Activation of yeaR-yoaG Operon Transcription by the Nitrate-Responsive Regulator NarL Is Independent of Oxygen- Responsive Regulator Fnr in Escherichia coli K-12

Abstract

The facultative aerobe Escherichia coli K-12 can use respiratory nitrate ammonification to generate energy during anaerobic growth. The toxic compound nitric oxide is a by-product of this metabolism. Previous transcript microarray studies identified the yeaR-yoaG operon, encoding proteins of unknown function, among genes whose transcription is induced in response to nitrate, nitrite, or nitric oxide. Nitrate and nitrite regulate anaerobic respiratory gene expression through the NarX-NarL and NarQ-NarP two-component systems. All known Nar-activated genes also require the oxygen-responsive Fnr transcription activator. However, previous studies indicated that yeaR-yoaG operon transcription does not require Fnr activation. Here, we report results from mutational analyses demonstrating that yeaR-yoaG operon transcription is activated by phospho-NarL protein independent of the Fnr protein. Escherichia coli K-12, a facultative aerobe, is able to respire with a variety of electron acceptors, including oxygen (O 2 ), nitrate (NO 3 Ϫ ), and nitrite (NO 2 Ϫ ). Synthesis of the corresponding respiratory enzymes is subject to hierarchical control to ensure use of the preferred electron acceptor. The top level of this control is mediated by the Fnr transcription activator, which senses the absence of oxygen through its iron-sulfur cluster (27). The second level of hierarchical control is mediated by the NarL and NarP response regulators, which, when phosphorylated, bind DNA to activate or repress transcription. The NarX and NarQ sensors control NarL and NarP phosphorylation in response to nitrate and nitrite (53).Several operons require both Fnr and phospho-NarL or -NarP proteins for maximal transcription. For the narGHJI, narK, and fdnGHI operons, Fnr protein, bound near position Ϫ41.5 with respect to the transcription initiation site, acts synergistically with phospho-NarL protein bound to sites further upstream (53). For the napFDAGHBC operon, Fnr protein, bound at position Ϫ64.5, acts synergistically with phospho-NarP protein bound at position Ϫ44.5 (15, 17). For the nirBDC and nrfABCDEFG operons, Fnr protein, bound near position Ϫ41.5, activates transcription maximally only when phospho-NarL or -NarP protein is bound further upstream to block inhibition by other proteins (2,8,59). Although transcription of many other operons is known to be activated by the Fnr protein acting alone (12, 26), to date there are no examples of Fnr-independent transcription activation by the phosphoNarL or -NarP protein.In preliminary transcript microarray experiments, we observed that the levels of yeaR-yoaG operon transcripts (encoding proteins of unknown function) are increased during growth with nitrate only in a narL ϩ strain (23). Sequence inspection revealed a likely binding site for phospho-NarL protein but no obvious site for binding of Fnr protein (Fig. 1A). Therefore, we were interested in characterizing the control of yeaR-yoaG operon transcription in more detail. Our results, reported here, suggest that this is an example of Fnr-...

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

“…1). A similar sequence can also be found in the yeaR promoter, which is regulated directly by NsrR (Lin et al, 2007).…”

“…The ygbA gene is of unknown function, while hmp encodes a well-characterized NO detoxification system (Poole and Hughes, 2000). Subsequently described targets for NsrR regulation include the hcp-hcr and yeaR-yoaG genes, and the nrf operon that encodes Nrf (Filenko et al, 2007;Lin et al, 2007). Known and predicted targets for NsrR regulation have in their promoter regions an 11 bp inverted repeat with a spacing of 1 bp (Rodionov et al, 2005;Bodenmiller and Spiro, 2006;Lin et al, 2007).…”

“…Subsequently described targets for NsrR regulation include the hcp-hcr and yeaR-yoaG genes, and the nrf operon that encodes Nrf (Filenko et al, 2007;Lin et al, 2007). Known and predicted targets for NsrR regulation have in their promoter regions an 11 bp inverted repeat with a spacing of 1 bp (Rodionov et al, 2005;Bodenmiller and Spiro, 2006;Lin et al, 2007). In this paper we confirm that this sequence is required for NsrRmediated repression of the ytfE promoter, but also present evidence to suggest that a single copy of the 11 bp motif may be sufficient for NsrR binding.…”

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

“…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. dSee text for details.…”

“…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).…”