Competition between Ammonia-Oxidizing Archaea and Bacteria from Freshwater Environments.

In the environment, nutrients are rarely available in a constant supply. Therefore, microorganisms require strategies to compete for limiting nutrients. In freshwater systems, ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) compete with heterotrophic bacteria, photosynthetic microorganisms, and each other for ammonium, which AOA and AOB utilize as their sole source of energy and nitrogen. We investigated the competition between highly enriched cultures of AOA (AOA-AC1) and AOB (AOB-G5-7) for ammonium. Based on the amoA gene, the newly enriched archaeal ammonia oxidizer in AOA-AC1 was closely related to Nitrosotenuis spp., and the bacterial ammonia oxidizer in AOB-G5-7, Nitrosomonas sp. strain Is79, belonged to the Nitrosomonas oligotropha group (Nitrosomonas cluster 6a). Growth experiments in batch cultures showed that AOB-G5-7 had higher growth rates than AOA-AC1 at higher ammonium concentrations. During chemostat competition experiments under ammonium-limiting conditions, AOA-AC1 dominated the cultures, while AOB-G5-7 decreased in abundance. In batch cultures, the outcome of the competition between AOA and AOB was determined by the initial ammonium concentrations. AOA-AC1 was the dominant ammonia oxidizer at an initial ammonium concentration of 50 µM, and AOB-G5-7 was dominant at 500 µM. These findings indicate that during direct competition, AOA-AC1 was able to use ammonium that was unavailable to AOB-G5-7, while AOB-G5-7 dominated at higher ammonium concentrations. The results are in strong accordance with environmental survey data suggesting that AOA are mainly responsible for ammonia oxidation under more oligotrophic conditions, whereas AOB dominate under eutrophic conditions. IMPORTANCE Nitrification is an important process in the global nitrogen cycle. The first step, ammonia oxidation to nitrite, can be carried out by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). In many natural environments, these ammonia oxidizers coexist. Therefore, it is important to understand the population dynamics in response to increasing ammonium concentrations. Here, we study the competition between AOA and AOB enriched from freshwater systems. The results demonstrate that AOA are more abundant in systems with low ammonium availabilities and that AOB are more abundant when the ammonium availability increases. These results will help to predict potential shifts in the community composition of ammonia oxidizers in the environment due to changes in ammonium availability.

Metagenomes from Coastal Marine Sediments Give Insights into the Ecological Role and Cellular Features of Loki- and Thorarchaeota.

The genomes of Asgard Archaea, a novel archaeal proposed superphylum, share an enriched repertoire of eukaryotic signature genes and thus promise to provide insights into early eukaryote evolution. However, the distribution, metabolisms, cellular structures, and ecology of the members within this superphylum are not well understood. Here we provide a meta-analysis of the environmental distribution of the Asgard archaea, based on available 16S rRNA gene sequences. Metagenome sequencing of samples from a salt-crusted lagoon on the Baja California Peninsula of Mexico allowed the assembly of a new Thorarchaeota and three Lokiarchaeota genomes. Comparative analyses of all known Lokiarchaeota and Thorarchaeota genomes revealed overlapping genome content, including central carbon metabolism. Members of both groups contained putative reductive dehalogenase genes, suggesting that these organisms might be able to metabolize halogenated organic compounds. Unlike the first report on Lokiarchaeota, we identified genes encoding glycerol-1-phosphate dehydrogenase in all Loki- and Thorarchaeota genomes, suggesting that these organisms are able to synthesize bona fide archaeal lipids with their characteristic glycerol stereochemistry.IMPORTANCE Microorganisms of the superphylum Asgard Archaea are considered to be the closest living prokaryotic relatives of eukaryotes (including plants and animals) and thus promise to give insights into the early evolution of more complex life forms. However, very little is known about their biology as none of the organisms has yet been cultivated in the laboratory. Here we report on the ecological distribution of Asgard Archaea and on four newly sequenced genomes of the Lokiarchaeota and Thorarchaeota lineages that give insight into possible metabolic features that might eventually help to identify these enigmatic groups of archaea in the environment and to culture them.

Comparative Proteomics of Three Species of Ammonia-Oxidizing Bacteria.

