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

Abstract

SummaryThe Escherichia coli NsrR protein is a nitric oxidesensitive repressor of transcription. The NsrRbinding 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 (ChIPchip) to show that NsrR binds to 62 sites close to the 5Ј 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 softagar 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.

Inefficient translation of nsrR constrains behaviour of the NsrR regulon in Escherichia coli

“…1). The tynA, feaB and fliA promoters are weakly regulated by NsrR, meaning that these are only modestly derepressed in an nsrR mutant (Rankin et al, 2008;Partridge et al, 2009). These promoters are more strongly repressed when the nsrR start codon is AUG than when it is GUG (Fig.…”

“…Thus, we speculate that inefficient translation maintains a low abundance of NsrR, while still allowing high-level expression of downstream genes. Furthermore, we have shown that the overexpression of nsrR severely represses some genes that are only modestly derepressed by deletion of nsrR (Rankin et al, 2008;Partridge et al, 2009). Thus, the abundance of NsrR seems to be poised in such a way that an increase in NsrR levels has measurable consequences for the expression of some NsrR targets.…”

“…Thus, assignment of an arbitrary threshold in the expression data would lead us to conclude that some genes are in the NsrR regulon when the nsrR start codon is AUG, but not when it is GUG. One example of this effect is tehA, which is 1.5-fold repressed in the microarray data when the nsrR start codon is GUG and (Partridge et al, 2009). Note that yeaR is a known NsrR target (Lin et al, 2007) and is a false-negative in ChIP-chip data.…”

“…The NsrR binding site is an 11-1-11 bp inverted repeat of the consensus motif AAGATGCYTTT (Bodenmiller & Spiro, 2006). Evidence from chromatin immunoprecipitation and microarray analysis (ChIP-chip) suggests that a single 11 bp copy of this motif can be bound by NsrR in vivo and that occupation of such a site (for example in the fliA promoter) can result in weak regulation by NsrR (Partridge et al, 2009).…”

“…The cluster is likely to be [4Fe-4S], and reaction with NO is accompanied by a decrease in DNA binding affinity and de-repression of NsrR-regulated promoters (Bodenmiller & Spiro, 2006;Tucker et al, 2008;Yukl et al, 2008;Crack et al, 2015). The previously characterized NsrR regulon includes genes encoding the NO-scavenging flavohaemoglobin (hmp), the RIC (repair of iron centres) protein that participates in the repair of NO-damaged [Fe-S] clusters (ytfE), the respiratory nitrite reductase Nrf that is also an NO reductase (nrfABCDEFG), genes involved in motility ( fliA) and aromatic amine metabolism (tynA, feaB), and genes of unknown function, for example ygbA, yccM and yeaR-yoaG (Bodenmiller & Spiro, 2006;Filenko et al, 2007;Lin et al, 2007;Rankin et al, 2008;Partridge et al, 2009). The NsrR binding site is an 11-1-11 bp inverted repeat of the consensus motif AAGATGCYTTT (Bodenmiller & Spiro, 2006).…”