▪ Abstract The eutrophication of many ecosystems in recent decades has led to an increased interest in the ecology of nitrogen transformation. Chemolitho-autotrophic ammonia-oxidizing bacteria are responsible for the rate-limiting step of nitrification in a wide variety of environments, making them important in the global cycling of nitrogen. These organisms are unique in their ability to use the conversion of ammonia to nitrite as their sole energy source. Because of the importance of this functional group of bacteria, understanding of their ecology and physiology has become a subject of intense research over recent years. The monophyletic nature of these bacteria in terrestrial environments has facilitated molecular biological approaches in studying their ecology, and progress in this field has been rapid. The ammonia-oxidizing bacteria of the β-subclass Proteobacteria have become somewhat of a model system within molecular microbial ecology, and this chapter reviews recent progress in our knowledge of ...

Ammonia-Oxidizing Bacteria: A Model for Molecular Microbial Ecology

Marine Crenarchaeota are the most abundant single group of prokaryotes in the ocean, but their physiology and role in marine biogeochemical cycles are unknown. Recently, a member of this clade was isolated from a sea aquarium and shown to be capable of nitrification, tentatively suggesting that Crenarchaeota may play a role in the oceanic nitrogen cycle. We enriched a crenarchaeote from North Sea water and showed that its abundance, and not that of bacteria, correlates with ammonium oxidation to nitrite. A time series study in the North Sea revealed that the abundance of the gene encoding for the archaeal ammonia monooxygenase alfa subunit (amoA) is correlated with a decline in ammonium concentrations and with the abundance of Crenarchaeota. Remarkably, the archaeal amoA abundance was 1–2 orders of magnitude higher than those of bacterial nitrifiers, which are commonly thought to mediate the oxidation of ammonium to nitrite in marine environments. Analysis of Atlantic waters of the upper 1,000 m, where most of the ammonium regeneration and oxidation takes place, showed that crenarchaeotal amoA copy numbers are also 1–3 orders of magnitude higher than those of bacterial amoA. Our data thus suggest a major role for Archaea in oceanic nitrification.

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Archaeal nitrification in the ocean

The current perception of evolutionary relationships and the natural diversity of ammonia-oxidizing bacteria (AOB) is mainly based on comparative sequence analyses of their genes encoding the 16S rRNA and the active site polypeptide of the ammonia monooxygenase (AmoA). However, only partial 16S rRNA sequences are available for many AOB species and most AOB have not yet been analyzed on the amoA level. In this study, the 16S rDNA sequence data of 10 Nitrosomonas species and Nitrosococcus mobilis were completed. Furthermore, previously unavailable 16S rRNA sequences were determined for three Nitrosomonas sp. isolates and for the gamma-subclass proteobacterium Nitrosococcus halophilus. These data were used to revaluate the specificities of published oligonucleotide primers and probes for AOB. In addition, partial amoA sequences of 17 AOB, including the above-mentioned 15 AOB, were obtained. Comparative phylogenetic analyses suggested similar but not identical evolutionary relationships of AOB by using 16S rRNA and AmoA as marker molecules, respectively. The presented 16S rRNA and amoA and AmoA sequence data from all recognized AOB species significantly extend the currently used molecular classification schemes for AOB and now provide a more robust phylogenetic framework for molecular diversity inventories of AOB. For 16S rRNA-independent evaluation of AOB species-level diversity in environmental samples, amoA and AmoA sequence similarity threshold values were determined which can be used to tentatively identify novel species based on cloned amoA sequences. Subsequently, 122 amoA sequences were obtained from 11 nitrifying wastewater treatment plants. Phylogenetic analyses of the molecular isolates showed that in all but two plants only nitrosomonads could be detected. Although several of the obtained amoA sequences were only relatively distantly related to known AOB, none of these sequences unequivocally suggested the existence of previously unrecognized species in the wastewater treatment environments examined.

