TPR domain of NrfG mediates complex formation between heme lyase and formate-dependent nitrite reductase in Escherichia coli O157:H7

Escherichia coli synthesize C-type cytochromes only during anaerobic growth in media supplemented with nitrate and nitrite. The reduction of nitrate to ammonium in the periplasm of Escherichia coli involves two separate periplasmic enzymes, nitrate reductase and nitrite reductase. The nitrite reductase involved, NrfA, contains cytochrome C and is synthesized coordinately with a membrane-associated cytochrome C, NrfB, during growth in the presence of nitrite or in limiting nitrate concentrations. The genes NrfE, NrfF, and NrfG are required for the formate-dependent nitrite reduction pathway, which involves at least two C-type cytochrome proteins, NrfA and NrfB. The NrfE, NrfF, and NrfG genes (heme lyase complex) are involved in the maturation of a special C-type cytochrome, apocytochrome C (apoNrfA), to cytochrome C (NrfA) by transferring a heme to the unusual heme binding motif of the Cys-Trp-Ser-Cys-Lys sequence in apoNrfA protein. Thus, in order to further investigate the roles of NrfG in the formation of heme lyase complex (NrfEFG) and in the interaction between heme lyase complex and formate-dependent nitrite reductase (NrfA), we determined the crystal structure of NrfG at 2.05 A. The structure of NrfG showed that the contact between heme lyase complex (NrfEFG) and NrfA is accomplished via a TPR domain in NrfG which serves as a binding site for the C-terminal motif of NrfA. The portion of NrfA that binds to TPR domain of NrfG has a unique secondary motif, a helix followed by about a six-residue C-terminal loop (the so called "hook conformation"). This study allows us to better understand the mechanism of special C-type cytochrome assembly during the maturation of formate-dependent nitrite reductase, and also adds a new TPR binding conformation to the list of TPR-mediated protein-protein interactions.     

Patterns and drivers of anaerobic nitrogen transformations in sediments of thermokarst lakes

Significant attention has been given to the way in which the soil nitrogen (N) cycle responds to permafrost thaw in recent years, yet little is known about anaerobic N transformations in thermokarst lakes, which account for more than one-third of thermokarst landforms across permafrost regions. Based on the N isotope dilution and tracing technique, combined with qPCR and high-throughput sequencing, we presented large-scale measurements of anaerobic N transformations of sediments across 30 thermokarst lakes over the Tibetan alpine permafrost region. Our results showed that gross N mineralization, ammonium immobilization, and dissimilatory nitrate reduction rates in thermokarst lakes were higher in the eastern part of our study area than in the west. Denitrification dominated in the dissimilatory nitrate reduction processes, being two and one orders of magnitude higher than anaerobic ammonium oxidation (anammox) and dissimilatory nitrate reduction to ammonium (DNRA), respectively. The abundances of the dissimilatory nitrate reduction genes (nirK, nirS, hzsB, and nrfA) exhibited patterns consistent with sediment N transformation rates, while α diversity did not. The inter-lake variability in gross N mineralization and ammonium immobilization was dominantly driven by microbial biomass, while the variability in anammox and DNRA was driven by substrate supply and organic carbon content, respectively. Denitrification was jointly affected by nirS abundance and organic carbon content. Overall, the patterns and drivers of anaerobic N transformation rates detected in this study provide a new perspective on potential N release, retention, and removal upon the formation and development of thermokarst lakes.     

