Structure and sequence conservation of hao cluster genes of autotrophic ammonia-oxidizing bacteria: evidence for their evolutionary history

Comparison of the organization and sequence of the hao (hydroxylamine oxidoreductase) gene clusters from the gammaproteobacterial autotrophic ammonia-oxidizing bacterium (aAOB) Nitrosococcus oceani and the betaproteobacterial aAOB Nitrosospira multiformis and Nitrosomonas europaea revealed a highly conserved gene cluster encoding the following proteins: hao, hydroxylamine oxidoreductase; orf2, a putative protein; cycA, cytochrome c(554); and cycB, cytochrome c(m)(552). The deduced protein sequences of HAO, c(554), and c(m)(552) were highly similar in all aAOB despite their differences in species evolution and codon usage. Phylogenetic inference revealed a broad family of multi-c-heme proteins, including HAO, the pentaheme nitrite reductase, and tetrathionate reductase. The c-hemes of this group also have a nearly identical geometry of heme orientation, which has remained conserved during divergent evolution of function. High sequence similarity is also seen within a protein family, including cytochromes c(m)(552), NrfH/B, and NapC/NirT. It is proposed that the hydroxylamine oxidation pathway evolved from a nitrite reduction pathway involved in anaerobic respiration (denitrification) during the radiation of the Proteobacteria. Conservation of the hydroxylamine oxidation module was maintained by functional pressure, and the module expanded into two separate narrow taxa after a lateral gene transfer event between gamma- and betaproteobacterial ancestors of extant aAOB. HAO-encoding genes were also found in six non-aAOB, either singly or tandemly arranged with an orf2 gene, whereas a c(554) gene was lacking. The conservation of the hao gene cluster in general and the uniqueness of the c(554) gene in particular make it a suitable target for the design of primers and probes useful for molecular ecology approaches to detect aAOB.     

Mutational Analysis of the Multicopy hao Gene Coding for Hydroxylamine Oxidoreductase in Nitrosomonas sp. Strain ENI-11

The ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 contains three copies of the hao gene (hao ₁, hao ₂, and hao ₃) coding for hydroxylamine oxidoreductase (HAO). Three single mutants (hao ₁::kan, hao ₂::kan, or hao ₃::kan) had 68 to 75% of the wild-type growth rate and 58 to 89% of the wild-type HAO activity when grown under the same conditions. A double mutant (hao ₁::kan and hao ₃::amp) also had 68% of the wild-type growth and 37% of the wild-type HAO activity.     

Electron transfer during the oxidation of ammonia by the chemolithotrophic bacterium Nitrosomonas europaea

The combined action of ammonia monooxygenase, AMO, (NH(3)+2e(-)+O(2)-->NH(2)OH) and hydroxylamine oxidoreductase, HAO, (NH(2)OH+H(2)O-->HNO(2)+4e(-)+4H(+)) accounts for ammonia oxidation in Nitrosomonas europaea. Pathways for electrons from HAO to O(2), nitrite, NO, H(2)O(2) or AMO are reviewed and some recent advances described. The membrane cytochrome c(M)552 is proposed to participate in the path between HAO and ubiquinone. A bc(1) complex is shown to mediate between ubiquinol and the terminal oxidase and is shown to be downstream of HAO. A novel, red, low-potential, periplasmic copper protein, nitrosocyanin, is introduced. Possible mechanisms for the inhibition of ammonia oxidation in cells by protonophores are summarized. Genes for nitrite- and NO-reductase but not N(2)O or nitrate reductase are present in the genome of Nitrosomonas. Nitrite reductase is not repressed by growth on O(2); the flux of nitrite reduction is controlled at the substrate level.     

Laboratoire de Chimie Bactérienne C.N.R.S., Marsielle, France.

Summary Mutants of E. coli, completely devoid of nitrite reductase activity with glucose or formate as donor were studied. Biochemical analysis indicates that they are simultaneously affected in nitrate reductase, nitrite reductase, fumarate reductase and hydrogenase activities as well as in cytochrome c₅₅₂ biosynthesis. The use of an antiserum specific for nitrate reductase shows that the nitrate reductase protein is probably missing. A single mutation is responsible for this phenotype: the gene affected, nir R, is located close to tyr R i.e. at 29 min on the chromosomal map.Abbreviations BV Benzyl-Viologen - NTG N-methyl-N-nitro-N-nitrosoguanidine - NR nitrate reductase - NIR nitrite reductase - FR fumarate reductase - HYD hydrogenase - CYT c₅₅₂ cytochrome c₅₅₂     

