Highly diverse nirK genes comprise two major clades that harbour ammonium-producing denitrifiers

Background: Copper dependent nitrite reductase, NirK, catalyses the key step in denitrification, i.e. nitrite reduction to nitric oxide. Distinct structural NirK classes and phylogenetic clades of NirK-type denitrifiers have previously been observed based on a limited set of NirK sequences, however, their environmental distribution or ecological strategies are currently unknown. In addition, environmental nirK-type denitrifiers are currently underestimated in PCR-dependent surveys due to primer coverage limitations that can be attributed to their broad taxonomic diversity and enormous nirK sequence divergence. Therefore, we revisited reported analyses on partial NirK sequences using a taxonomically diverse, full-length NirK sequence dataset. Results: Division of NirK sequences into two phylogenetically distinct clades was confirmed, with Clade I mainly comprising Alphaproteobacteria (plus some Gamma- and Betaproteobacteria) and Clade II harbouring more diverse taxonomic groups like Archaea, Bacteroidetes, Chloroflexi, Gemmatimonadetes, Nitrospirae, Firmicutes, Actinobacteria, Planctomycetes and Proteobacteria (mainly Beta and Gamma). Failure of currently available primer sets to target diverse NirK-type denitrifiers in environmental surveys could be attributed to mismatches over the whole length of the primer binding regions including the 3' site, with Clade II sequences containing higher sequence divergence than Clade I sequences. Simultaneous presence of both the denitrification and DNRA pathway could be observed in 67 % of all NirK-type denitrifiers. Conclusion: The previously reported division of NirK into two distinct phylogenetic clades was confirmed using a taxonomically diverse set of full-length NirK sequences. Enormous sequence divergence of nirK gene sequences, probably due to variable nirK evolutionary trajectories, will remain an issue for covering diverse NirK-type denitrifiers in amplicon-based environmental surveys. The potential of a single organism to partition nitrate to either denitrification or dissimilatory nitrate reduction to ammonium appeared to be more widespread than originally anticipated as more than half of all NirK-type denitrifiers were shown to contain both pathways in their genome

Nitrous oxide metabolism in nitrate-reducing bacteria: Physiology and regulatory mechanisms

Nitrous oxide (N2O) is an important greenhouse gas (GHG) with substantial global warming potential and also contributes to ozone depletion through photochemical nit- ric oxide (NO) production in the stratosphere. The negative effects of N2O on climate and stratospheric ozone make N2O mitigation an international challenge. More than 60% of global N2O emissions are emitted from agricultural soils mainly due to the appli- cation of synthetic nitrogen-containing fertilizers. Thus, mitigation strategies must be developed which increase (or at least do not negatively impact) on agricultural effi- ciency whilst decrease the levels of N2O released. This aim is particularly important in the context of the ever expanding population and subsequent increased burden on the food chain. More than two-thirds of N2O emissions from soils can be attributed to bacterial and fungal denitrification and nitrification processes. In ammonia-oxidizing bacteria, N2O is formed through the oxidation of hydroxylamine to nitrite. In denitrifiers, nitrate is reduced to N2 via nitrite, NO and N2O production. In addition to denitrification, respiratory nitrate ammonification (also termed dissimilatory nitrate reduction to ammonium) is another important nitrate-reducing mechanism in soil, responsible for the loss of nitrate and production of N2O from reduction of NO that is formed as a by-product of the reduction process. This review will synthesize our current understand- ing of the environmental, regulatory and biochemical control of N2O emissions by nitrate-reducing bacteria and point to new solutions for agricultural GHG mitigation

An investigation of carbon and nitrogen metabolism through a genomic analysis of the genus Nitrobacter

