Ammonia-oxidising archaea living at low pH: Insights from comparative genomics.

Obligate acidophilic members of the thaumarchaeotal genus Candidatus Nitrosotalea play an important role in nitrification in acidic soils, but their evolutionary and physiological adaptations to acidic environments are still poorly understood, with only a single member of this genus (Ca. N. devanaterra) having its genome sequenced. In this study, we sequenced the genomes of two additional cultured Ca. Nitrosotalea strains, extracted an almost complete Ca. Nitrosotalea metagenome-assembled genome from an acidic fen, and performed comparative genomics of the four Ca. Nitrosotalea genomes with 19 other archaeal ammonia oxidiser genomes. Average nucleotide and amino acid identities revealed that the four Ca. Nitrosotalea strains represent separate species within the genus. The four Ca. Nitrosotalea genomes contained a core set of 103 orthologous gene families absent from all other ammonia-oxidizing archaea and, for most of these gene families, expression could be demonstrated in laboratory culture or the environment via proteomic or metatranscriptomic analyses respectively. Phylogenetic analyses indicated that four of these core gene families were acquired by the Ca. Nitrosotalea common ancestor via horizontal gene transfer from acidophilic representatives of Euryarchaeota. We hypothesize that gene exchange with these acidophiles contributed to the competitive success of the Ca. Nitrosotalea lineage in acidic environments.

Acyl-Homoserine Lactone Production in Nitrifying Bacteria of the Genera Nitrosospira, Nitrobacter, and Nitrospira Identified via a Survey of Putative Quorum-Sensing Genes.

The genomes of many bacteria that participate in nitrogen cycling through the process of nitrification contain putative genes associated with acyl-homoserine lactone (AHL) quorum sensing (QS). AHL QS or bacterial cell-cell signaling is a method of bacterial communication and gene regulation and may be involved in nitrogen oxide fluxes or other important phenotypes in nitrifying bacteria. Here, we carried out a broad survey of AHL production in nitrifying bacteria in three steps. First, we analyzed the evolutionary history of AHL synthase and AHL receptor homologs in sequenced genomes and metagenomes of nitrifying bacteria to identify AHL synthase homologs in ammonia-oxidizing bacteria (AOB) of the genus Nitrosospira and nitrite-oxidizing bacteria (NOB) of the genera Nitrococcus, Nitrobacter, and Nitrospira Next, we screened cultures of both AOB and NOB with uncharacterized AHL synthase genes and AHL synthase-negative nitrifiers by a bioassay. Our results suggest that an AHL synthase gene is required for, but does not guarantee, cell density-dependent AHL production under the conditions tested. Finally, we utilized mass spectrometry to identify the AHLs produced by the AOB Nitrosospira multiformis and Nitrosospira briensis and the NOB Nitrobacter vulgaris and Nitrospira moscoviensis as N-decanoyl-l-homoserine lactone (C10-HSL), N-3-hydroxy-tetradecanoyl-l-homoserine lactone (3-OH-C14-HSL), a monounsaturated AHL (C10:1-HSL), and N-octanoyl-l-homoserine lactone (C8-HSL), respectively. Our survey expands the list of AHL-producing nitrifiers to include a representative of Nitrospira lineage II and suggests that AHL production is widespread in nitrifying bacteria.IMPORTANCE Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite by nitrifying microorganisms, plays an important role in environmental nitrogen cycling from agricultural fertilization to wastewater treatment. The genomes of many nitrifying bacteria contain genes associated with bacterial cell-cell signaling or quorum sensing (QS). QS is a method of bacterial communication and gene regulation that is well studied in bacterial pathogens, but less is known about QS in environmental systems. Our previous work suggested that QS might be involved in the regulation of nitrogen oxide gas production during nitrite metabolism. This study characterized putative QS signals produced by different genera and species of nitrifiers. Our work lays the foundation for future experiments investigating communication between nitrifying bacteria, the purpose of QS in these microorganisms, and the manipulation of QS during nitrification.

Steady-State Growth under Inorganic Carbon Limitation Conditions Increases Energy Consumption for Maintenance and Enhances Nitrous Oxide Production in Nitrosomonas europaea.