Parallel Pathways for Nitrite Reduction during Anaerobic Growth in Thermus thermophilus

Respiratory reduction of nitrate and nitrite is encoded in Thermus thermophilus by the respective transferable gene clusters. Nitrate is reduced by a heterotetrameric nitrate reductase (Nar) encoded along transporters and regulatory signal transduction systems within the nitrate respiration conjugative element (NCE). The nitrite respiration cluster (nic) encodes homologues of nitrite reductase (Nir) and nitric oxide reductase (Nor). The expression and role of the nirSJM genes in nitrite respiration were analyzed. The three genes are expressed from two promoters, one (nirSp) producing a tricistronic mRNA under aerobic and anaerobic conditions and the other (nirJp) producing a bicistronic mRNA only under conditions of anoxia plus a nitrogen oxide. As for its nitrite reductase homologues, NirS is expressed in the periplasm, has a covalently bound heme c, and conserves the heme d₁ binding pocket. NirJ is a cytoplasmic protein likely required for heme d₁ synthesis and NirS maturation. NirM is a soluble periplasmic homologue of cytochrome c₅₅₂. Mutants defective in nirS show normal anaerobic growth with nitrite and nitrate, supporting the existence of an alternative Nir in the cells. Gene knockout analysis of different candidate genes did not allow us to identify this alternative Nir protein but revealed the requirement for Nar in NirS-dependent and NirS-independent nitrite reduction. As the likely role for Nar in the process is in electron transport through its additional cytochrome c periplasmic subunit (NarC), we concluded all the Nir activity takes place in the periplasm by parallel pathways.     

Multiple copies of genes coding for electron transport proteins in the bacterium Nitrosomonas europaea.

The genome of Nitrosomonas europaea contains at least three copies each of the genes coding for hydroxylamine oxidoreductase (HAO) and cytochrome c554. A copy of an HAO gene is always located within 2.7 kb of a copy of a cytochrome c554 gene. Cytochrome P-460, a protein that shares very unusual spectral features with HAO, was found to be encoded by a gene separate from the HAO genes.     

A myxothiazol-sensitive Q-binding protein isolated from Chromatium vinosum

A quinol: ferricytochrome c oxidoreductase has been isolated from chromatophores of Chromatium vinosum by two procedures, involving extraction by bile salts and methanol, respectively. The steady-state kinetics indicate a random mechanism, with a K_m for 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol of 1.1 μM and for the acceptor cytochrome c 1.75 μM. The enzyme is inhibited by myxothiazol, competitively with respect to quinol, with a K_i of about 2.3 μM. The protein reacts with ubiquinol produced by the succinate: Q oxidoreductase in submitochondrial particles or isolated succinate: cytochrome c reductase and can partially restore activity to myxothiazol-inhibited, antimycin-sensitive ubiquinol: cytochrome c oxidoreductase. The protein is considered to be analogous to the postulated myxothiazol-sensitive Q-binding protein in ubiquinol: cytochrome c oxidoreductase.     

Evidence for a crosslink between c-heme and a lysine residue in cytochrome P460 of Nitrosomonas europaea

Cytochrome P460 and hydroxylamine oxidoreductase (HAO) of Nitrosomonas europaea catalyze the oxidation of hydroxylamine. Cytochrome P460 contains an unidentified heme-like chromophore whose distinctive spectroscopic properties are similar to those for the P460 heme found in HAO. The heme P460 of HAO has previously been shown by protein chemistry and NMR structural analysis to be a c-heme with an additional covalent crosslink between the C2 ring carbon of a tyrosine residue of the polypeptide chain and a meso carbon of the porphyrin [Arciero, D.M. et al. (1993) Biochemistry 32, 9370–9378]. The recent determination of the gene sequence for cytochrome P460 [Bergmann, D.J. and Hooper, A.B. (1994) FEBS Lett. 353, 324–326] indicates that the heme in this protein also possesses a c-heme binding site and provides the basis for determining whether an HAO-like crosslink exists to the porphyrin.Sequence analysis of a purified heme-containing tryptic chromopeptide from cytochrome P460 revealed two predominant amino acid residues per cycle. Two peptides present in the chromopeptide with the sequences NLPTAEXAAXHK and DGTVTVXELVSV. Comparison of the data to the gene sequence for the protein revealed that the gaps in the first peptide (indicated by X's) code for C residues, confirming the prediction of a c-heme binding motif. The gap in the sequence in the second peptide at cycle 7 is predicted by the gene sequence to be a K. The results suggest that the lysine residue is crosslinked in some manner to the porphyrin macrocycle, possibly mimicking the tyrosine crosslink found for the heme P460 of HAO. While a common role for the crosslinked residues in HAO and cytochrome P460 is difficult to ascertain due to the dissimilarities in side chain structure, it may be related to the similar pK_a values for lysine and tyrosine.     

