Nitrite Reductase NrfA Research Articles

Metabolic mechanism of Cr(VI) pollution remediation by Alicycliphilus denitrificans Ylb10

Cr(VI) is a well-known toxic pollutant and its remediation has attracted great attention. It is important to continuously discover and explore new high-efficiency Cr(VI) reducing bacteria to further improve the efficiency of Cr(VI) pollution remediation. In this paper, metabolic mechanism of Cr(VI) reduction in a new highly efficient Cr(VI) reducing bacterium, Alicycliphilus denitrificans Ylb10, was investigated. The results showed that Ylb10 could tolerate and completely reduce 450 mg/L Cr(VI). Cr(VI) can be reduced in the intracellular compartment, membrane and the extracellular compartment, with the plasma membrane being the main active site for Cr(VI) reduction. With the addition of NADH, the reduction efficiency of cell membrane components for Cr(VI) increased 2.3-fold. The omics data analysis showed that sulfite reductase CysJ, thiosulfate dehydrogenase TsdA, nitrite reductase NrfA, nitric oxide reductase NorB, and quinone oxidoreductase ChrR play important roles in the reduction of Cr(VI), in the intracellular, and the extracellular compartment, and the membrane of Ylb10, and therefore Cr(VI) was reduced by the combined action of several reductases at these three locations.

An in-field assembled hierarchical mesoporous electroenzymatic sensor for sensitive and real-time monitoring of nitrite

Electroenzymatic sensors are promising for nitrite detection but their application in real environmental scenarios is restricted due to the challenges in fabrication complexity and storage stability. Herein, we proposed a “drop & go” strategy to allow non-professionals to fabricate the electroenzymatic sensors in-field for monitoring the nitrite in real-time. To achieve this, hierarchical mesoporous ITO (mesoITO) electrode was prepared via a sol-gel process and a nitrite reductase NrfA originated from Shewanella oneidensis MR-1 was immobilized via a simple dropping process. Cyclic voltammetric (CV) analyses confirmed the direct electron transfer between NrfA and mesoITO electrode. After optimizing the enzyme dosage and electrochemical parameters, both CV and amperometric assays for nitrite detection were developed and a linear detection range of 10–130 μM and 1–140 μM nitrite were determined, respectively. Besides, these electroenzymatic sensors exhibited excellent resistance to the common anions in the water and good performance stability under working conditions. With this basis, the fabricated biosensors were applied for continuously monitoring the nitrite in a denitrifying process and the results were well-matched with the colorimetric method reference. More importantly, the presence of bacterial cells created favorable microenvironment for electroenzymatic nitrite reduction, and promoted the detection of trace nitrite in the bioreactor. This work developed a new methodology for fast electroenzymatic biosensor assembling, facilitate sensitive nitrite quantification in the real environment scenario, and may bring new concept for environment contaminants monitoring in real-time.

An unusual active site architecture in cytochrome c nitrite reductase NrfA-1 from Geobacter metallireducens.

Dissimilatory nitrate reduction to ammonia (DNRA) is a central pathway in the biogeochemical nitrogen cycle, allowing for the utilisation of nitrate or nitrite as terminal electron acceptors. In contrast to the competing denitrification to N2, a major part of the essential nutrient nitrogen in DNRA is retained within the ecosystem and made available as ammonium to serve as a nitrogen source for other organisms. The second step of DNRA is mediated by the pentahaem cytochrome c nitrite reductase NrfA that catalyses the six-electron reduction of nitrite to ammonium and is widely distributed among bacteria. A recent crystal structure of a NrfA ortholog from Geobacter lovleyi was the first characterised representative of a novel subclass of NrfA enzymes that lacked the canonical Ca2+ ion close to the active site haem 1. Here we report the structural and functional characterisation of NrfA from the closely related Geobacter metallireducens. We established the recombinant production of catalytically active NrfA with its unique, lysine-coordinated active site haem heterologously in Escherichia coli and determined its three-dimensional structure by X-ray crystallography to 1.9Å resolution. The structure confirmed GmNrfA as a further calcium-independent NrfA protein, and it also shows an altered active site that contained an unprecedented aspartate residue, D80, close to the substrate-binding site. This residue formed part of a loop that also caused a changed arrangement of the conserved substrate/product channel relative to other NrfA proteins and rendered the protein insensitive to the inhibitor sulphate. To elucidate the relevance of D80, we produced and studied the variants D80A and D80N that showed significantly reduced catalytic activity.

A New Paradigm of Multiheme Cytochrome Evolution by Grafting and Pruning Protein Modules.

