Alan B. Hooper

Complete Genome Sequence of the Ammonia-Oxidizing Bacterium and Obligate Chemolithoautotroph Nitrosomonas europaea

Nitrosomonas europaea (ATCC 19718) is a gram-negative obligate chemolithoautotroph that can derive all its energy and reductant for growth from the oxidation of ammonia to nitrite. Nitrosomonas europaea participates in the biogeochemical N cycle in the process of nitrification. Its genome consists of a single circular chromosome of 2,812,094 bp. The GC skew analysis indicates that the genome is divided into two unequal replichores. Genes are distributed evenly around the genome, with approximately 47% transcribed from one strand and approximately 53% transcribed from the complementary strand. A total of 2,460 protein-encoding genes emerged from the modeling effort, averaging 1,011 bp in length, with intergenic regions averaging 117 bp. Genes necessary for the catabolism of ammonia, energy and reductant generation, biosynthesis, and CO(2) and NH(3) assimilation were identified. In contrast, genes for catabolism of organic compounds are limited. Genes encoding transporters for inorganic ions were plentiful, whereas genes encoding transporters for organic molecules were scant. Complex repetitive elements constitute ca. 5% of the genome. Among these are 85 predicted insertion sequence elements in eight different families. The strategy of N. europaea to accumulate Fe from the environment involves several classes of Fe receptors with more than 20 genes devoted to these receptors. However, genes for the synthesis of only one siderophore, citrate, were identified in the genome. This genome has provided new insights into the growth and metabolism of ammonia-oxidizing bacteria.

Enzymology of the oxidation of ammonia to nitrite by bacteria

The enzymes which catalyze the oxidation of ammonia to nitrite by autotrophic bacteria are reviewed. A comparison is made with enzymes which catalyze the same reactions in methylotrophs and organotrophic heterotrophic bacteria.

O2 and H2O are each the source of one O in NO− 2 produced from NH3 by Nitrosomonas: 15N-NMR evidence

The exchange of 18O between H2 18O and exogeneously added 15N16O− 2 which occurs during oxidation of ammonia by Nitrosomonas is shown to occur one oxygen at a time. Conditions in which the exchange is diminished (notably the presence of 14NO– 2 and CCCP) allowed demonstration that water and dioxygen are each the source of one oxygen in nitrite produced from 15NH3. The nitrate produced in the presence of 18O2 consisted of 67 and 0% 15N18O16O− and 15N18O18O−, respectively. Analysis was made using the 18O‐isotope shift in 15N‐NMR.

Hydroxylamine oxidoreductase of Nitrosomonas. Production of nitric oxide from hydroxylamine.

As shown previously, higly purified hydroxylamine oxidoreductase (hydroxylamine:oxygen oxidoreductase, EC 1.7.3.4) from Nitrosomonas catalyzes the aerobic oxidation of NH2OH to a mixture of NO2− and NO3− in the presence of phenazine methosulfate. The present work shows that N2O and NO are also products of the oxidation of NH2OH. A nitrite reductase, present in less purified samples of the hydroxylamine oxidoreductase is shown to catalyze the reduction of NO2− to a mixture of NO and N2O with leucopyocyanine as electron donor. The possible reduction, by purified hydroxylamine oxidoreductase, of NO2− or NO3− to form NO (with NH2OH-reduced enzyme or phenazine methosulfate as potential electron donors) was eliminated; 15NO was shown not to be produced in the presence of 15NO2− and 15NO3−. NO thus appears to be a product of NH2OH oxidation by purified hydroxylamine oxidoreductase and is thus a possible intermediate in the production of nitrite. Mn2+ in the concentration range 10−6–10−5 M progessively and completely inhibits the formation of nitrite and nitrate by purified hydroxylamine oxidoreductase while concomitantly stimulating the rate of dehydrogenation of hydroxylamine three-fold. The presence of Mn(II) results in decreased formation of NO and increased formation of N2O. The present results are consistent with a mechanism of NH2OH oxidation in which the oxidation of an intermediate compound of the oxidation state of (HNO) occurs by dehydrogenation rather than direct addition of O. The dehydrogenation step is inhibited in the presence of Mn(II).

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.

Nitrite Reductase of Nitrosomonas europaea Is Not Essential for Production of Gaseous Nitrogen Oxides and Confers Tolerance to Nitrite

A gene that encodes a periplasmic copper-type nitrite reductase (NirK) was identified in Nitrosomonas europaea. Disruption of this gene resulted in the disappearance of Nir activity in cell extracts. The nitrite tolerance of NirK-deficient cells was lower than that of wild-type cells. Unexpectedly, NirK-deficient cells still produced nitric oxide (NO) and nitrous oxide (N(2)O), the latter in greater amounts than that of wild-type cells. This demonstrates that NirK is not essential for the production of NO and N(2)O by N. europaea. Inactivation of the putative fnr gene showed that Fnr is not essential for the expression of nirK.