Microbially facilitated nitrogen cycling in tropical corals

Tropical scleractinian corals support a diverse assemblage of microbial symbionts. This ‘microbiome’ possesses the requisite functional diversity to conduct a range of nitrogen (N) transformations including denitrification, nitrification, nitrogen fixation and dissimilatory nitrate reduction to ammonium (DNRA). Very little direct evidence has been presented to date verifying that these processes are active within tropical corals. Here we use a combination of stable isotope techniques, nutrient uptake calculations and captured metagenomics to quantify rates of nitrogen cycling processes in a selection of tropical scleractinian corals. Denitrification activity was detected in all species, albeit with very low rates, signifying limited importance in holobiont N removal. Relatively greater nitrogen fixation activity confirms that corals are net N importers to reef systems. Low net nitrification activity suggests limited N regeneration capacity; however substantial gross nitrification activity may be concealed through nitrate consumption. Based on nrfA gene abundance and measured inorganic N fluxes, we calculated significant DNRA activity in the studied corals, which has important implications for coral reef N cycling and warrants more targeted investigation. Through the quantification and characterisation of all relevant N-cycling processes, this study provides clarity on the subject of tropical coral-associated biogeochemical N-cycling.Subject terms: Stable isotope analysis, Biogeochemistry, Biogeochemistry, Biogeochemistry, Microbial ecology     

The nrfI gene is essential for the attachment of the active site haem group of Wolinella succinogenes cytochrome c nitrite reductase

The cytochrome c nitrite reductase complex (NrfHA) is the terminal enzyme in the electron transport chain catalysing nitrite respiration of Wolinella succinogenes. The catalytic subunit NrfA is a pentahaem cytochrome c containing an active site haem group which is covalently bound via the cysteine residues of a unique CWTCK motif. The lysine residue serves as the axial ligand of the haem iron. The other four haem groups of NrfA are bound by conventional haem-binding motifs (CXXCH). The nrfHAIJ locus was restored on the genome of the W. succinogenes DeltanrfAIJ deletion mutant by integration of a plasmid, thus enabling the expression of modified alleles of nrfA and nrfI. A mutant (K134H) was constructed which contained a nrfA gene encoding a CWTCH motif instead of CWTCK. NrfA of strain K134H was found to be synthesized with five bound haem groups, as judged by matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry. Its nitrite reduction activity with reduced benzyl viologen was 40% of the wild-type activity. Ammonia was formed as the only product of nitrite reduction. The mutant did not grow by nitrite respiration and its electron transport activity from formate to nitrite was 5% of that of the wild-type strain. The predicted nrfI gene product is similar to proteins involved in system II cytochrome c biogenesis. A mutant of W. succinogenes (stopI) was constructed that contained a nrfHAIJ gene cluster with the nrfI codons 47 and 48 altered to stop codons. The NrfA protein of this mutant did not catalyse nitrite reduction and lacked the active site haem group, whereas the other four haem groups were present. Mutant (K134H/stopI) which contained the K134H modification in NrfA in addition to the inactivated nrfI gene had essentially the same properties as strain K134H. NrfA from strain K134H/stopI contained five haem groups. It is concluded that NrfI is involved in haem attachment to the CWTCK motif in NrfA but not to any of the CXXCH motifs. The nrfI gene is obviously dispensable for maturation of a modified NrfA protein containing a CWTCH motif instead of CWTCK. Therefore, NrfI might function as a specific haem lyase that recognizes the active site lysine residue of NrfA. A similar role has been proposed for NrfE, F and G of Escherichia coli, although these proteins share no overall sequence similarity to NrfI and belong to system I cytochrome c biogenesis, which differs fundamentally from system II.     

A NapC/NirT-type cytochrome c (NrfH) is the mediator between the quinone pool and the cytochrome c nitrite reductase of Wolinella succinogenes

