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 ϳ47% transcribed from one strand and ϳ53% 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.

Nitrosomonas europaea uses only NH(3), CO(2) and mineral salts for growth and as such it is an obligate chemo-lithoautotroph. The oxidation of NH(3) is a two-step process catalyzed by ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO). AMO catalyzes the oxidation of NH(3) to NH(2)OH and HAO catalyzes the oxidation of NH(2)OH to NO(2)(-). AMO is a membrane-bound enzyme composed of three subunits. HAO is located in the periplasm and is a homotrimer with each subunit containing eight c-type hemes. The electron flow from HAO is channeled through cytochrome c(554) to cytochrome c(m552), where it is partitioned for further utilization. Among the ammonia-oxidizing bacteria, the genes for AMO, these cytochromes, and HAO are present in up to three highly similar copies. Mutants with mutations in the copies of amoCAB and hao in N. europaea have been isolated. All of the amoCAB and hao gene copies are functional. N. europaea was selected by the United States Department of Energy for a whole-genome sequencing project. In this article, we review recent research on the molecular biology and biochemistry of NH(3) oxidation in nitrifiers.

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.

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The gammaproteobacterium Nitrosococcus oceani (ATCC 19707) is a gram-negative obligate chemolithoautotroph capable of extracting energy and reducing power from the oxidation of ammonia to nitrite. Sequencing and annotation of the genome revealed a single circular chromosome (3,481,691 bp; G؉C content of 50.4%) and a plasmid (40,420 bp) that contain 3,052 and 41 candidate protein-encoding genes, respectively. The genes encoding proteins necessary for the function of known modes of lithotrophy and autotrophy were identified. Contrary to betaproteobacterial nitrifier genomes, the N. oceani genome contained two complete rrn operons. In contrast, only one copy of the genes needed to synthesize functional ammonia monooxygenase and hydroxylamine oxidoreductase, as well as the proteins that relay the extracted electrons to a terminal electron acceptor, were identified. The N. oceani genome contained genes for 13 complete two-component systems. The genome also contained all the genes needed to reconstruct complete central pathways, the tricarboxylic acid cycle, and the Embden-Meyerhof-Parnass and pentose phosphate pathways. The N. oceani genome contains the genes required to store and utilize energy from glycogen inclusion bodies and sucrose. Polyphosphate and pyrophosphate appear to be integrated in this bacterium's energy metabolism, stress tolerance, and ability to assimilate carbon via gluconeogenesis. One set of genes for type I ribulose-1,5-bisphosphate carboxylase/oxygenase was identified, while genes necessary for methanotrophy and for carboxysome formation were not identified. The N. oceani genome contains two copies each of the genes or operons necessary to assemble functional complexes I and IV as well as ATP synthase (one H ؉ -dependent F 0 F 1 type, one Na ؉ -dependent V type).

Hydroxylamine oxidoreductase (HAO) catalyzes the oxidation of hydroxylamine to nitrite in Nitrosomonas europaea. The electrons released in the reaction are partitioned to ammonium monooxygenase and to the respiratory chain. The immediate acceptor of electrons from HAO is believed to be cytochrome c-554 (Cyt c-554). We have isolated a genomic DNA fragment containing the structural gene encoding HAO (hao) and a part of the gene for Cyt c-554. The nucleotide sequence of hao was determined, and its transcription was analyzed. The open reading frame (ORF) encodes amino acid sequences matching the purified peptides of HAO. A 64.28-kDa protein is encoded in this ORF, in close agreement with the empirically determined molecular mass of 63 kDa. The N terminus was located 24 amino acids from the start codon, suggesting the presence of a leader sequence. The putative eight heme-binding peptides were localized in this ORF. The gene for Cyt c-554 was located 1,200 bp downstream from the 3' end of hao. An ORF was identified in the upstream region from hao and may encode a protein of unknown function. Data bank searches did not reveal proteins with substantial similarities to HAO, but they did reveal similarities between Cyt c-554 and other c-type cytochromes.

In Nitrosomonas europaea, ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) catalyse the oxidation of ammonia (NH3) to nitrite (NO2-). A transcript of 3500 bases hybridizes to probes for amoA and amoB (genes that code for AMO proteins). A transcript of 2100 bases hybridizes to probes for hao (the gene that codes for HAO). Induction of the mRNAs detected by amo and hao probes required the presence of ammonium (NH4+). To correlate new levels of mRNA with de novo activity, existent mRNA pools and AMO activity were depleted prior to induction by NH4+. The mRNAs of AMO and HAO were depleted by depriving the cells of energy for at least 8 h; AMO activity was inactivated with acetylene (C2H2) after mRNA depletion. In cells treated this way, levels of new AMO mRNA and de novo AMO enzyme activity were correlated with increased NH4+ concentrations up to 1 mM after 3 h of incubation. HAO mRNA also increased in the NH4(+)-treated cells. Other proteins and RNAs induced by NH4+ were detected in 14CO2-labelling experiments. The AMO and HAO mRNAs were preferentially synthesized during energy-limiting conditions.

The transcriptome of Nitrosomonas europaea was analyzed with whole-genome microarrays. Growing cells were compared to cells deprived of (NH4)2SO4 and Na2CO3. Hybridization signals were detected for 76% of the genes represented on the array under either or both conditions. Transcript levels for 68% of the genes were at least twofold higher in growing cells than in deprived cells, while only 0.42% of the genes were present at more than twofold higher levels in deprived cells. Transcript levels for the remaining 7% of the genes did not change significantly with the treatments. These trends were confirmed for selected genes by Northern hybridizations and quantitative RT-PCR. Compared to heterotrophic bacteria, N. europaea downregulates a greater proportion of its genes and fewer genes appear to be associated with the adaptation to starvation.