Heme packing motifs revealed by the crystal structure of the tetra-heme cytochrome c554 from Nitrosomonas europaea.

Cytochrome c554 (cyt c554), a tetra-heme cytochrome from Nitrosomonas europaea, is an essential component in the biological nitrification pathway. In N. europaea, ammonia is converted to hydroxylamine, which is then oxidized to nitrite by hydroxylamine oxidoreductase (HAO). Cyt c554 functions in the latter process by accepting pairs of electrons from HAO and transferring them to a cytochrome acceptor. The crystal structure of cyt c554 at 2.6 Å resolution shows a predominantly α-helical protein with four covalently attached hemes. The four hemes are arranged in two pairs such that the planes of the porphyrin rings are almost parallel and overlapping at the edge; corresponding heme arrangements are observed in other multi-heme proteins. Striking structural similarities are evident between the tetra-heme core of cyt c554 and hemes 3–6 of HAO, which suggests an evolutionary relationship between these redox partners.

Structure and Sequence Conservation of hao Cluster Genes of Autotrophic Ammonia-Oxidizing Bacteria: Evidence for Their Evolutionary History

ABSTRACT 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 c554; and cycB, cytochrome cm552. The deduced protein sequences of HAO, c554, and cm552 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 cm552, 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 c554 gene was lacking. The conservation of the hao gene cluster in general and the uniqueness of the c554 gene in particular make it a suitable target for the design of primers and probes useful for molecular ecology approaches to detect aAOB.

A ‘blue’ copper oxidase from Nitrosomonas europaea

Abstract A soluble copper-containing protein with p -phenylenediamine oxidase activity was purified from Nitrosomonas europaea by flat-bed isoelectric focusing and chromatography on Sephacryl S-300. The native and subunit molecular weights were 127 500 and 40 100, respectively; the isoelectric point was pH 4.63. The protein had an absorption maximum at 607 nm in the oxidized form with an extinction coefficient of 6.0 cm −1 · mM −1 at pH 7.5. On reduction with dithionite, the absorbance was abolished. The electron paramagnetic resonance spectrum of the protein showed evidence for both Type 1 and Type 2 copper in a 1:1 ratio. The protein catalyzed the aerobic oxidation of p -phenylenediamine, cytochrome c -554, and hydroxylamine oxidoreductase. The enzyme catalyzed the reduction of nitrite with cytochrome c -552 as electron donor. In contrast, p -phenylenediamine, reduced cytochrome c -554, and hydroxylamine oxidoreductase were not active as electron donors for nitrite reduction.

Evidence for an iron center in the ammonia monooxygenase from Nitrosomonas europaea

Binding of the ligand, nitric oxide, in the presence of reductant was used to identify a ferrous S=3/2 signal, characteristic of a ferrous nitrosyl complex, and a g=2.03 copper of iron signal in membranes of the ammonia‐oxidizing bacterium, Nitrosomonas europaea. The same ferrous S=3/2 signal is thought to be a component of the membrane‐associated methane monooxygenase (pMMO) of Methylococcus capsulatus Bath, since it is seen in the membrane fraction of cells expressing pMMO and in the purified enzyme, but not in the membrane fraction of cells expressing the soluble MMO [Zahn, J.A. and DiSpirito, A.A. (1996) J. Bacteriol. 178, 1018–1029]. Treatment of resting membranes or cells of N. europaea with nitrapyrin, 2‐chloro,6‐trichloromethylpyridine, resulted in the increase in magnitude of a g = 6, high‐spin ferric iron signal. In the presence of NO and reductant, nitrapyrin prevented the formation of the S=3/2 nitrosyl‐iron complex while increasing the intensity of the g=6 signal. Nitrapyrin is a specific inhibitor of, and is reduced by, the ammonia monoxygenase (AMO) [Bédard, C. and Knowles, R. (1989) Microbiol. Rev. 53, 38–83]. Taken together the data suggest that iron capable of forming the S=3/2 complex is a catalytic component of AMO of N. europaea, possibly a part of the oxygen‐activating center. Inactivation of the membrane‐associated AMO with acetylene did not diminish the S=3/2 nitrosyl‐iron signal, the g=6 signal, or the g=6 signal.