Stephen Spiro

The NsrR Regulon of Escherichia coli K-12 Includes Genes Encoding the Hybrid Cluster Protein and the Periplasmic, Respiratory Nitrite Reductase

Successful pathogens must be able to protect themselves against reactive nitrogen species generated either as part of host defense mechanisms or as products of their own metabolism. The regulatory protein NsrR (a member of the Rrf2 family of transcription factors) plays key roles in this stress response. Microarray analysis revealed that NsrR represses nine operons encoding 20 genes in Escherichia coli MG1655, including the hmpA, ytfE, and ygbA genes that were previously shown to be regulated by NsrR. Novel NsrR targets revealed by this study include hcp-hcr (which were predicted in a recent bioinformatic study to be NsrR regulated) and the well-studied nrfA promoter that directs the expression of the periplasmic respiratory nitrite reductase. Conversely, transcription from the ydbC promoter is strongly activated by NsrR. Regulation of the nrf operon by NsrR is consistent with the ability of the periplasmic nitrite reductase to reduce nitric oxide and hence protect against reactive nitrogen species. Gel retardation assays were used to show that both FNR and NarL bind to the hcp promoter. The expression of hcp and the contiguous gene hcr is not induced by hydroxylamine. As hmpA and ytfE encode a nitric oxide reductase and a mechanism to repair iron-sulfur centers damaged by nitric oxide, the demonstration that hcp-hcr, hmpA, and ytfE are the three transcripts most tightly regulated by NsrR highlights the possibility that the hybrid cluster protein, HCP, might also be part of a defense mechanism against reactive nitrogen stress.

A combination of cytochrome c nitrite reductase (NrfA) and flavorubredoxin (NorV) protects Salmonella enterica serovar Typhimurium against killing by NO in anoxic environments.

The enteric bacterium Salmonella enterica serovar Typhimurium is a pathogen that is highly adapted for both intracellular and extracellular survival in a range of oxic and anoxic environments. The cytotoxic radical nitric oxide (NO) is encountered in many of these environments. Protection against NO may involve reductive detoxification in low-oxygen environments, and three enzymes, flavorubredoxin (NorV), flavohaemoglobin (HmpA) and cytochrome c nitrite reductase (NrfA), have been shown to reduce NO in vitro. In this work we determined the role of these three enzymes in NO detoxification by Salmonella by assessing the effects of all eight possible combinations of norV, hmpA and nrfA single, double and triple mutations. The mutant strains were cultured and exposed to NO following either glucose fermentation (when nitrite reductase activity is low), or anaerobic respiration (when nitrite reductase activity is high). Wild-type cultures were more sensitive to the addition of a pulse of NO when grown under fermentative conditions compared with anaerobic respiratory conditions. Analysis of the mutant strains suggested an important additive role for both NorV and NrfA in both environments, since the norV nrfA mutant could not grow after NO addition. The results also suggested a minor role for HmpA in anaerobic detoxification of NO under the two growth conditions, and a larger role for HmpA in aerobic NO detoxification was confirmed. Activity assays and measurements of NO consumption showed that increased nitrite reductase activity correlates with an elevated capacity for NO reduction by intact cells. Taken together, the results reveal a combined role for NorV and NrfA in NO detoxification under anaerobic conditions, and highlight the influence that growth conditions have on the sensitivity to NO of this pathogenic bacterium.

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 11u2003bp motif arranged as an inverted repeat with 1u2003bp spacing. By mutagenesis we confirmed that both 11u2003bp 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 11u2003bp 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 11u2003bp 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.u2003coli K12 and in a uropathogenic strain; surface attachment assays revealed decreased levels of attached growth in the absence of NsrR.

Nitrous oxide production and consumption: regulation of gene expression by gas- sensitive transcription factors

Several biochemical mechanisms contribute to the biological generation of nitrous oxide (N2O). N2O generating enzymes include the respiratory nitric oxide (NO) reductase, an enzyme from the flavo-diiron family, and flavohaemoglobin. On the other hand, there is only one enzyme that is known to use N2O as a substrate, which is the respiratory N2O reductase typically found in bacteria capable of denitrification (the respiratory reduction of nitrate and nitrite to dinitrogen). This article will briefly review the properties of the enzymes that make and consume N2O, together with the accessory proteins that have roles in the assembly and maturation of those enzymes. The expression of the genes encoding the enzymes that produce and consume N2O is regulated by environmental signals (typically oxygen and NO) acting through regulatory proteins, which, either directly or indirectly, control the frequency of transcription initiation. The roles and mechanisms of these proteins, and the structures of the regulatory networks in which they participate will also be reviewed.