Ammonia-oxidizing bacteria (AOB) are important members of terrestrial, marine, and industrial microbial communities and play a fundamental role in the Nitrogen cycle within these systems. They are responsible for the first step of nitrification, ammonia oxidation to nitrite. Although AOB are widespread and essential to environmental and industrial systems, where they regularly experience fluctuations in ammonia availability, no comparative studies of the physiological response of diverse AOB species at the protein level exist. In the present study, we used 1D-LC-MS/MS proteomics to compare the metabolism and physiology of three species of ammonia AOB, Nitrosomonas europaea, Nitrosospira multiformis, and Nitrosomonas ureae, under ammonia replete and ammonia starved conditions. Additionally, we compared the expression of orthologous genes to determine the major differences in the proteome composition of the three species. We found that approximately one-third of the predicted proteome was expressed in each species and that proteins for the key metabolic processes, ammonia oxidation and carbon fixation, were among the most abundant. The red copper protein, nitrosocyanin was highly abundant in all three species hinting toward its possible role as a central metabolic enzyme in AOB. The proteomic data also allowed us to identify pyrophosphate-dependent 6-phosphofructokinase as the potential enzyme replacing the Calvin-Benson-Bassham cycle enzyme Fructose-1,6-bisphosphatase missing in N. multiformis and N. ureae. Additionally, between species, there were statistically significant differences in the expression of many abundant proteins, including those related to nitrogen metabolism (nitrite reductase), motility (flagellin), cell growth and division (FtsH), and stress response (rubrerythrin). The three species did not exhibit a starvation response at the proteome level after 24 h of ammonia starvation, however, the levels of the RuBisCO enzyme were consistently reduced after the starvation period, suggesting a decrease in capacity for biomass accumulation. This study presents the first published proteomes of N. ureae and N. multiformis, and the first comparative proteomics study of ammonia-oxidizing bacteria, which gives new insights into consistent metabolic features and differences between members of this environmentally and industrially important group.

Comparison of Nitrogen Oxide Metabolism among Diverse Ammonia-Oxidizing Bacteria.

Ammonia-oxidizing bacteria (AOB) have well characterized genes that encode and express nitrite reductases (NIR) and nitric oxide reductases (NOR). However, the connection between presence or absence of these and other genes for nitrogen transformations with the physiological production of nitric oxide (NO) and nitrous oxide (N2O) has not been tested across AOB isolated from various trophic states, with diverse phylogeny, and with closed genomes. It is therefore unclear if genomic content for nitrogen oxide metabolism is predictive of net N2O production. Instantaneous microrespirometry experiments were utilized to measure NO and N2O emitted by AOB during active oxidation of ammonia (NH3) or hydroxylamine (NH2OH) and through a period of anoxia. This data was used in concert with genomic content and phylogeny to assess whether taxonomic factors were predictive of nitrogen oxide metabolism. Results showed that two oligotrophic AOB strains lacking annotated NOR-encoding genes released large quantities of NO and produced N2O abiologically at the onset of anoxia following NH3-oxidation. Furthermore, high concentrations of N2O were measured during active O2-dependent NH2OH oxidation by the two oligotrophic AOB in contrast to non-oligotrophic strains that only produced N2O at the onset of anoxia. Therefore, complete nitrifier denitrification did not occur in the two oligotrophic strains, but did occur in meso- and eutrophic strains, even in Nitrosomonas communis Nm2 that lacks an annotated NIR-encoding gene. Regardless of mechanism, all AOB strains produced measureable N2O under tested conditions. This work further confirms that AOB require NOR activity to enzymatically reduce NO to N2O in the nitrifier denitrification pathway, and also that abiotic reactions play an important role in N2O formation, in oligotrophic AOB lacking NOR activity.

Complete Genome Sequence of Nitrosomonas ureae Strain Nm10, an Oligotrophic Group 6a Nitrosomonad.

The complete genome of Nitrosomonas ureae strain Nm10, a mesophilic betaproteobacterial ammonia oxidizer isolated from Mediterranean soils in Sardinia, Italy, is reported here. This genome represents a cluster 6a nitrosomonad.

Pathways and key intermediates required for obligate aerobic ammonia-dependent chemolithotrophy in bacteria and Thaumarchaeota.