https://www.researchgate.net/profile/Michael_Wagner5/publication/12229852_Phylogeny_of_All_Recognized_Species_of_Ammonia_Oxidizers_Based_on_Comparative_16S_rRNA_and_amoA_Sequence_Analysis_Implications_for_Molecular_Diversity_Surveys/links/02e7e520a467c0e4c7000000.pdf

Phylogeny of All Recognized Species of Ammonia Oxidizers Based on Comparative 16S rRNA and amoA Sequence Analysis: Implications for Molecular Diversity Surveys

https://www.cell.com/trends/microbiology/pdf/S0966-842X(12)00134-5.pdf

Archaeal and bacterial ammonia-oxidisers in soil: the quest for niche specialisation and differentiation

Active expression of putative ammonia monooxygenase gene subunit A (amoA) of marine group I Crenarchaeota has been detected in the Black Sea water column. It reached its maximum, as quantified by reverse-transcription quantitative PCR, exactly at the nitrate maximum or the nitrification zone modeled in the lower oxic zone. Crenarchaeal amoA expression could explain 74.5% of the nitrite variations in the lower oxic zone. In comparison, amoA expression by gamma-proteobacterial ammonia-oxidizing bacteria (AOB) showed two distinct maxima, one in the modeled nitrification zone and one in the suboxic zone. Neither the amoA expression by crenarchaea nor that by beta-proteobacterial AOB was significantly elevated in this latter zone. Nitrification in the suboxic zone, most likely microaerobic in nature, was verified by (15)NO(2)(-) and (15)N(15)N production in (15)NH(4)(+) incubations with no measurable oxygen. It provided a direct local source of nitrite for anammox in the suboxic zone. Both ammonia-oxidizing crenarchaea and gamma-proteobacterial AOB were important nitrifiers in the Black Sea and were likely coupled to anammox in indirect and direct manners respectively. Each process supplied about half of the nitrite required by anammox, based on (15)N-incubation experiments and modeled calculations. Because anammox is a major nitrogen loss in marine suboxic waters, such nitrification-anammox coupling potentially occurring also in oceanic oxygen minimum zones would act as a short circuit connecting regenerated ammonium to direct nitrogen loss, thus reducing the presumed direct contribution from deep-sea nitrate.

/pdf/linking-crenarchaeal-and-bacterial-nitrification-to-anammox-2yxddtndzu.pdf

Linking crenarchaeal and bacterial nitrification to anammox in the Black Sea

Autotrophic ammonia-oxidizing bacteria use the essential enzyme ammonia monooxygenase (AMO) to transform ammonia to hydroxylamine. The amo operon consists of at least three genes, amoC, amoA, and amoB; amoA encodes the subunit containing the putative enzyme active site. The use of the amo genes as functional markers for ammonia-oxidizing bacteria in environmental applications requires knowledge of the diversity of the amo operon on several levels: (1) the copy number of the operon in the genome, (2) the arrangement of the three genes in an individual operon, and (3) the primary sequence of the individual genes. We present a database of amo gene sequences for pure cultures of ammonia-oxidizing bacteria representing both the β- and the γ-subdivision of Proteobacteria in the following genera: Nitrosospira (6 strains), Nitrosomonas (5 strains) and Nitrosococcus (2 strains). The amo operon was found in multiple (2–3) nearly identical copies in the β-subdivision representatives but in single copies in the γ-subdivision ammonia oxidizers. The analysis of the deduced amino acid sequence revealed strong conservation for all three Amo peptides in both primary and secondary structures. For the amoA gene within the β-subdivision, nucleotide identity values are approximately 85% within the Nitrosomonas or the Nitrosospira groups, but approximately 75% when comparing between these groups. Conserved regions in amoA and amoC were identified and used as primer sites for PCR amplification of amo genes from pure cultures, enrichments and the soil environment. The intergenic region between amoC and amoA is variable in length and may be used to profile the community of ammonia-oxidizing bacteria in environmental samples. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00203-001-0369-z.

Diversity of ammonia monooxygenase operon in autotrophic ammonia-oxidizing bacteria.