Isolation of Paracoccus denitrificans cytochrome cd1: comparative kinetics with other nitrite reductases

The dissimilatory nitrite reductase of the cytochrome cd₁ type was purified from Paracoccus denitrificans (ATCC 13543) by a novel procedure that avoided conventional ion-exchange techniques. The characterization of this enzyme was extended to include amino acid composition, extinction coefficients, and kinetic properties not previously reported. Cytochromes cd₁ from Alicaligenes faecalis and Pseudomonas aeruginosa were also isolated and assayed with electron donor proteins. The enzymes from all three sources were shown to obey the same integrated rate law. Cross-reactivities were measured in which a reduced donor protein from one strain was assayed with cytochrome cd₁ from another strain using nitrite as ultimate acceptor. Donors included c-type cytochromes and azurins. In general, the enzymes showed specificity for a donor from the same strain; interspecies cross-reactions were typically slower on the order of 10-fold than corresponding native rates. Notable exceptions were Paracoccus cytochrome cd₁, which alone reacted with eukaryotic horse cytochrome c at appreciable rates, and the Pseudomonas cd₁-Alcaligenesc554 reaction, which was 4-fold faster than the native Alcaligenes cd₁-Alcaligenesc554 reaction. For all three enzymes, competitive kinetics were measured in which the alternative substrates, nitrite and oxygen, competed for enzyme in the same assay. It was found that the competitive kinetics were dominated by nonenzymatic reactions involving an enzyme product, nitric oxide.     

Epsilonproteobacterial hydroxylamine oxidoreductase (εHao): characterization of a ‘missing link’ in the multihaem cytochrome c family

Members of the multihaem cytochrome c family such as pentahaem cytochrome c nitrite reductase (NrfA) or octahaem hydroxylamine oxidoreductase (Hao) are involved in various microbial respiratory electron transport chains. Some members of the Hao subfamily, here called εHao proteins, have been predicted from the genomes of nitrate/nitrite‐ammonifying bacteria that usually lack NrfA. Here, εHao proteins from the host‐associated Epsilonproteobacteria Campylobacter fetus and Campylobacter curvus and the deep‐sea hydrothermal vent bacteria Caminibacter mediatlanticus and Nautilia profundicola were purified as εHao‐maltose binding protein fusions produced in Wolinella succinogenes. All four proteins were able to catalyze reduction of nitrite (yielding ammonium) and hydroxylamine whereas hydroxylamine oxidation was negligible. The introduction of a tyrosine residue at a position known to cause covalent trimerization of Hao proteins did neither stimulate hydroxylamine oxidation nor generate the Hao‐typical absorbance maximum at 460 nm. In most cases, the εHao‐encoding gene haoA was situated downstream of haoC, which predicts a tetrahaem cytochrome c of the NapC/NrfH family. This suggested the formation of a membrane‐bound HaoCA assembly reminiscent of the menaquinol‐oxidizing NrfHA complex. The results indicate that εHao proteins form a subfamily of ammonifying cytochrome c nitrite reductases that represents a ‘missing link’ in the evolution of NrfA and Hao enzymes.     

Cytochrome components of denitrifyingPseudomonas stutzeri

Cytochromesc-551,c-552,c-554,cd, ac type with low α/β peaks, and an acidicc-type cytochrome were detected in extracts ofPseudomonas stutzeri. The first four were purified and physically characterized. Light absorption spectra indicate a probably histidine-methionine liganding of heme iron inc-551 andc-554, but an absence of methionine in the ligand ofc-552 heme iron. In displaying two separately reduciblec-hemes, thec-552 appears homologous with that fromP. perfectomarinus.     