Wolinella succinogenes can grow by anaerobic respiration with nitrate or nitrite using formate as electron donor. Two forms of nitrite reductase were isolated from the membrane fraction of W. succinogenes. One form consisted of a 58 kDa polypeptide (NrfA) that was identical to the periplasmic nitrite reductase. The other form consisted of NrfA and a 22 kDa polypeptide (NrfH). Both forms catalysed nitrite reduction by reduced benzyl viologen, but only the dimeric form catalysed nitrite reduction by dimethylnaphthoquinol. Liposomes containing heterodimeric nitrite reductase, formate dehydrogenase and menaquinone catalysed the electron transport from formate to nitrite; this was coupled to the generation of an electrochemical proton potential (positive outside) across the liposomal membrane. It is concluded that the electron transfer from menaquinol to the catalytic subunit (NrfA) of W. succinogenes nitrite reductase is mediated by NrfH. The structural genes nrfA and nrfH were identified in an apparent operon (nrfHAIJ) with two additional genes. The gene nrfA encodes the precursor of NrfA carrying an N-terminal signal peptide (22 residues). NrfA (485 residues) is predicted to be a hydrophilic protein that is similar to the NrfA proteins of Sulfurospirillum deleyianum and of Escherichia coli. NrfH (177 residues) is predicted to be a membrane-bound tetrahaem cytochrome c belonging to the NapC/NirT family. The products of nrfI and nrfJ resemble proteins involved in cytochrome c biogenesis. The C-terminal third of NrfI (902 amino acid residues) is similar to CcsA proteins from Gram-positive bacteria, cyanobacteria and chloroplasts. The residual N-terminal part of NrfI resembles Ccs1 proteins. The deduced NrfJ protein resembles the thioredoxin-like proteins (ResA) of Helicobacter pylori and of Bacillus subtilis, but lacks the common motif CxxC of ResA. The properties of three deletion mutants of W. succinogenes (DeltanrfJ, DeltanrfIJ and DeltanrfAIJ) were studied. Mutants DeltanrfAIJ and DeltanrfIJ did not grow with nitrite as terminal electron acceptor or with nitrate in the absence of NH4+ and lacked nitrite reductase activity, whereas mutant DeltanrfJ showed wild-type properties. The NrfA protein formed by mutant DeltanrfIJ seemed to lack part of the haem C, suggesting that NrfI is involved in NrfA maturation.     

Habitat Segregation of a Functional Gene Encoding Nitrate Ammonification in Estuarine Sediments

Distribution and diversity of nrfA gene encoding dissimilatory nitrite reduction to ammonium (DNRA) in the sediments of the Colne River, North Essex, UK, were investigated. Sequencing cloned nrfA fragments amplified from environmental DNA enabled structure analysis of the bacterial community responsible for this pathway. The DNA was extracted from the sediment samples at different depths from the estuary ranging from freshwater to seawater regions, and amplified using specific PCR primer pairs targeting for the nrfA gene. Analysis of the nrfA clones showed two distinct clusters corresponding to their origins, namely, divided into the stable sites (marine and freshwater regions) and the unstable sites (brackish water region), where the tidal rise and fall constantly disturbs the environmental conditions. In addition, the nrfA clones from the deeper layer of the sediment formed a more homogenous community than those from the surface layer of the sediment. This may be due to more isolated and anaerobic conditions kept in the deeper sediment less influenced by the overlying water and other environmental factors. Most of the nrfA clones from the Colne estuarine sediments formed several distinct clusters including known nitrate ammonifiers such as Aeromonas, Shewanella, Desulfovibrio and Sulfurospillum. One of which was, however, related to Bacteroides but still quite divergent (～70% identity) and the rest forming unknown clusters of supposedly uncultured members of bacteria. This is the first trial to describe the nrfA partial sequences derived from a natural environment, with reference to their habitat-specific community structure.     

On the control mechanisms of the nitrite level in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells: the mathematical model

Background

Due to a high toxicity of nitrite and its metabolites, it is of high interest to study mechanisms underlying the low NO₂ level maintenance in the cell. During anaerobic growth of Escherichia coli the main nitrite-reducing enzymes are NrfA and NirB nitrite reductases. NrfA reductase is localized in the cell periplasm and uses NO₂ as an electron acceptor to create a proton gradient; NirB reductase is restricted to the cytoplasm and metabolizes excessive nitrite inside the cell, the uptake of which is mediated by the transporter protein NirC. While it is known that these three systems, periplasmic, cytoplasmic and transport, determine nitrite uptake and assimilation in the cell as well as its excretion, little is known about their co-ordination.