UNLABELLED: Nitrosomonas europaea is a chemolithoautotrophic bacterium that oxidizes ammonia (NH3) to obtain energy for growth on carbon dioxide (CO2) and can also produce nitrous oxide (N2O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses of N. europaea to conditions of replete (>5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na2CO3) supplemented with atmospheric CO2 IC-limited cultures oxidized 25 to 58% of available NH3 to nitrite, depending on the dilution rate and Na2CO3 concentration. IC limitation resulted in a 2.3-fold increase in cellular maintenance energy requirements compared to those for NH3-limited cultures. Rates of N2O production increased 2.5- and 6.3-fold under the two IC-limited conditions, increasing the percentage of oxidized NH3-N that was transformed to N2O-N from 0.5% (replete) up to 4.4% (0.2 mM Na2CO3). Transcriptome analysis showed differential expression (P ≤ 0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in a decreased expression of ammonium/ammonia transporter and ammonia monooxygenase subunits and increased the expression of genes involved in C1 metabolism, including the genes for RuBisCO (cbb gene cluster), carbonic anhydrase, folate-linked metabolism of C1 moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924 to NE0927) correlated with increased production of N2O. Together, these data suggest that N. europaea adapts physiologically during IC-limited steady-state growth, which leads to the uncoupling of NH3 oxidation from growth and increased N2O production. IMPORTANCE: Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, is an important process in the global nitrogen cycle. This process is generally dependent on ammonia-oxidizing microorganisms and nitrite-oxidizing bacteria. Most nitrifiers are chemolithoautotrophs that fix inorganic carbon (CO2) for growth. Here, we investigate how inorganic carbon limitation modifies the physiology and transcriptome of Nitrosomonas europaea, a model ammonia-oxidizing bacterium, and report on increased production of N2O, a potent greenhouse gas. This study, along with previous work, suggests that inorganic carbon limitation may be an important factor in controlling N2O emissions from nitrification in soils and wastewater treatment.

Identifying Potential Mechanisms Enabling Acidophily in the Ammonia-Oxidizing Archaeon "Candidatus Nitrosotalea devanaterra".

Ammonia oxidation is the first and rate-limiting step in nitrification and is dominated by two distinct groups of microorganisms in soil: ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). AOA are often more abundant than AOB and dominate activity in acid soils. The mechanism of ammonia oxidation under acidic conditions has been a long-standing paradox. While high rates of ammonia oxidation are frequently measured in acid soils, cultivated ammonia oxidizers grew only at near-neutral pH when grown in standard laboratory culture. Although a number of mechanisms have been demonstrated to enable neutrophilic AOB growth at low pH in the laboratory, these have not been demonstrated in soil, and the recent cultivation of the obligately acidophilic ammonia oxidizer "Candidatus Nitrosotalea devanaterra" provides a more parsimonious explanation for the observed high rates of activity. Analysis of the sequenced genome, transcriptional activity, and lipid content of "Ca Nitrosotalea devanaterra" reveals that previously proposed mechanisms used by AOB for growth at low pH are not essential for archaeal ammonia oxidation in acidic environments. Instead, the genome indicates that "Ca Nitrosotalea devanaterra" contains genes encoding both a predicted high-affinity substrate acquisition system and potential pH homeostasis mechanisms absent in neutrophilic AOA. Analysis of mRNA revealed that candidate genes encoding the proposed homeostasis mechanisms were all expressed during acidophilic growth, and lipid profiling by high-performance liquid chromatography-mass spectrometry (HPLC-MS) demonstrated that the membrane lipids of "Ca Nitrosotalea devanaterra" were not dominated by crenarchaeol, as found in neutrophilic AOA. This study for the first time describes a genome of an obligately acidophilic ammonia oxidizer and identifies potential mechanisms enabling this unique phenotype for future biochemical characterization.

Hydroxylamine as an intermediate in ammonia oxidation by globally abundant marine archaea.

The ammonia-oxidizing archaea have recently been recognized as a significant component of many microbial communities in the biosphere. Although the overall stoichiometry of archaeal chemoautotrophic growth via ammonia (NH(3)) oxidation to nitrite (NO(2)(-)) is superficially similar to the ammonia-oxidizing bacteria, genome sequence analyses point to a completely unique biochemistry. The only genomic signature linking the bacterial and archaeal biochemistries of NH(3) oxidation is a highly divergent homolog of the ammonia monooxygenase (AMO). Although the presumptive product of the putative AMO is hydroxylamine (NH(2)OH), the absence of genes encoding a recognizable ammonia-oxidizing bacteria-like hydroxylamine oxidoreductase complex necessitates either a novel enzyme for the oxidation of NH(2)OH or an initial oxidation product other than NH(2)OH. We now show through combined physiological and stable isotope tracer analyses that NH(2)OH is both produced and consumed during the oxidation of NH(3) to NO(2)(-) by Nitrosopumilus maritimus, that consumption is coupled to energy conversion, and that NH(2)OH is the most probable product of the archaeal AMO homolog. Thus, despite their deep phylogenetic divergence, initial oxidation of NH(3) by bacteria and archaea appears mechanistically similar. They however diverge biochemically at the point of oxidation of NH(2)OH, the archaea possibly catalyzing NH(2)OH oxidation using a novel enzyme complex.

Nitrite-Oxidizing Bacterium Nitrobacter winogradskyi Produces N-Acyl-Homoserine Lactone Autoinducers.

