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.
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.
Partridge
Jonathan D
JD
Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, TX 75080, USA.
Bodenmiller
Diane M
DM
Humphrys
Michael S
MS
Spiro
Stephen
S
D001483:Base Sequence; D001665:Binding Sites; D047369:Chromatin Immunoprecipitation; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D016296:Mutagenesis; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C529899:NsrR protein, E coli; D012097:Repressor Proteins; D014157:Transcription Factors