Results

Using a mathematical model describing the nitrite utilization in E. coli cells cultured in a flow chemostat, the role of enzymes involved in nitrite metabolism and transport in controlling nitrite intracellular levels was investigated. It was demonstrated that the model adapted to the experimental data on expression of nrfA and nirB genes encoding NrfA and NirB nitrite reductases, can describe nitrite accumulation kinetics in the chemostat in the millimolar range of added substrate concentrations without any additional assumptions. According to the model, in this range, low intracellular nitrite level, weakly dependent on its concentration in the growth media, is maintained (mcM). It is not sufficient to consider molecular-genetic mechanisms of NrfA reductase activity regulation to describe the nitrite accumulation dynamics in the chemostat in the micromolar range (≤1 mM) of added nitrite concentrations. Analysis of different hypotheses has shown that the mechanism of local enzyme concentration change due to membrane potential-induced diffusion from the cytoplasm to the periplasm at low nitrite levels is sufficient to explain the nitrite accumulation dynamics in the chemostat.

Conclusions

At nitrite concentrations in the media more than 2 mM, the model adapted to the experimental data on nitrite utilization dynamics in E. coli cells cultured in the flow chemostat demonstrates the largest contribution of genetic mechanisms involved in nrf and nir operons activity regulation to the control of nitrite intracellular levels. The model predicts a significant contribution of the membrane potential to the periplasmic NrfA nitrite reductase activity regulation and nitrite utilization dynamics at substrate concentrations ≤1 mM.

     

Characterization of the active site and calcium binding in cytochrome c nitrite reductases

The decahaem homodimeric cytochrome c nitrite reductase (NrfA) is expressed within the periplasm of a wide range of Gamma-, Delta- and Epsilon-proteobacteria and is responsible for the six-electron reduction of nitrite to ammonia. This allows nitrite to be used as a terminal electron acceptor, facilitating anaerobic respiration while allowing nitrogen to remain in a biologically available form. NrfA has also been reported to reduce nitric oxide (a reaction intermediate) and sulfite to ammonia and sulfide respectively, suggesting a potential secondary role as a detoxification enzyme. The protein sequences and crystal structures of NrfA from different bacteria and the closely related octahaem nitrite reductase from Thioalkalivibrio nitratireducens (TvNir) reveal that these enzymes are homologous. The NrfA proteins contain five covalently attached haem groups, four of which are bis-histidine-co-ordinated, with the proximal histidine being provided by the highly conserved CXXCH motif. These haems are responsible for intraprotein electron transfer. The remaining haem is the site for nitrite reduction, which is ligated by a novel lysine residue provided by a CXXCK haem-binding motif. The TvNir nitrite reductase has five haems that are structurally similar to those of NrfA and three extra bis-histidine-coordinated haems that precede the NrfA conserved region. The present review compares the protein sequences and structures of NrfA and TvNir and discusses the subtle differences related to active-site architecture and Ca2+ binding that may have an impact on substrate reduction.     

Nitrate respiration and diel migration patterns of diatoms are linked in sediments underneath a microbial mat

Diatoms are among the few eukaryotes known to store nitrate (NO₃⁻) and to use it as an electron acceptor for respiration in the absence of light and O₂. Using microscopy and ¹⁵N stable isotope incubations, we studied the relationship between dissimilatory nitrate/nitrite reduction to ammonium (DNRA) and diel vertical migration of diatoms in phototrophic microbial mats and the underlying sediment of a sinkhole in Lake Huron (USA). We found that the diatoms rapidly accumulated NO₃⁻ at the mat-water interface in the afternoon and 40% of the population migrated deep into the sediment, where they were exposed to dark and anoxic conditions for ~75% of the day. The vertical distribution of DNRA rates and diatom abundance maxima coincided, suggesting that DNRA was the main energy generating metabolism of the diatom population. We conclude that the illuminated redox-dynamic ecosystem selects for migratory diatoms that can store nitrate for respiration in the absence of light. A major implication of this study is that the dominance of DNRA over denitrification is not explained by kinetics or thermodynamics. Rather, the dynamic conditions select for migratory diatoms that perform DNRA and can outcompete sessile denitrifiers.     