Multiheme cytochromes play key roles in diverse biogeochemical cycles, but understanding the origin and evolution of these proteins is a challenge due to their ancient origin and complex structure. Up until now, the evolution of multiheme cytochromes composed by multiple redox modules in a single polypeptide chain was proposed to occur by gene fusion events. In this context, the pentaheme nitrite reductase NrfA and the tetraheme cytochrome c554 were previously proposed to be at the origin of the extant octa- and nonaheme cytochrome c involved in metabolic pathways that contribute to the nitrogen, sulfur, and iron biogeochemical cycles by a gene fusion event. Here, we combine structural and character-based phylogenetic analysis with an unbiased root placement method to refine the evolutionary relationships between these multiheme cytochromes. The evidence show that NrfA and cytochrome c554 belong to different clades, which suggests that these two multiheme cytochromes are products of truncation of ancestral octaheme cytochromes related to extant octaheme nitrite reductase and MccA, respectively. From our phylogenetic analysis, the last common ancestor is predicted to be an octaheme cytochrome with nitrite reduction ability. Evolution from this octaheme framework led to the great diversity of extant multiheme cytochromes analyzed here by pruning and grafting of protein modules and hemes. By shedding light into the evolution of multiheme cytochromes that intervene in different biogeochemical cycles, this work contributes to our understanding about the interplay between biology and geochemistry across large time scales in the history of Earth.

Optimization of PCR primers to detect phylogenetically diverse nrfA genes associated with nitrite ammonification

Dissimilatory nitrate reduction to ammonium (DNRA) is now known to be a more prevalent process in terrestrial ecosystems than previously thought. The key enzyme, a pentaheme cytochrome c nitrite reductase NrfA associated with respiratory nitrite ammonification, is encoded by the nrfA gene in a broad phylogeny of bacteria. The lack of reliable and comprehensive molecular tools to detect diverse nrfA from environmental samples has hampered efforts to meaningfully characterize the genetic potential for DNRA in environmental systems. In this study, modifications were made to optimize the amplification efficiency of previously-designed PCR primers, targeting the diagnostic region of NrfA between the conserved third- and fourth heme binding domains, and to increase coverage to include detection of environmentally relevant Geobacteraceae-like nrfA. Using an alignment of the primers to >270 bacterial nrfA genes affiliated with 18 distinct clades, modifications to the primer sequences improved coverage, minimized amplification artifacts, and yielded the predicted product sizes from reference-, soil-, and groundwater DNA. Illumina sequencing of amplicons showed the successful recovery of nrfA gene fragments from environmental DNA based on alignments of the translated sequences. The new primers developed in this study are more efficient in PCR reactions, although gene targets with high GC content affect efficiency. Furthermore, the primers have a broader spectrum of detection and were validated rigorously for use in detecting nrfA from natural environments. These are suitable for conventional PCR, qPCR, and use in PCR access array technologies that allow multiplex gene amplification for downstream high throughput sequencing platforms.

Respiratory Ammonification of Nitrate Coupled to Anaerobic Oxidation of Elemental Sulfur in Deep-Sea Autotrophic Thermophilic Bacteria.

Respiratory ammonification of nitrate is the microbial process that determines the retention of nitrogen in an ecosystem. To date, sulfur-dependent dissimilatory nitrate reduction to ammonium has been demonstrated only with sulfide as an electron donor. We detected a novel pathway that couples the sulfur and nitrogen cycles. Thermophilic anaerobic bacteria Thermosulfurimonas dismutans and Dissulfuribacter thermophilus, isolated from deep-sea hydrothermal vents, grew autotrophically with elemental sulfur as an electron donor and nitrate as an electron acceptor producing sulfate and ammonium. The genomes of both bacteria contain a gene cluster that encodes a putative nitrate ammonification enzyme system. Nitrate reduction occurs via a Nap-type complex. The reduction of produced nitrite to ammonium does not proceed via the canonical Nrf system because nitrite reductase NrfA is absent in the genomes of both microorganisms. The genome of D. thermophilus encodes a complete sulfate reduction pathway, while the Sox sulfur oxidation system is missing, as shown previously for T. dismutans. Thus, in high-temperature environments, nitrate ammonification with elemental sulfur may represent an unrecognized route of primary biomass production. Moreover, the anaerobic oxidation of sulfur compounds coupled to growth has not previously been demonstrated for the members of Thermodesulfobacteria or Deltaproteobacteria, which were considered exclusively as participants of the reductive branch of the sulfur cycle.