Chemolithotrophic ammonia-oxidizing bacteria and Thaumarchaeota are central players in the global nitrogen cycle. Obligate ammonia chemolithotrophy has been characterized for bacteria; however, large gaps remain in the Thaumarchaeotal pathway. Using batch growth experiments and instantaneous microrespirometry measurements of resting biomass, we show that the terrestrial Thaumarchaeon Nitrososphaera viennensis EN76(T) exhibits tight control over production and consumption of nitric oxide (NO) during ammonia catabolism, unlike the ammonia-oxidizing bacterium Nitrosospira multiformis ATCC 25196(T). In particular, pulses of hydroxylamine into a microelectrode chamber as the sole substrate for N. viennensis resulted in iterative production and consumption of NO followed by conversion of hydroxylamine to nitrite. In support of these observations, oxidation of ammonia in growing cultures of N. viennensis, but not of N. multiformis, was inhibited by the NO-scavenger PTIO. When based on the marginal nitrous oxide (N2O) levels detected in cell-free media controls, the higher levels produced by N. multiformis were explained by enzyme activity, whereas N2O in N. viennensis cultures was attributed to abiotic reactions of released N-oxide intermediates with media components. Our results are conceptualized in a pathway for ammonia-dependent chemolithotrophy in Thaumarchaea, which identifies NO as an essential intermediate in the pathway and implements known biochemistry to be executed by a proposed but still elusive copper enzyme. Taken together, this work identifies differences in ammonia-dependent chemolithotrophy between bacteria and the Thaumarchaeota, advances a central catabolic role of NO only in the Thaumarchaeotal pathway and reveals stark differences in how the two microbial cohorts contribute to N2O emissions.

Genome Sequence of Nitrosomonas communis Strain Nm2, a Mesophilic Ammonia-Oxidizing Bacterium Isolated from Mediterranean Soil.

The complete genome sequence of Nitrosomonas communis strain Nm2, a mesophilic betaproteobacterial ammonia oxidizer isolated from Mediterranean soils in Corfu, Greece, is reported here. This is the first genome to describe a cluster 8 Nitrosomonas species and represents an ammonia-oxidizing bacterium commonly found in terrestrial ecosystems.

Nitrosospira lacus sp. nov., a psychrotolerant, ammonia-oxidizing bacterium from sandy lake sediment.

A Gram-negative, spiral-shaped, chemolithotrophic, ammonia-oxidizing bacterium, designated APG3(T), was isolated into pure culture from sandy lake sediment collected from Green Lake, Seattle, WA, USA. Phylogenetic analyses based on the 16S rRNA gene sequence showed that strain APG3(T) belongs to cluster 0 of the genus Nitrosospira, which is presently not represented by described species, with Nitrosospira multiformis (cluster 3) as the closest species with a validly published name (identity of 98.6 % to the type strain). Strain APG3(T) grew at 4 °C but could not grow at 35 °C, indicating that this bacterium is psychrotolerant. Remarkably, the strain was able to grow over a wide range of pH (pH 5-9), which was greater than the pH range of any studied ammonia-oxidizing bacteria in pure culture. The DNA G+C content of the APG3(T) genome is 53.5 %, which is similar to that of Nitrosospira multiformis ATCC 25196(T) (53.9 %) but higher than that of Nitrosomonas europaea ATCC 19718 (50.7 %) and Nitrosomonas eutropha C71 (48.5 %). The average nucleotide identity (ANI) calculated for the genomes of strain APG3(T) and Nitrosospira multiformis ATCC 25196(T) was 75.45 %, significantly lower than the value of 95 % ANI that corresponds to the 70 % species-level cut-off based on DNA-DNA hybridization. Overall polyphasic taxonomy study indicated that strain APG3(T) represents a novel species in the genus Nitrosospira, for which the name Nitrosospira lacus sp. nov. is proposed (type strain APG3(T) = NCIMB 14869(T) = LMG 27536(T) = ATCC BAA-2542(T)).

Revision of N2O-producing pathways in the ammonia-oxidizing bacterium Nitrosomonas europaea ATCC 19718.

