovich [1], using microarrays described in Grainger et al. [2]. This array platform, from Oxford Gene Technologies, is deposited in GEO as GPL6754
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- ↑ Efromovich, S et al. (2008) Genome-wide identification of binding sites for the nitric oxide-sensitive transcriptional regulator NsrR. Meth. Enzymol. 437 211-33 PubMed
- ↑ Grainger, DC et al. (2005) Studies of the distribution of Escherichia coli cAMP-receptor protein and RNA polymerase along the E. coli chromosome. Proc. Natl. Acad. Sci. U.S.A. 102 17693-8 PubMed