The cytochrome bd-I respiratory oxidase augments survival of multidrug-resistant Escherichia coli during infection.

Nitric oxide (NO) is a toxic free radical produced by neutrophils and macrophages in response to infection. Uropathogenic Escherichia coli (UPEC) induces a variety of defence mechanisms in response to NO, including direct NO detoxification (Hmp, NorVW, NrfA), iron-sulphur cluster repair (YtfE), and the expression of the NO-tolerant cytochrome bd-I respiratory oxidase (CydAB). The current study quantifies the relative contribution of these systems to UPEC growth and survival during infection. Loss of the flavohemoglobin Hmp and cytochrome bd-I elicit the greatest sensitivity to NO-mediated growth inhibition, whereas all but the periplasmic nitrite reductase NrfA provide protection against neutrophil killing and promote survival within activated macrophages. Intriguingly, the cytochrome bd-I respiratory oxidase was the only system that augmented UPEC survival in a mouse model after 2 days, suggesting that maintaining aerobic respiration under conditions of nitrosative stress is a key factor for host colonisation. These findings suggest that while UPEC have acquired a host of specialized mechanisms to evade nitrosative stresses, the cytochrome bd-I respiratory oxidase is the main contributor to NO tolerance and host colonisation under microaerobic conditions. This respiratory complex is therefore of major importance for the accumulation of high bacterial loads during infection of the urinary tract.

Molecular Underpinnings of Nitrite Effect on CymA-Dependent Respiration in Shewanella oneidensis.

Shewanella exhibit a remarkable versatility of respiration, with a diverse array of electron acceptors (EAs). In environments where these bacteria thrive, multiple EAs are usually present. However, we know little about strategies by which these EAs and their interaction affect ecophysiology of Shewanella. In this study, we demonstrate in the model strain, Shewanella oneidensis MR-1, that nitrite, not through nitric oxide to which it may convert, inhibits respiration of fumarate, and probably many other EAs whose reduction depends on quinol dehydrogenase CymA. This is achieved via the repression of cyclic adenosine monophosphate (cAMP) production, a second messenger required for activation of cAMP-receptor protein (Crp) which plays a primary role in regulation of respiration. If nitrite is not promptly removed, intracellular cAMP levels drop, and this impairs Crp activity. As a result, the production of nitrite reductase NrfA, CymA, and fumarate reductase FccA is substantially reduced. In contrast, nitrite can be simultaneously respired with trimethylamine N-oxide, resulting in enhanced biomass.

Seasonal variation in denitrification and dissimilatory nitrate reduction to ammonia process rates and corresponding key functional genes along an estuarine nitrate gradient.

This research investigated spatial-temporal variation in benthic bacterial community structure, rates of denitrification and dissimilatory nitrate reduction to ammonium (DNRA) processes and abundances of corresponding genes and transcripts at three sites—the estuary-head, mid-estuary and the estuary mouth (EM) along the nitrate gradient of the Colne estuary over an annual cycle. Denitrification rates declined down the estuary, while DNRA rates were higher at the estuary head and middle than the EM. In four out of the six 2-monthly time-points, rates of DNRA were greater than denitrification at each site. Abundance of gene markers for nitrate-reduction (nitrate reductase narG and napA), denitrification (nitrite reductase nirS) and DNRA (DNRA nitrite reductase nrfA) declined along the estuary with significant relationships between denitrification and nirS abundance, and DNRA and nrfA abundance. Spatially, rates of denitrification, DNRA and corresponding functional gene abundances decreased along the estuary. However, temporal correlations between rate processes and functional gene and transcript abundances were not observed.

Shewanella oneidensis cytochrome c maturation component CcmI is essential for heme attachment at the non-canonical motif of nitrite reductase NrfA.

Shewanella oneidensis is renowned for its respiratory versatility, which is largely due to abundant c-type cytochromes. Maturation of these proteins depends on a Ccm system encoded by genes in an unusual chromosomal arrangement, but the detailed mechanism is not understood. In this study, we identify SO0265 as CcmI, an apocytochrome c chaperone that is important and essential for maturation of c-type cytochromes with the canonical heme binding motif(s) (HBM; CX(2)CH) and nitrite reductase NrfA carrying a non-canonical CX(2)CK motif respectively. We show that the N-terminal transmembrane segment of CcmI, CcmI-1, is sufficient for maturation of the former but the entire protein is required for maturation of the latter. Although S. oneidensis possesses a heme lyase, SirEFG, dedicated for non-canonical HBMs, it is specific for SirA, a sulfite reductase with a CX(15)CH motif. By presenting evidence that the periplasmic portion of CcmI, CcmI-2, interacts with NrfA, we suggest that CcmI also takes the role of Escherichia coli NrfG for chaperoning apo-NrfA for maturation at CX(2)CK. Moreover, intact CcmI is required for maturation of NrfA, presumably by ensuring that heme attachment at canonical HBMs occurs before apoprotein degradation.