Dissimilatory nitrate reduction by Pseudomonas alcaliphila with an electrode as the sole electron donor

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) were considered two alternative pathways of dissimilatory nitrate reduction. In this study, we firstly reported that both denitrification and DNRA occurred in Pseudomonas alcaliphila strain MBR with an electrode as the sole electron donor in a double chamber bio‐electrochemical system (BES). The initial concentration of nitrate appeared as a factor determining the type of nitrate reduction with electrode as the sole electron donor at the same potential (?500 mV). As the initial concentration of nitrate increased, the fraction of nitrate reduced through denitrification also increased. While nitrite (1.38 ± 0.04 mM) was used as electron acceptor instead of nitrate, the electrons recovery via DNRA and denitrification were 43.06 ± 1.02% and 50.51 ± 1.37%, respectively. The electrochemical activities and surface topography of the working electrode catalyzed by strain MBR were evaluated by cyclic voltammetry and scanning electron microscopy. The results suggested that cells of strain MBR were adhered to the electrode, playing the role of electron transfer media for nitrate and nitrite reduction. Thus, for the first time, the results that DNRA and denitrification occurred simultaneously were confirmed by powering the strain with electricity. The study further expanded the range of metabolic reactions and had potential value for the recognization of dissimilatory nitrate reduction in various ecosystems. Biotechnol. Bioeng. 2012; 109: 2904–2910. © 2012 Wiley Periodicals, Inc.     

Nitrogen‐cycling bacteria and archaea in the carbonate sediment of a coral reef

In the coarse‐grained carbonate sediments of coral reefs, advective porewater flow and the respiration of organic matter establish redox zones that are the scene of microbially mediated transformations of N compounds. To investigate the geobiology of N cycling in reef sediments, the benthic microbiota of Checker Reef in Kaneohe Bay, Hawaii, were surveyed for candidate nitrate reducers, ammonifying nitrite reducers, aerobic and anaerobic ammonia oxidizers (anammox) by identifying phylotypes of their key metabolic genes (napA, narG, nrfA, amoA) and ribotypes (unique RNA sequences) of anammox‐like 16S rRNA. Putative proteobacteria with the catalytic potential for nitrate reduction were identified in oxic, interfacial and anoxic habitats. The estimated richness of napA (≥202 in anoxic sediment) and narG (≥373 and ≥441 in oxic and interfacial sediment, respectively) indicates a diverse guild of nitrate reducers. The guild of nrfA hosts in interfacial reef sediment was dominated by Vibrio species. The identified members of the aerobic ammonium oxidizing guild (amoA hosts) were Crenarchaeota or close relatives of Nitrosomonadales. Putative anammox bacteria were detected in the RNA pool of Checker Reef sediment. More than half of these ribotypes show ≥90% identity with homologous sequences of Scalindua spp., while no evidence was found for members of the genera Brocadia or Kuenenia. In addition to exploring the diversity of these four nitrogen‐cycling microbial guilds in coral reef sediments, the abundances of aerobic ammonium oxidizers (amoA), nitrite oxidizers (nxrAB), ammonifying nitrite reducers (nrfA) and denitrifiers (nosZ) were estimated using real‐time PCR. Representatives of all targeted guilds were detected, suggesting that most processes of the biogeochemical N cycle can be catalyzed by the benthic microbiota of tropical coral reefs.     

On the control mechanisms of the nitrite level in Escherichia coli cells: the mathematical model

Background

Due to a high toxicity of nitrite and its metabolites, it is of high interest to study mechanisms underlying the low NO₂ level maintenance in the cell. During anaerobic growth of Escherichia coli the main nitrite-reducing enzymes are NrfA and NirB nitrite reductases. NrfA reductase is localized in the cell periplasm and uses NO₂ as an electron acceptor to create a proton gradient; NirB reductase is restricted to the cytoplasm and metabolizes excessive nitrite inside the cell, the uptake of which is mediated by the transporter protein NirC. While it is known that these three systems, periplasmic, cytoplasmic and transport, determine nitrite uptake and assimilation in the cell as well as its excretion, little is known about their co-ordination.

