Lisa Y. Stein

Metabolism of Inorganic N Compounds by Ammonia-Oxidizing Bacteria

ABSTRACT Ammonia oxidizing bacteria extract energy for growth from the oxidation of ammonia to nitrite. Ammonia monooxygenase, which initiates ammonia oxidation, remains enigmatic given the lack of purified preparations. Genetic and biochemical studies support a model for the enzyme consisting of three subunits and metal centers of copper and iron. Knowledge of hydroxylamine oxidoreductase, which oxidizes hydroxylamine formed by ammonia monooxygenase to nitrite, is informed by a crystal structure and detailed spectroscopic and catalytic studies. Other inorganic nitrogen compounds, including NO, N2O, NO2, and N2 can be consumed and/or produced by ammonia-oxidizing bacteria. NO and N2O can be produced as byproducts of hydroxylamine oxidation or through nitrite reduction. NO2 can serve as an alternative oxidant in place of O2 in some ammonia-oxidizing strains. Our knowledge of the diversity of inorganic N metabolism by ammonia-oxidizing bacteria continues to grow. Nonetheless, many questions remain regarding the enzymes and genes involved in these processes and the role of these pathways in ammonia oxidizers.

Complete Genome Sequence of Nitrosospira multiformis, an Ammonia-Oxidizing Bacterium from the Soil Environment

ABSTRACT The complete genome of the ammonia-oxidizing bacterium Nitrosospira multiformis (ATCC 25196T) consists of a circular chromosome and three small plasmids totaling 3,234,309 bp and encoding 2,827 putative proteins. Of the 2,827 putative proteins, 2,026 proteins have predicted functions and 801 are without conserved functional domains, yet 747 of these have similarity to other predicted proteins in databases. Gene homologs from Nitrosomonas europaea and Nitrosomonas eutropha were the best match for 42% of the predicted genes in N. multiformis. The N. multiformis genome contains three nearly identical copies of amo and hao gene clusters as large repeats. The features of N. multiformis that distinguish it from N. europaea include the presence of gene clusters encoding urease and hydrogenase, a ribulose-bisphosphate carboxylase/oxygenase-encoding operon of distinctive structure and phylogeny, and a relatively small complement of genes related to Fe acquisition. Systems for synthesis of a pyoverdine-like siderophore and for acyl-homoserine lactone were unique to N. multiformis among the sequenced genomes of ammonia-oxidizing bacteria. Gene clusters encoding proteins associated with outer membrane and cell envelope functions, including transporters, porins, exopolysaccharide synthesis, capsule formation, and protein sorting/export, were abundant. Numerous sensory transduction and response regulator gene systems directed toward sensing of the extracellular environment are described. Gene clusters for glycogen, polyphosphate, and cyanophycin storage and utilization were identified, providing mechanisms for meeting energy requirements under substrate-limited conditions. The genome of N. multiformis encodes the core pathways for chemolithoautotrophy along with adaptations for surface growth and survival in soil environments.

Nitrous oxide production by lithotrophic ammonia-oxidizing bacteria and implications for engineered nitrogen-removal systems

Chemolithoautotrophic AOB (ammonia-oxidizing bacteria) form a crucial component in microbial nitrogen cycling in both natural and engineered systems. Under specific conditions, including transitions from anoxic to oxic conditions and/or excessive ammonia loading, and the presence of high nitrite (NO₂⁻) concentrations, these bacteria are also documented to produce nitric oxide (NO) and nitrous oxide (N₂O) gases. Essentially, ammonia oxidation in the presence of non-limiting substrate concentrations (ammonia and O₂) is associated with N₂O production. An exceptional scenario that leads to such conditions is the periodical switch between anoxic and oxic conditions, which is rather common in engineered nitrogen-removal systems. In particular, the recovery from, rather than imposition of, anoxic conditions has been demonstrated to result in N₂O production. However, applied engineering perspectives, so far, have largely ignored the contribution of nitrification to N₂O emissions in greenhouse gas inventories from wastewater-treatment plants. Recent field-scale measurements have revealed that nitrification-related N₂O emissions are generally far higher than emissions assigned to heterotrophic denitrification. In the present paper, the metabolic pathways, which could potentially contribute to NO and N₂O production by AOB have been conceptually reconstructed under conditions especially relevant to engineered nitrogen-removal systems. Taken together, the reconstructed pathways, field- and laboratory-scale results suggest that engineering designs that achieve low effluent aqueous nitrogen concentrations also minimize gaseous nitrogen emissions.

Surveying N2O-producing pathways in bacteria.