Contribution of the NO-detoxifying enzymes HmpA, NorV and NrfA to nitrosative stress protection of Salmonella Typhimurium in raw sausages

The antimicrobial action of the curing agent sodium nitrite (NaNO2) in raw sausage fermentation is thought to mainly depend on the release of cytotoxic nitric oxide (NO) at acidic pH. Salmonella Typhimurium is capable of detoxifying NO via the flavohemoglobin HmpA, the flavorubredoxin NorV and the periplasmic cytochrome C nitrite reductase NrfA. In this study, the contribution of these systems to nitrosative stress tolerance in raw sausages was investigated. In vitro growth assays of the S. Typhimurium 14028 deletion mutants ΔhmpA, ΔnorV and ΔnrfA revealed a growth defect of ΔhmpA in the presence of acidified NaNO2. Transcriptional analysis of the genes hmpA, norV and nrfA in the wild-type showed a 41-fold increase in hmpA transcript levels in the presence of 150 mg/l acidified NaNO2, whereas transcription of norV and nrfA was not enhanced. However, challenge assays performed with short-ripened spreadable sausages produced with 0 or 150 mg/kg NaNO2 failed to reveal a phenotype for any of the mutants compared to the wild-type. Hence, none of the NO detoxification systems HmpA, NorV and NrfA is solely responsible for nitrosative stress tolerance of S. Typhimurium in raw sausages. Whether these systems act cooperatively, or if there are other yet undescribed mechanisms involved is currently unknown.

Refined NrfA phylogeny improves PCR-based nrfA gene detection.

Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are contrasting microbial processes in the terrestrial nitrogen (N) cycle, in that the former promotes N retention and the latter leads to N loss (i.e., the formation of gaseous products). The nitrite reductase NrfA catalyzes nitrite reduction to ammonium, the enzyme associated with respiratory nitrite ammonification and the key step in DNRA. Although well studied biochemically, the diversity and phylogeny of this enzyme had not been rigorously analyzed. A phylogenetic analysis of 272 full-length NrfA protein sequences distinguished 18 NrfA clades with robust statistical support (>90% Bayesian posterior probabilities). Three clades possessed a CXXCH motif in the first heme-binding domain, whereas all other clades had a CXXCK motif in this location. The analysis further identified a KXRH or KXQH motif between the third and fourth heme-binding motifs as a conserved and diagnostic feature of all pentaheme NrfA proteins. PCR primers targeting a portion of the heme-binding motifs that flank this diagnostic region yielded the expected 250-bp-long amplicons with template DNA from eight pure cultures and 16 new nrfA-containing isolates. nrfA amplicons obtained with template DNA from two geomorphically distinct agricultural soils could be assigned to one of the 18 NrfA clades, providing support for this expanded classification. The extended NrfA phylogeny revealed novel diagnostic features of DNRA populations and will be useful to assess nitrate/nitrite fate in natural and engineered ecosystems.

Do Globins in Microaerophilic Campylobacter jejuni Confer Nitrosative Stress Tolerance Under Oxygen Limitation?

The microaerophilic pathogen Campylobacter jejuni possesses inducible systems for resisting NO. Two globins--Cgb (a single-domain globin) and Ctb (a truncated globin)--are up-regulated in response to NO via the positively acting transcription factor NssR. Our aims were to determine whether these oxygen-binding globins also function in severely oxygen-limited environments, as in the host. At growth-limiting oxygen transfer rates, bacteria were more S-nitrosoglutathione (GSNO) sensitive, irrespective of the presence of Cgb, Ctb, or NssR. Pregrowth of cells with GSNO enhanced GSNO resistance, even in nssR and cgb mutants, but transcriptomic profiling of oxygen-limited, NO-exposed cells failed to reveal the NssR regulon. Nevertheless, globin expression in an Escherichia coli mutant lacking the NO-detoxifying flavohemoglobin Hmp showed that Cgb and Ctb consume NO aerobically or anoxically and offer some protection to respiratory inhibition by NO. The constitutively expressed nitrite reductase NrfA does not provide resistance under oxygen-limited conditions. We, therefore, hypothesize that, although Cgb and NrfA can detoxify NO, even anoxically, they are neither up-regulated nor functional under physiologically relevant oxygen-limited conditions and, second, responses to NO do not stem from trancriptional regulation.