Results

Using a mathematical model describing the nitrite utilization in E. coli cells cultured in a flow chemostat, the role of enzymes involved in nitrite metabolism and transport in controlling nitrite intracellular levels was investigated. It was demonstrated that the model adapted to the experimental data on expression of nrfA and nirB genes encoding NrfA and NirB nitrite reductases, can describe nitrite accumulation kinetics in the chemostat in the millimolar range of added substrate concentrations without any additional assumptions. According to the model, in this range, low intracellular nitrite level, weakly dependent on its concentration in the growth media, is maintained (mcM). It is not sufficient to consider molecular-genetic mechanisms of NrfA reductase activity regulation to describe the nitrite accumulation dynamics in the chemostat in the micromolar range (≤1 mM) of added nitrite concentrations. Analysis of different hypotheses has shown that the mechanism of local enzyme concentration change due to membrane potential-induced diffusion from the cytoplasm to the periplasm at low nitrite levels is sufficient to explain the nitrite accumulation dynamics in the chemostat.

Conclusions

At nitrite concentrations in the media more than 2 mM, the model adapted to the experimental data on nitrite utilization dynamics in E. coli cells cultured in the flow chemostat demonstrates the largest contribution of genetic mechanisms involved in nrf and nir operons activity regulation to the control of nitrite intracellular levels. The model predicts a significant contribution of the membrane potential to the periplasmic NrfA nitrite reductase activity regulation and nitrite utilization dynamics at substrate concentrations ≤1 mM.

     

Enzymology and bioenergetics of respiratory nitrite ammonification

Nitrite is widely used by bacteria as an electron acceptor under anaerobic conditions. In respiratory nitrite ammonification an electrochemical proton potential across the membrane is generated by electron transport from a non-fermentable substrate like formate or H(2) to nitrite. The corresponding electron transport chain minimally comprises formate dehydrogenase or hydrogenase, a respiratory quinone and cytochrome c nitrite reductase. The catalytic subunit of the latter enzyme (NrfA) catalyzes nitrite reduction to ammonia without liberating intermediate products. This review focuses on recent progress that has been made in understanding the enzymology and bioenergetics of respiratory nitrite ammonification. High-resolution structures of NrfA proteins from different bacteria have been determined, and many nrf operons sequenced, leading to the prediction of electron transfer pathways from the quinone pool to NrfA. Furthermore, the coupled electron transport chain from formate to nitrite of Wolinella succinogenes has been reconstituted by incorporating the purified enzymes into liposomes. The NrfH protein of W. succinogenes, a tetraheme c-type cytochrome of the NapC/NirT family, forms a stable complex with NrfA in the membrane and serves in passing electrons from menaquinol to NrfA. Proteins similar to NrfH are predicted by open reading frames of several bacterial nrf gene clusters. In gamma-proteobacteria, however, NrfH is thought to be replaced by the nrfBCD gene products. The active site heme c group of NrfA proteins from different bacteria is covalently bound via the cysteine residues of a unique CXXCK motif. The lysine residue of this motif serves as an axial ligand to the heme iron thus replacing the conventional histidine residue. The attachment of the lysine-ligated heme group requires specialized proteins in W. succinogenes and Escherichia coli that are encoded by accessory nrf genes. The proteins predicted by these genes are unrelated in the two bacteria but similar to proteins of the respective conventional cytochrome c biogenesis systems.     

Nitrate Reduction Functional Genes and Nitrate Reduction Potentials Persist in Deeper Estuarine Sediments. Why?