The gene cluster encoding ammonia monooxygenase (AMO) in the chemolithotrophic soil bacterium Nitrosospira sp. NpAV was found to contain a third open reading frame, termed amoC, upstream of the genes amoA and amoB that encode the subunits of AMO. The amoC gene and its flanking regions were isolated and sequenced from a 4.4 kb EcoRI fragment that contains one of three copies of the ammonia monooxygenase gene cluster. The presence of this gene upstream of the other two amoA gene copies in Nitrosospira NpAV as well as upstream of amoA genes in the genomes of other ammonia oxidizing nitrifiers (strains in the genera Nitrosomonas, Nitrosospira, Nitrosolobus and Nitrosovibrio) was confirmed using genomic DNA, oligodeoxyribonucleotide primers and the PCR. The amoC gene in Nitrosospira sp. NpAV encodes a 270 amino acid polypeptide of approximately 36 kDa. Topological analysis of the predicted primary structure revealed 6 membrane spanning domains. The amoC gene was expressed in recombinant Escherichia coli from its indigenous promoter.

A gene encoding a membrane protein exists upstream of the amoA/amoB genes in ammonia oxidizing bacteria: a third member of the amo operon?

Nitrifying bacteria such as Nitrosomonas europaea and Nitrosospira sp. NpAV use ammonia monooxygenase (AMO) for oxidation of their primary growth substrate, ammonia. Two polypeptides of AMO are coded for by contiguous genes, amoA and amoB, which are preceded by a third gene, amoC. The amoCAB clusters are present in multiple copies in nitrifying bacteria of the beta subdivision. These bacteria also have one amoC copy that is not adjacent to a copy of amoAB. The seven known amoC genes in different nitrifiers code for similar polypeptides (> 68%). Reverse transcriptase-polymerase chain reactions and Northern blots indicated that amoC from the amoCAB cluster is contained on a transcript with amoAB. Two other transcripts were detected with amo probes and may be a product of processing of the amoCAB mRNA or independent transcripts.

Transcription of the amoC, amoA and amoB genes in Nitrosomonas europaea and Nitrosospira sp. NpAV

A gene encoding a membrane protein exists upstream of the genes in ammonia oxidizing bacteria: a third member of the operon?

While there is an extensive database of genes encoding ammonia monooxygenase (amo) found in the ammonia-oxidizing beta-proteobacteria, few amo sequences are available representing the gamma-proteobacteria. We sequenced the complete amo operon (amoCAB) for Nitrosococcus oceani (ATCC 19707), a marine, autotrophic, ammonia-oxidizing bacterium belonging to the gamma-subdivision of the proteobacteria. An additional autotrophic, ammonia-oxidizing bacterium isolated from a marine environment (strain C-113) was identified as belonging to the Nitrosococcus genus by 16S rDNA analysis and its amo operon was sequenced. This is the first report of a full-length sequence for the amo operon from a gamma-subdivision autotrophic ammonia-oxidizing bacterium. The N. oceani and C-113 amo genes were 88-90% identical to each other, 49-53% identical to the pmo genes encoding the related particulate methane monooxygenase of Methylococcus capsulatus (Bath), and 39-42% identical to the amo genes of the beta-subdivision autotrophic ammonia-oxidizing bacteria. In both Nitrosococcus strains, the amo operon was found as a single copy and contained three genes, amoC, amoA, amoB, with intergenic spacer regions between amoC and amoA (286 bp) and between amoA and amoB (65 bp). We conclude that the amo genes will allow for a finer scale phylogenetic differentiation than 16S rDNA within the gamma-subdivision AOB.

J.Javier Alzerreca

Papers

Diversity of ammonia monooxygenase operon in autotrophic ammonia-oxidizing bacteria.

A gene encoding a membrane protein exists upstream of the amoA/amoB genes in ammonia oxidizing bacteria: a third member of the amo operon?

Transcription of the amoC, amoA and amoB genes in Nitrosomonas europaea and Nitrosospira sp. NpAV

A gene encoding a membrane protein exists upstream of the genes in ammonia oxidizing bacteria: a third member of the operon?

The amo operon in marine, ammonia-oxidizing γ-proteobacteria