Molecular interactions between multihaem cytochromes: probing the protein–protein interactions between pentahaem cytochromes of a nitrite reductase complex

The cytochrome c nitrite reductase NrfA is a 53kDa pentahaem enzyme that crystallizes as a decahaem homodimer. NrfA catalyses the reduction of NO2- to NH4+ through a six electron reduction pathway that is of major physiological significance to the anaerobic metabolism of enteric and sulfate reducing bacteria. NrfA receives electrons from the 21kDa pentahaem NrfB donor protein. This requires that redox complexes form between the NrfA and NrfB pentahaem cytochromes. The formation of these complexes can be monitored using a range of methodologies for studying protein-protein interactions, including dynamic light scattering, gel filtration, analytical ultracentrifugation and visible spectroscopy. These methods have been used to show that oxidized NrfA exists in dynamic monomer-dimer equilibrium with a Kd (dissociation constant) of 4 μM. Significantly, the monomeric and dimeric forms of NrfA are equally active for either the six electron reduction of NO2- or HSO3-. When mixed together, NrfA and NrfB exist in equilibrium with NrfAB, which is described by a Kd of 50nM. Thus, since NrfA and NrfB are present in micromolar concentrations in the periplasmic compartment, it is likely that NrfB remains tightly associated with its NrfA redox partner under physiological conditions.

The Nitric Oxide Reductase Activity of Cytochrome c Nitrite Reductase from Escherichia coli

Cytochrome c nitrite reductase (NrfA) from Escherichia coli has a well established role in the respiratory reduction of nitrite to ammonium. More recently the observation that anaerobically grown E. coli nrf mutants were more sensitive to NO. than the parent strain led to the proposal that NrfA might also participate in NO. detoxification. Here we describe protein film voltammetry that presents a quantitative description of NrfA NO. reductase activity. NO. reduction is initiated at similar potentials to NrfA-catalyzed reduction of nitrite and hydroxylamine. All three activities are strongly inhibited by cyanide. Together these results suggest a common site for reduction of all three substrates as axial ligands to the lysine-coordinated NrfA heme rather than nonspecific NO. reduction at one of the four His-His coordinated hemes also present in each NrfA subunit. NO. reduction by NrfA is described by a K(m) of the order of 300 microm. The predicted turnover number of approximately 840 NO. s(-1) is much higher than that of the dedicated respiratory NO. reductases of denitrification and the flavorubredoxin and flavohemoglobin of E. coli that are also proposed to play roles in NO. detoxification. In considering the manner by which anaerobically growing E. coli might detoxify exogenously generated NO. encountered during invasion of a human host it appears that the periplasmically located NrfA should be effective in maintaining low NO. levels such that any NO. reaching the cytoplasm is efficiently removed by flavorubredoxin (K(m) approximately 0.4 microm).

The crystal structure of the pentahaem c-type cytochrome NrfB and characterization of its solution-state interaction with the pentahaem nitrite reductase NrfA

NrfB is a small pentahaem electron-transfer protein widely involved in the respiratory reduction of nitrite or nitric oxide to ammonia, processes that provide energy for anaerobic metabolism in many enteric bacteria and also serve to detoxify these reactive nitrogen species. The X-ray crystal structure of Escherichia coli NrfB is presented at 1.74 A (1 A=0.1 nm) resolution. The architecture of the protein is that of a 40 A 'nanowire' in which the five haems are positioned within 6 A of each other along a polypeptide scaffold. During nitrite reduction, the physiological role of NrfB is to mediate electron transfer to another pentahaem protein, NrfA, the enzyme that catalyses periplasmic nitrite or nitric oxide reduction. Protein-protein interaction studies suggest NrfA and NrfB can form a 20-haem NrfA2-NrfB2 heterotetrameric complex.