Vibrio fischeri flavohaemoglobin protects against nitric oxide during initiation of the squid–Vibrio symbiosis

Nitric oxide (NO) is implicated in a wide range of biological processes, including innate immunity against pathogens, signal transduction and protection against oxidative stress. However, its possible roles in beneficial host–microbe associations are less well recognized. During the early stages of the squid–vibrio symbiosis, the bacterial symbiont Vibrio fischeri encounters host‐derived NO, which has been hypothesized to serve as a specificity determinant. We demonstrate here that the flavohaemoglobin, Hmp, of V. fischeri protects against NO, both in culture and during colonization of the squid host. Transcriptional analyses indicate that hmp expression is highly responsive to NO, principally through the repressor, NsrR. Hmp protects V. fischeri from NO inhibition of aerobic respiration, and removes NO under both oxic and anoxic conditions. A Δhmp mutant of V. fischeri initiates squid colonization less effectively than wild type, but is rescued by the presence of an NO synthase inhibitor. The hmp promoter is activated during the initial stage of colonization, during which the Δhmp strain fails to form normal‐sized aggregates of colonizing cells. Taken together, these results suggest that the sensing of host‐derived NO by NsrR, and the subsequent removal of NO by Hmp, influence aggregate size and, thereby, V. fischeri colonization efficiency.

Escherichia coli NsrR Regulates a Pathway for the Oxidation of 3-Nitrotyramine to 4-Hydroxy-3-Nitrophenylacetate

Chromatin immunoprecipitation and microarray (ChIP-chip) analysis showed that the nitric oxide (NO)-sensitive repressor NsrR from Escherichia coli binds in vivo to the promoters of the tynA and feaB genes. These genes encode the first two enzymes of a pathway that is required for the catabolism of phenylethylamine (PEA) and its hydroxylated derivatives tyramine and dopamine. Deletion of nsrR caused small increases in the activities of the tynA and feaB promoters in cultures grown on PEA. Overexpression of nsrR severely retarded growth on PEA and caused a marked repression of the tynA and feaB promoters. Both the growth defect and the promoter repression were reversed in the presence of a source of NO. These results are consistent with NsrR mediating repression of the tynA and feaB genes by binding (in an NO-sensitive fashion) to the sites identified by ChIP-chip. E. coli was shown to use 3-nitrotyramine as a nitrogen source for growth, conditions which partially induce the tynA and feaB promoters. Mutation of tynA (but not feaB) prevented growth on 3-nitrotyramine. Growth yields, mutant phenotypes, and analyses of culture supernatants suggested that 3-nitrotyramine is oxidized to 4-hydroxy-3-nitrophenylacetate, with growth occurring at the expense of the amino group of 3-nitrotyramine. Accordingly, enzyme assays showed that 3-nitrotyramine and its oxidation product (4-hydroxy-3-nitrophenylacetaldehyde) could be oxidized by the enzymes encoded by tynA and feaB, respectively. The results suggest that an additional physiological role of the PEA catabolic pathway is to metabolize nitroaromatic compounds that may accumulate in cells exposed to NO.

Analysis of the nitric oxide-sensing non-heme iron center in the NorR regulatory protein.

The NorR regulatory protein senses nitric oxide (NO) to activate genes required for NO detoxification under anaerobic and microaerobic conditions in Escherichia coli. NorR belongs to the σ54-dependent family of transcriptional activators and contains an N-terminal regulatory GAF (cGMP phosphodiesterase, adenylate cyclase, FhlA) domain that controls the ATPase activity of the central AAA+ domain to regulate productive interactions with σ54. Binding of NO to a non-heme iron center in the GAF domain results in the formation of a mononitrosyl-iron complex and releases intramolecular repression of the AAA+ domain to enable activation of transcription. In this study, we have further characterized NorR spectroscopically and substituted conserved residues in the GAF domain. This analysis, in combination with structural modeling of the GAF domain, has identified five candidate ligands to the non-heme iron and suggests a model in which the metal ion is coordinated in a pseudo-octahedral environment by three aspartate residues, an arginine, and a cysteine.

Escherichia coli cytochrome c nitrite reductase NrfA

The periplasmic cytochrome c nitrite reductase (Nrf) system of Escherichia coli utilizes nitrite as a respiratory electron acceptor by reducing it to ammonium. Nitric oxide (NO) is a proposed intermediate in this six-electron reduction and NrfA can use exogenous NO as a substrate. This chapter describes the method used to assay Nrf-catalyzed NO reduction in whole cells of E. coli and the procedures for preparing highly purified NrfA suitable for use in kinetic, spectroscopic, voltammetric, and crystallization studies.

Genome-Wide Identification of Binding Sites for the Nitric Oxide-Sensitive Transcriptional Regulator NsrR

NsrR is a nitric oxide-sensitive regulator of transcription. In Escherichia coli, NsrR is a repressor of the hmp gene encoding the flavohemoglobin that detoxifies nitric oxide. Three other transcription units (ytfE, ygbA, and hcp-hcr) are known to be subject to regulation by NsrR. This chapter describes experimental and statistical protocols used to identify NsrR-binding sites in the E. coli chromosome using chromatin immunoprecipitation and microarray analysis. The methods are applicable, with suitable modifications, to any regulatory protein and any organism.

Down-Regulation of the Escherichia coli K-12 nrf Promoter by Binding of the NsrR Nitric Oxide-Sensing Transcription Repressor to an Upstream Site

FNR-dependent activation of the Escherichia coli K-12 nrf promoter is downregulated by the nitric oxide-sensitive NsrR protein together with the nucleoid-associated protein IHF, which bind to overlapping targets adjacent to the DNA site for FNR. The NsrR target is inactivated by mutation at the Salmonella enterica serovar Typhimurium nrf promoter.