Millimeter-scale vertical partitioning of nitrogen cycling in hypersaline mats reveals prominence of genes encoding multi-heme and prismane proteins

Microbial mats are modern analogues of the first ecosystems on the Earth. As extant representatives of microbial communities where free oxygen may have first been available on a changing planet, they offer an ecosystem within which to study the evolution of biogeochemical cycles requiring and inhibited by oxygen. Here, we report the distribution of genes involved in nitrogen metabolism across a vertical oxygen gradient at 1 mm resolution in a microbial mat using quantitative PCR (qPCR), retro-transcribed qPCR (RT-qPCR) and metagenome sequencing. Vertical patterns in the presence and expression of nitrogen cycling genes, corresponding to oxygen requiring and non-oxygen requiring nitrogen metabolism, could be seen across gradients of dissolved oxygen and ammonium. Metagenome analysis revealed that genes annotated as hydroxylamine dehydrogenase (proper enzyme designation EC 1.7.2.6, hao) and hydroxylamine reductase (hcp) were the most abundant nitrogen metabolism genes in the mat. The recovered hao genes encode hydroxylamine dehydrogenase EC 1.7.2.6 (HAO) proteins lacking the tyrosine residue present in aerobic ammonia oxidizing bacteria (AOB). Phylogenetic analysis confirmed that those proteins were more closely related to ɛHao protein present in Campylobacterota lineages (previously known as Epsilonproteobacteria) rather than oxidative HAO of AOB. The presence of hao sequences related with ɛHao protein, as well as numerous hcp genes encoding a prismane protein, suggest the presence of a nitrogen cycling pathway previously described in Nautilia profundicola as ancestral to the most commonly studied present day nitrogen cycling pathways.Subject terms: Microbial ecology, Population dynamics, Population genetics, Metagenomics     

NADPH-cytochrome c reductase in outer membrane of kidney mitochondria. Purification and properties.

NADPH-cytochrome c reductase of vitamin D₃-deficient chick kidney mitochondria has been purified approximately 1100-fold to a specific activity of 788 nmol cytochrome c reduced/min/mg protein. Analytical gel electrophoresis of the purified enzyme revealed two bands when stained for protein, but only the more anodic band demonstrated NADPH-tetrazolium reductase activity. The relative ease of solubilization of the reductase without the use of lipases, proteases, or detergents was the first line of evidence that suggested a novel mitochondrial localization for this previously unreported NADPH-linked cytochrome c reductase. The apparent properties of the reductase suggest that the enzyme is a component of kidney mitochondrial outer membrane. The kinetic determination of Michaelis constants with respect to NADPH, cytochrome c, and NADH gave the following values: K_mNADPH = 1.7 μM, K_mcytc = 3.4 μM, and K_mNADH = 20 mM. These constants were different from those of the intact kidney microsomal reductase: K_mNADPH = 5.5 μM, K_mcytc = 10.5 μM, and K_mNADH = 13.3 μM. The mitochondrial as well as the intact microsomal reductases exhibited a ping-pong kinetic mechanism for NADPH-mediated cytochrome c reduction. Spectrofluorometric measurements demonstrated the presence of equimolar amounts of FAD and FMN. The oxidized enzyme has absorption maxima at 280 and 450 nm with a shoulder at 415 nm. Upon reduction with NADPH a distinct loss in the 450-nm absorption was observed. Ouchterlony immunodiffusion studies with rabbit antiserum to chick renal mitochondrial ferredoxin did not reveal cross-reactivity when the purified reductase was the antigen. This excludes the involvement of a ferredoxin-type iron-sulfur protein in the NADPH-mediated reduction of cytochrome c by the purified reductase.     

A Critical Role for the cccA Gene Product,Cytochrome c2, in Diverting Electrons from Aerobic Respiration to Denitrification in Neisseria gonorrhoeae