Nitrous oxide (N(2)O) is produced by bacteria as an intermediate of both dissimilatory and detoxification pathways under a range of oxygen levels, although the majority of N(2)O is released in suboxic to anoxic environments. N(2)O production under physiologically relevant conditions appears to require the reduction of nitric oxide (NO) produced from the oxidation of hydroxylamine (nitrification), reduction of nitrite (denitrification), or by host cells of pathogenic bacteria. In a single bacterial isolate, N(2)O-producing pathways can be complex, overlapping, involve multiple enzymes with the same function, and require multiple layers of regulatory machinery. This overview discusses how to identify known N(2)O-producing inventory and regulatory sequences within bacterial genome sequences and basic physiological approaches for investigating the function of that inventory. A multitude of review articles have been published on individual enzymes, pathways, regulation, and environmental significance of N(2)O-production encompassing a large diversity of bacterial isolates. The combination of next-generation deep sequencing platforms, emerging proteomics technologies, and basic microbial physiology can be used to expand what is known about N(2)O-producing pathways in individual bacterial species to discover novel inventory and unifying features of pathways. A combination of approaches is required to understand and generalize the function and control of N(2)O production across a range of temporal and spatial scales within natural and host environments.

Adaptations to Submarine Hydrothermal Environments Exemplified by the Genome of Nautilia profundicola

Submarine hydrothermal vents are model systems for the Archaean Earth environment, and some sites maintain conditions that may have favored the formation and evolution of cellular life. Vents are typified by rapid fluctuations in temperature and redox potential that impose a strong selective pressure on resident microbial communities. Nautilia profundicola strain Am-H is a moderately thermophilic, deeply-branching Epsilonproteobacterium found free-living at hydrothermal vents and is a member of the microbial mass on the dorsal surface of vent polychaete, Alvinella pompejana. Analysis of the 1.7-Mbp genome of N. profundicola uncovered adaptations to the vent environment—some unique and some shared with other Epsilonproteobacterial genomes. The major findings included: (1) a diverse suite of hydrogenases coupled to a relatively simple electron transport chain, (2) numerous stress response systems, (3) a novel predicted nitrate assimilation pathway with hydroxylamine as a key intermediate, and (4) a gene (rgy) encoding the hallmark protein for hyperthermophilic growth, reverse gyrase. Additional experiments indicated that expression of rgy in strain Am-H was induced over 100-fold with a 20°C increase above the optimal growth temperature of this bacterium and that closely related rgy genes are present and expressed in bacterial communities residing in geographically distinct thermophilic environments. N. profundicola, therefore, is a model Epsilonproteobacterium that contains all the genes necessary for life in the extreme conditions widely believed to reflect those in the Archaean biosphere—anaerobic, sulfur, H2- and CO2-rich, with fluctuating redox potentials and temperatures. In addition, reverse gyrase appears to be an important and common adaptation for mesophiles and moderate thermophiles that inhabit ecological niches characterized by rapid and frequent temperature fluctuations and, as such, can no longer be considered a unique feature of hyperthermophiles.

Complete genome sequence of Nitrobacter hamburgensis X14 and comparative genomic analysis of species within the genus Nitrobacter.

ABSTRACT The alphaproteobacterium Nitrobacter hamburgensis X14 is a gram-negative facultative chemolithoautotroph that conserves energy from the oxidation of nitrite to nitrate. Sequencing and analysis of the Nitrobacter hamburgensis X14 genome revealed four replicons comprised of one chromosome (4.4 Mbp) and three plasmids (294, 188, and 121 kbp). Over 20% of the genome is composed of pseudogenes and paralogs. Whole-genome comparisons were conducted between N. hamburgensis and the finished and draft genome sequences of Nitrobacter winogradskyi and Nitrobacter sp. strain Nb-311A, respectively. Most of the plasmid-borne genes were unique to N. hamburgensis and encode a variety of functions (central metabolism, energy conservation, conjugation, and heavy metal resistance), yet ∼21 kb of a ∼28-kb “autotrophic” island on the largest plasmid was conserved in the chromosomes of Nitrobacter winogradskyi Nb-255 and Nitrobacter sp. strain Nb-311A. The N. hamburgensis chromosome also harbors many unique genes, including those for heme-copper oxidases, cytochrome b561, and putative pathways for the catabolism of aromatic, organic, and one-carbon compounds, which help verify and extend its mixotrophic potential. A Nitrobacter “subcore” genome was also constructed by removing homologs found in strains of the closest evolutionary relatives, Bradyrhizobium japonicum and Rhodopseudomonas palustris. Among the Nitrobacter subcore inventory (116 genes), copies of genes or gene clusters for nitrite oxidoreductase (NXR), cytochromes associated with a dissimilatory nitrite reductase (NirK), PII-like regulators, and polysaccharide formation were identified. Many of the subcore genes have diverged significantly from, or have origins outside, the alphaproteobacterial lineage and may indicate some of the unique genetic requirements for nitrite oxidation in Nitrobacter.