The chemolithoautotrophic nitrite oxidizing bacteria (NOB) participate in the biogeochemical cycling of nitrogen by catalyzing and conserving energy from the oxidation of nitrite (NO₂-) to nitrate (NO₃-) via a nitrite oxidoreductase (NXR). The main objective of this work was to comparatively annotate and analyze the genome sequences of Nitrobacter winogradskyi NB255 and Nitrobacter hamburgensis X14 and use this information to extend our understanding of nitrogen and carbon metabolism in NOB. Through the analysis of the N. winogradskyi genome, genes encoding pathways for known modes of lithotrophic and heterotrophic growth were identified, including multiple enzymes involved in anapleurotic reactions centered on C2 to C4 metabolism. N. winogradskyi lacked genes encoding a complete glycolysis pathway and for the active transport of sugars. The N. hamburgensis genome harbored many genes not found in N. winogradskyi, including a complete glycolysis pathway, unique electron transport components, and putative pathways for the catabolism of aromatic, organic and one-carbon compounds. FAD-dependent oxidases were identified in the genome of N. hamburgensis which suggested that lactate could be metabolized, providing reductant and carbon to the cell. Indeed, D-lactate enhanced the growth rate and yield of N. hamburgensis in the presence of NO₂- and served as a sole energy and carbon source in the absence of NO₂-. Although lactate consumption occurred constitutively in lithoautotrophically grown cells, evidence was obtained for physiological adaptation to lactate. D-lactate grown cells consumed and assimilated lactate at a faster rate than NO₂- grown cells, and D-lactate-dependent O₂ uptake was significantly greater in cells grown heterotrophically or mixotrophically compared to cells grown lithoautotrophically. However, D-lactate could not substitute for CO₂ as the sole carbon source(lithoheterotrophy) during growth in the presence of NO₂-. Through a comparative analysis of the Nitrobacter 'core' genome, many genes involved in NO₂- metabolism were identified, including a dissimilatory nitrite reductase (NirK). The putative nirK in N. winogradskyi was maximally transcribed under low oxygen in the presence of NO₂- and transcription was not detected under anaerobic conditions. Although production of NO under aerobic conditions was not detected, NO was consumed in a cyanide sensitive process and reversibly inhibited NO₂-dependent O₂ uptake

The Respiratory Chain In Neisseria Species

This work presents the organization of respiratory chain in Neisseria species. The localization of redox proteins was determined. Lipid-modified azurin (Laz) and nitrite reductase (AniA) are mainly associated with outer membrane. All c-type cytochrome proteins are mainly associated with inner membrane. Cytochrome c5 is the major electron donor to AniA. Reduced form cytochrome c5 is able to donate electrons to AniA at a physiologically relevant rate. In addition, the second haem domain of cytochrome c5 is the direct donor to AniA. It presents a potential problem for inter-electron transfer between c5 and AniA, which are associated with inner and outer membrane respectively. Trihaem CcoP is the alternative electron donor to AniA in N. gonorrhoeae. The 3rd haem domain of N. gonorrhoeae CcoP is able to donate electrons to AniA at a physiologically relevant rate, suggesting there is alternative route for nitrite reduction in N gonorrhoeae. N. elongata cytochrome is an electron donor to AniA. N. elongata cytochrome which has high degree of similarity with c5, is confirmed to donate electrons to AniA at a physiologically relevant rate, suggesting N. elongata has one other route for nitrite reduction. Laz is not involved in nitrite reduction. Laz is able to receive electrons from cytochrome c5 at physiological relevant rate, but cannot donate electrons to AniA. Based on laz mutagenesis study, laz mutant strain has limited affect on growth and nitrite usage compared to the wild type strain. Cytochrome cx is not involved in oxygen reduction. Cytochrome cx has presumably been found to be involved in oxygen reduction in N. meningitidis, but not in N. gonorrhoeae. N. meningitidis carrying an N. gonorrhoeae ccoP gene has a similar growth rate as the growth rate of the wild type strain and also cx mutant strains

Producción de óxidos de nitrógeno gaseosos rn Thermus thermophilus

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 26-10-201