Neisseria gonorrhoeae is a microaerophile that, when oxygen availability is limited, supplements aerobic respiration with a truncated denitrification pathway, nitrite reduction to nitrous oxide. We demonstrate that the cccA gene of Neisseria gonorrhoeae strain F62 (accession number NG0292) is expressed, but the product, cytochrome c₂, accumulates to only low levels. Nevertheless, a cccA mutant reduced nitrite at about half the rate of the parent strain. We previously reported that cytochromes c₄ and c₅ transfer electrons to cytochrome oxidase cbb₃ by two independent pathways and that the CcoP subunit of cytochrome oxidase cbb₃ transfers electrons to nitrite. We show that mutants defective in either cytochrome c₄ or c₅ also reduce nitrite more slowly than the parent. By combining mutations in cccA (Δc₂), cycA (Δc₄), cycB (Δc₅), and ccoP (ccoP-C368A), we demonstrate that cytochrome c₂ is required for electron transfer from cytochrome c₄ via the third heme group of CcoP to the nitrite reductase, AniA, and that cytochrome c₅ transfers electrons to nitrite reductase by an independent pathway. We propose that cytochrome c₂ forms a complex with cytochrome oxidase. If so, the redox state of cytochrome c₂ might regulate electron transfer to nitrite or oxygen. However, our data are more consistent with a mechanism in which cytochrome c₂ and the CcoQ subunit of cytochrome oxidase form alternative complexes that preferentially catalyze nitrite and oxygen reduction, respectively. Comparison with the much simpler electron transfer pathway for nitrite reduction in the meningococcus provides fascinating insights into niche adaptation within the pathogenic neisseriae.     

Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3

The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b ₅₅₄ (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b ₅₅₆ (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth. Received: 21 August 1996 / Accepted: 24 October 1996     

Electron transport chains and bioenergetics of respiratory nitrogen metabolism in Wolinella succinogenes and other Epsilonproteobacteria

Recent phylogenetic analyses have established that the Epsilonproteobacteria form a globally ubiquitous group of ecologically significant organisms that comprises a diverse range of free-living bacteria as well as host-associated organisms like Wolinella succinogenes and pathogenic Campylobacter and Helicobacter species. Many Epsilonproteobacteria reduce nitrate and nitrite and perform either respiratory nitrate ammonification or denitrification. The inventory of epsilonproteobacterial genomes from 21 different species was analysed with respect to key enzymes involved in respiratory nitrogen metabolism. Most ammonifying Epsilonproteobacteria employ two enzymic electron transport systems named Nap (periplasmic nitrate reductase) and Nrf (periplasmic cytochrome c nitrite reductase). The current knowledge on the architecture and function of the corresponding proton motive force-generating respiratory chains using low-potential electron donors are reviewed in this article and the role of membrane-bound quinone/quinol-reactive proteins (NapH and NrfH) that are representative of widespread bacterial electron transport modules is highlighted. Notably, all Epsilonproteobacteria lack a napC gene in their nap gene clusters. Possible roles of the Nap and Nrf systems in anabolism and nitrosative stress defence are also discussed. Free-living denitrifying Epsilonproteobacteria lack the Nrf system but encode cytochrome cd₁ nitrite reductase, at least one nitric oxide reductase and a characteristic cytochrome c nitrous oxide reductase system (cNosZ). Interestingly, cNosZ is also found in some ammonifying Epsilonproteobacteria and enables nitrous oxide respiration in W. succinogenes.     

Arsenite Oxidase from Ralstonia sp. 22: CHARACTERIZATION OF THE ENZYME AND ITS INTERACTION WITH SOLUBLE CYTOCHROMES

We characterized the aro arsenite oxidation system in the novel strain Ralstonia sp. 22, a β-proteobacterium isolated from soil samples of the Salsigne mine in southern France. The inducible aro system consists of a heterodimeric membrane-associated enzyme reacting with a dedicated soluble cytochrome c₅₅₄. Our biochemical results suggest that the weak association of the enzyme to the membrane probably arises from a still unknown interaction partner. Analysis of the phylogeny of the aro gene cluster revealed that it results from a lateral gene transfer from a species closely related to Achromobacter sp. SY8. This constitutes the first clear cut case of such a transfer in the Aro phylogeny. The biochemical study of the enzyme demonstrates that it can accommodate in vitro various cytochromes, two of which, c₅₅₂ and c_554, are from the parent species. Cytochrome c₅₅₂ belongs to the sox and not the aro system. Kinetic studies furthermore established that sulfite and sulfide, substrates of the sox system, are both inhibitors of Aro activity. These results reinforce the idea that sulfur and arsenic metabolism are linked.     

Generation of polymerase chain reaction-specific probes for library screening using single degenerate primers

Degenerate oligonucleotide primers were made to peptide sequences from hydroxylamine oxidoreductase (HAO) from Nitrosomonas europaea. The primers were used singly in PCR reactions to amplify portions of the gene for HAO from genomic DNA. Southern hybridizations using fragments amplified with each primer showed that they labeled the same genomic DNA fragments. The PCR-amplified fragments were successfully used to screen a gene library for clones containing the HAO gene. The method of isolating genes by PCR with single primers has general utility.