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 EN76T exhibits tight control over production and consumption of nitric oxide (NO) during ammonia catabolism, unlike the ammonia-oxidizing bacterium Nitrosospira multiformis ATCC 25196T. 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.

Ammonia cometabolism and product inhibition vary considerably among species of methanotrophic bacteria

Ecological studies have indicated that relative abundances of Gammaproteobacteria methanotrophs (Gamma-MOB) vs. Alphaproteobacteria methanotrophs (Alpha-MOB) in nitrogen (N) impacted soils are dictated in part by their abilities to tolerate inhibitory effects of ammonium and nitrite. In particular, ammonia is a cometabolic substrate and competitive inhibitor of methane monooxygenase. The rates of ammonia and hydroxylamine oxidation and inhibition of methane-oxidizing activity by ammonium and nitrite were compared among two Gamma-MOB and two Alpha-MOB to determine whether methanotrophs of the same class shared similar physiological profiles. Each isolate exhibited unique K(m(app)) for ammonia and V(max) for nitrite production with or without reductant (methane or sodium formate). The rates of nitrite production from hydroxylamine followed similar trends to rates of ammonia oxidation, indicating that hydroxylamine-oxidizing enzymes were central participants in the ammonia-oxidizing pathway. Methylomonas methanica was incapable of either ammonia or hydroxylamine oxidation. A broad range of sensitivities to ammonium and nitrite inhibition were measured with little consistency between isolates of the same class. The results indicate that physiological responses, and perhaps environmental adaptations, to N compounds are organism specific for methanotrophs.

Nitrifying and denitrifying pathways of methanotrophic bacteria.

Nitrous oxide, a potent greenhouse gas and ozone-depleting molecule, continues to accumulate in the atmosphere as a product of anthropogenic activities and land-use change. Nitrogen oxides are intermediates of nitrification and denitrification and are released as terminal products under conditions such as high nitrogen load and low oxygen tension among other factors. The rapid completion and public availability of microbial genome sequences has revealed a high level of enzymatic redundancy in pathways terminating in nitrogen oxide metabolites, with few enzymes involved in returning nitrogen oxides to dinitrogen. The aerobic methanotrophic bacteria are particularly useful for discovering and analysing diverse mechanisms for nitrogen oxide production, as these microbes both nitrify (oxidize ammonia to nitrite) and denitrify (reduce nitrate/nitrite to nitrous oxide via nitric oxide), and yet do not rely on these pathways for growth. The fact that methanotrophs have a rich inventory for nitrogen oxide metabolism is, in part, a consequence of their evolutionary relatedness to ammonia-oxidizing bacteria. Furthermore, the ability of individual methanotrophic taxa to resist toxic intermediates of nitrogen metabolism affects the relative abundance of nitrogen oxides released into the environment, the composition of their community, and the balance between nitrogen and methane cycling.

Autotrophic Ammonia-Oxidizing Bacteria Contribute Minimally to Nitrification in a Nitrogen-Impacted Forested Ecosystem

ABSTRACT Deposition rates of atmospheric nitrogenous pollutants to forests in the San Bernardino Mountains range east of Los Angeles, California, are the highest reported in North America. Acidic soils from the west end of the range are N-saturated and have elevated rates of N-mineralization, nitrification, and nitrate leaching. We assessed the impact of this heavy nitrogen load on autotrophic ammonia-oxidizing communities by investigating their composition, abundance, and activity. Analysis of 177 cloned β-Proteobacteria ammonia oxidizer 16S rRNA genes from highly to moderately N-impacted soils revealed similar levels of species composition; all of the soils supported the previously characterized Nitrosospira clusters 2, 3, and 4. Ammonia oxidizer abundance measured by quantitative PCR was also similar among the soils. However, rates of potential nitrification activity were greater for N-saturated soils than for soils collected from a less impacted site, but autotrophic (i.e., acetylene-sensitive) activity was low in all soils examined. N-saturated soils incubated for 30 days with ammonium accumulated additional soluble ammonium, whereas less-N-impacted soils had a net loss of ammonium. Lastly, nitrite production by cultivated Nitrosospira multiformis, an autotrophic ammonia-oxidizing bacterium adapted to relatively high ammonium concentrations, was significantly inhibited in pH-controlled slurries of sterilized soils amended with ammonium despite the maintenance of optimal ammonia-oxidizing conditions. Together, these results showed that factors other than autotrophic ammonia oxidizers contributed to high nitrification rates in these N-impacted forest soils and, unlike many other environments, differences in nitrogen content and soil pH did not favor particular autotrophic ammonia oxidizer groups.