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are processes occurring simultaneously under oxygen-limited or anaerobic conditions, where both compete for nitrate and organic carbon. Despite their ecological importance, there has been little investigation of how denitrification and DNRA potentials and related functional genes vary vertically with sediment depth. Nitrate reduction potentials measured in sediment depth profiles along the Colne estuary were in the upper range of nitrate reduction rates reported from other sediments and showed the existence of strong decreasing trends both with increasing depth and along the estuary. Denitrification potential decreased along the estuary, decreasing more rapidly with depth towards the estuary mouth. In contrast, DNRA potential increased along the estuary. Significant decreases in copy numbers of 16S rRNA and nitrate reducing genes were observed along the estuary and from surface to deeper sediments. Both metabolic potentials and functional genes persisted at sediment depths where porewater nitrate was absent. Transport of nitrate by bioturbation, based on macrofauna distributions, could only account for the upper 10 cm depth of sediment. A several fold higher combined freeze-lysable KCl-extractable nitrate pool compared to porewater nitrate was detected. We hypothesised that his could be attributed to intracellular nitrate pools from nitrate accumulating microorganisms like Thioploca or Beggiatoa. However, pyrosequencing analysis did not detect any such organisms, leaving other bacteria, microbenthic algae, or foraminiferans which have also been shown to accumulate nitrate, as possible candidates. The importance and bioavailability of a KCl-extractable nitrate sediment pool remains to be tested. The significant variation in the vertical pattern and abundance of the various nitrate reducing genes phylotypes reasonably suggests differences in their activity throughout the sediment column. This raises interesting questions as to what the alternative metabolic roles for the various nitrate reductases could be, analogous to the alternative metabolic roles found for nitrite reductases.     

Enrichment of DNRA bacteria in a continuous culture

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are competing microbial nitrate-reduction processes. The occurrence of DNRA has been shown to be effected qualitatively by various parameters in the environment. A more quantitative understanding can be obtained using enrichment cultures in a laboratory reactor, yet no successful DNRA enrichment culture has been described. We showed that a stable DNRA-dominated enrichment culture can be obtained in a chemostat system. The enrichment was based on the hypothesis that nitrate limitation is the dominant factor in selecting for DNRA. First, a conventional denitrifying culture was enriched from activated sludge, with acetate and nitrate as substrates. Next, the acetate concentration in the medium was increased to obtain nitrate-limiting conditions. As a result, conversions shifted from denitrification to DNRA. In this selection of a DNRA culture, two important factors were the nitrate limitation and a relatively low dilution rate (0.026 h⁻¹). The culture was a highly enriched population of Deltaproteobacteria most closely related to Geobacter lovleyi, based on 16S rRNA gene sequencing (97% similarity). We established a stable and reproducible cultivation method for the enrichment of DNRA bacteria in a continuously operated reactor system. This enrichment method allows to further investigate the DNRA process and address the factors for competition between DNRA and denitrification, or other N-conversion pathways.     

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.     

Modeling nitrogen cycling in a coastal fresh water sediment

Increased nitrogen (N) loading to coastal marine and freshwater systems is occurring worldwide as a result of human activities. Diagenetic processes in sediments can change the N availability in these systems, by supporting removal through denitrification and burial of organic N (N_org) or by enhancing N recycling. In this study, we use a reactive transport model (RTM) to examine N transformations in a coastal fresh water sediment and quantify N removal rates. We also assess the response of the sediment N cycle to environmental changes that may result from increased salinity which is planned to occur at the site as a result of an estuarine restoration project. Field results show that much of the N_org deposited on the sediment is currently remineralized to ammonium. A rapid removal of nitrate is observed in the sediment pore water, with the resulting nitrate reduction rate estimated to be 130 μmol N cm⁻² yr⁻¹. A model sensitivity study was conducted altering the distribution of nitrate reduction between dissimilatory nitrate reduction to ammonium (DNRA) and denitrification. These results show a 40% decline in sediment N removal as NO ₃ ⁻ reduction shifts from denitrification to DNRA. This decreased N removal leads to a shift in sediment-water exchange flux of dissolved inorganic nitrogen (DIN) from near zero with denitrification to 133 μmol N cm⁻² yr⁻¹ if DNRA is the dominant pathway. The response to salinization includes a short-term release of adsorbed ammonium. Additional changes expected to result from the estuarine restoration include: lower NO ₃ ⁻ concentrations and greater SO ₄ ²⁻ concentrations in the bottom water, decreased nitrification rates, and increased sediment mixing. The effect of these changes on net DIN flux and N removal vary based on the distribution of DNRA versus denitrification, illustrating the need for a better understanding of factors controlling this competition.