Nitrite reductase (NirK) and nitric oxide reductase (NorB) have long been thought to play an essential role in nitrous oxide (N2O) production by ammonia-oxidizing bacteria. However, essential gaps remain in our understanding of how and when NirK and NorB are active and functional, putting into question their precise roles in N2O production by ammonia oxidizers. The growth phenotypes of the Nitrosomonas europaea ATCC 19718 wild-type and mutant strains deficient in expression of NirK, NorB, and both gene products were compared under atmospheric and reduced O2 tensions. Anoxic resting-cell assays and instantaneous nitrite (NO2 (-)) reduction experiments were done to assess the ability of the wild-type and mutant N. europaea strains to produce N2O through the nitrifier denitrification pathway. Results confirmed the role of NirK for efficient substrate oxidation of N. europaea and showed that NorB is involved in N2O production during growth at both atmospheric and reduced O2 tensions. Anoxic resting-cell assays and measurements of instantaneous NO2 (-) reduction using hydrazine as an electron donor revealed that an alternate nitrite reductase to NirK is present and active. These experiments also clearly demonstrated that NorB was the sole nitric oxide reductase for nitrifier denitrification. The results of this study expand the enzymology for nitrogen metabolism and N2O production by N. europaea and will be useful to interpret pathways in other ammonia oxidizers that lack NirK and/or NorB genes.

Ecophysiological characterization of ammonia-oxidizing archaea and bacteria from freshwater.

Aerobic biological ammonia oxidation is carried out by two groups of microorganisms, ammonia-oxidizing bacteria (AOB) and the recently discovered ammonia-oxidizing archaea (AOA). Here we present a study using cultivation-based methods to investigate the differences in growth of three AOA cultures and one AOB culture enriched from freshwater environments. The strain in the enriched AOA culture belong to thaumarchaeal group I.1a, with the strain in one enrichment culture having the highest identity with "Candidatus Nitrosoarchaeum koreensis" and the strains in the other two representing a new genus of AOA. The AOB strain in the enrichment culture was also obtained from freshwater and had the highest identity to AOB from the Nitrosomonas oligotropha group (Nitrosomonas cluster 6a). We investigated the influence of ammonium, oxygen, pH, and light on the growth of AOA and AOB. The growth rates of the AOB increased with increasing ammonium concentrations, while the growth rates of the AOA decreased slightly. Increasing oxygen concentrations led to an increase in the growth rate of the AOB, while the growth rates of AOA were almost oxygen insensitive. Light exposure (white and blue wavelengths) inhibited the growth of AOA completely, and the AOA did not recover when transferred to the dark. AOB were also inhibited by blue light; however, growth recovered immediately after transfer to the dark. Our results show that the tested AOB have a competitive advantage over the tested AOA under most conditions investigated. Further experiments will elucidate the niches of AOA and AOB in more detail.

Model of the molecular basis for hydroxylamine oxidation and nitrous oxide production in methanotrophic bacteria.

Many methane-oxidizing bacteria (MOB) have been shown to aerobically oxidize ammonia and hydroxylamine (NH(2)OH) to produce nitrite and nitrous oxide (N(2)O). Genome sequences of alphaproteobacterial, gammaproteobacterial, and verrucomicrobial methanotrophs revealed the presence of haoAB, cytL, cytS, nirS or nirK, and norCB genes that may be responsible for N(2)O production, and additional haoAB genes were sequenced from two strains of Methylomicrobium album. The haoAB genes of M. album ATCC 33003 were inducible by ammonia and NH(2)OH, similar to haoAB induction by ammonia in Methylococcus capsulatus Bath. Increased expression of genes encoding nitric oxide reductase (cNOR; norCB) was measured upon exposure of M. capsulatus Bath to NaNO(2) and NO-releasing sodium nitroprusside. Only incubations of M. capsulatus Bath with methane, ammonia, and nitrite produced N(2)O. The data suggest a possible pathway of nitrite reduction to NO by reversely operating NH(2)OH oxidoreductase and NO reduction to N(2)O by cNOR independently or in conjunction with ammonia-induced enzymes (i.e. HAO or cytochrome c'-ß). Results of this study show that MOB likely have diverse mechanisms for nitrogen oxide metabolism and detoxification of NH(2)OH that involve conventional and unconventional enzymes.