X-ray structure of the membrane-bound cytochrome c quinol dehydrogenase NrfH reveals novel haem coordination

Oxidation of membrane-bound quinol molecules is a central step in the respiratory electron transport chains used by biological cells to generate ATP by oxidative phosphorylation. A novel family of cytochrome c quinol dehydrogenases that play an important role in bacterial respiratory chains was recognised in recent years. Here, we describe the first structure of a cytochrome from this family, NrfH from Desulfovibrio vulgaris, which forms a stable complex with its electron partner, the cytochrome c nitrite reductase NrfA. One NrfH molecule interacts with one NrfA dimer in an asymmetrical manner, forming a large membrane-bound complex with an overall alpha(4)beta(2) quaternary arrangement. The menaquinol-interacting NrfH haem is pentacoordinated, bound by a methionine from the CXXCHXM sequence, with an aspartate residue occupying the distal position. The NrfH haem that transfers electrons to NrfA has a lysine residue from the closest NrfA molecule as distal ligand. A likely menaquinol binding site, containing several conserved and essential residues, is identified.

Growth of Campylobacter jejuni on nitrate and nitrite: electron transport to NapA and NrfA via NrfH and distinct roles for NrfA and the globin Cgb in protection against nitrosative stress

Pathways of electron transport to periplasmic nitrate (NapA) and nitrite (NrfA) reductases have been investigated in Campylobacter jejuni, a microaerophilic food-borne pathogen. The nap operon is unusual in lacking napC (encoding a tetra-haem c-type cytochrome) and napF, but contains a novel gene of unknown function, napL. The iron-sulphur protein NapG has a major role in electron transfer to the NapAB complex, but we show that slow nitrate-dependent growth of a napG mutant can be sustained by electron transfer from NrfH, the electron donor to the nitrite reductase NrfA. A napL mutant possessed approximately 50% lower NapA activity than the wild type but showed normal growth with nitrate as the electron acceptor. NrfA was constitutive and was shown to play a role in protection against nitrosative stress in addition to the previously identified NO-inducible single domain globin, Cgb. However, nitrite also induced cgb expression in an NssR-dependent manner, suggesting that growth of C. jejuni with nitrite causes nitrosative stress. This was confirmed by lack of growth of cgb and nssR mutants, and slow growth of the nrfA mutant, in media containing nitrite. Thus, NrfA and Cgb together provide C. jejuni with constitutive and inducible components of a robust defence against nitrosative stress.

The histidine of the c-type cytochrome CXXCH haem-binding motif is essential for haem attachment by the Escherichia coli cytochrome c maturation (Ccm) apparatus

c-type cytochromes are characterized by covalent attachment of haem to the protein by two thioether bonds formed between the haem vinyl groups and the cysteine sulphurs in a CXXCH peptide motif. In Escherichia coli and many other Gram-negative bacteria, this post-translational haem attachment is catalysed by the Ccm (cytochrome c maturation) system. The features of the apocytochrome substrate required and recognized by the Ccm apparatus are uncertain. In the present study, we report investigations of maturation of cytochrome b562 variants containing CXXCR, CXXCK or CXXCM haem-binding motifs. None of them showed any evidence for correct maturation by the Ccm system. However, we have determined, for each variant, that the proteins (i) were expressed in large amounts, (ii) could bind haem in vivo and/or in vitro and (iii) were not degraded in the cell. Together with previous observations, these results strongly suggest that the apocytochrome substrate feature recognized by the Ccm system is simply the two cysteine residues and the histidine of the CXXCH haem-binding motif. Using the same experimental approach, we have also investigated a cytochrome b562 variant containing the special CWSCK motif that binds the active-site haem of E. coli nitrite reductase NrfA. Whereas a CWSCH analogue was matured by the Ccm apparatus in large amounts, the CWSCK form was not detectably matured either by the Ccm system or by the dedicated Nrf biogenesis proteins, implying that the substrate recognition features for haem attachment in NrfA may be more extensive than the CWSCK motif.

New Genes Implicated in the Protection of Anaerobically Grown Escherichia coli against Nitric Oxide

Nitric oxide produced by activated macrophages plays a key role as one of the immune system's weapons against pathogens. Because the lifetime of nitric oxide is short in aerobic conditions, whereas in anaerobic conditions the cytotoxic effects of nitric oxide are greatly increased as in the infection/inflammation processes, it is important to establish which systems are able to detoxify nitric oxide under anaerobic conditions. In the present work a new set of Escherichia coli K-12 genes conferring anaerobic resistance to nitric oxide is presented, namely the gene product of YtfE and a potential transcriptional regulator of the helix-turn-helix LysR-type (YidZ). The crucial role of flavohemoglobin for anaerobic nitric oxide protection is also demonstrated. Furthermore, nitric oxide is shown to cause a significant alteration of the global E. coli gene transcription profile that includes the increase of the transcript level of genes encoding for detoxification enzymes, iron-sulfur cluster assembly systems, DNA-repairing enzymes, and stress response regulators.