Nitrobacter winogradskyi is a chemolithotrophic bacterium that plays a role in the nitrogen cycle by oxidizing nitrite to nitrate. Here, we demonstrate a functional N-acyl-homoserine lactone (acyl-HSL) synthase in this bacterium. The N. winogradskyi genome contains genes encoding a putative acyl-HSL autoinducer synthase (nwi0626, nwiI) and a putative acyl-HSL autoinducer receptor (nwi0627, nwiR) with amino acid sequences 38 to 78% identical to those in Rhodopseudomonas palustris and other Rhizobiales. Expression of nwiI and nwiR correlated with acyl-HSL production during culture. N. winogradskyi produces two distinct acyl-HSLs, N-decanoyl-l-homoserine lactone (C10-HSL) and a monounsaturated acyl-HSL (C10:1-HSL), in a cell-density- and growth phase-dependent manner, during batch and chemostat culture. The acyl-HSLs were detected by bioassay and identified by ultraperformance liquid chromatography with information-dependent acquisition mass spectrometry (UPLC-IDA-MS). The C=C bond in C10:1-HSL was confirmed by conversion into bromohydrin and detection by UPLC-IDA-MS.

Interactions of Nitrosomonas europaea and Nitrobacter winogradskyi grown in co-culture.

Nitrosomonas europaea and Nitrobacter winogradskyi were grown singly and in co-culture in chemostats to probe for physiological differences between the two growth conditions. Co-culture growth medium containing 60 mM NH4 (+) resulted in a cell density (0.20-0.29 OD600) greater than the sum of the densities in single chemostat cultures, i.e., 0.09-0.14 OD600 for N. europaea with 60 mM NH4 (+)and 0.04-0.06 OD600 for N. winogradskyi with 60 mM NO2 (-). The NO2 (-)- and NH4 (+)-dependent O2 uptake rates, qRT-PCR, and microscopic observations indicated that in co-culture, N. europaea contributed ~0.20 OD600 (~80 %) and N. winogradskyi ~0.05 OD600 (~20 %). In co-culture, the transcriptomes showed that the mRNA levels of 773 genes in N. europaea (30.2 % of the genes) and of 372 genes in N. winogradskyi (11.8 % of the genes) changed significantly. Total cell growth and the analysis of the transcriptome revealed that in co-culture, N. europaea benefits more than N. winogradskyi.

Characterization of a nitrite reductase involved in nitrifier denitrification.

Nitrifier denitrification is the conversion of nitrite to nitrous oxide by ammonia-oxidizing organisms. This process, which is distinct from denitrification, is active under aerobic conditions in the model nitrifier Nitrosomonas europaea. The central enzyme of the nitrifier dentrification pathway is a copper nitrite reductase (CuNIR). To understand how a CuNIR, typically inactivated by oxygen, functions in this pathway, the enzyme isolated directly from N. europaea (NeNIR) was biochemically and structurally characterized. NeNIR reduces nitrite at a similar rate to other CuNIRs but appears to be oxygen tolerant. Crystal structures of oxidized and reduced NeNIR reveal a substrate channel to the active site that is much more restricted than channels in typical CuNIRs. In addition, there is a second fully hydrated channel leading to the active site that likely acts a water exit pathway. The structure is minimally affected by changes in pH. Taken together, these findings provide insight into the molecular basis for NeNIR oxygen tolerance.

Global analysis of the Nitrosomonas europaea iron starvation stimulon.

The importance of iron to the metabolism of the ammonia-oxidizing bacterium Nitrosomonas europaea is well known. However, the mechanisms by which N. europaea acquires iron under iron limitation are less well known. To obtain insight into these mechanisms, transcriptional profiling of N. europaea was performed during growth under different iron availabilities. Of 2,355 N. europaea genes on DNA microarrays, transcripts for 247 genes were identified as differentially expressed when cells were grown under iron limitation compared to cells grown under iron-replete conditions. Genes with higher transcript levels in response to iron limitation included those with confirmed or assigned roles in iron acquisition. Genes with lower transcript levels included those encoding iron-containing proteins. Our analysis identified several potentially novel iron acquisition systems in N. europaea and provided support for the primary involvement of a TonB-dependent heme receptor gene in N. europaea iron homeostasis. We demonstrated that hemoglobin can act as an iron source under iron-depleted conditions for N. europaea. In addition, we identified a hypothetical protein carrying a lipocalin-like domain that may have the ability to chelate iron for growth in iron-limited media.

Role of a Fur homolog in iron metabolism in Nitrosomonas europaea.

BACKGROUND: In response to environmental iron concentrations, many bacteria coordinately regulate transcription of genes involved in iron acquisition via the ferric uptake regulation (Fur) system. The genome of Nitrosomonas europaea, an ammonia-oxidizing bacterium, carries three genes (NE0616, NE0730 and NE1722) encoding proteins belonging to Fur family. RESULTS: Of the three N. europaea fur homologs, only the Fur homolog encoded by gene NE0616 complemented the Escherichia coli H1780 fur mutant. A N. europaea fur:kanP mutant strain was created by insertion of kanamycin-resistance cassette in the promoter region of NE0616 fur homolog. The total cellular iron contents of the fur:kanP mutant strain increased by 1.5-fold compared to wild type when grown in Fe-replete media. Relative to the wild type, the fur:kanP mutant exhibited increased sensitivity to iron at or above 500 µM concentrations. Unlike the wild type, the fur:kanP mutant was capable of utilizing iron-bound ferrioxamine without any lag phase and showed over expression of several outer membrane TonB-dependent receptor proteins irrespective of Fe availability. CONCLUSIONS: Our studies have clearly indicated a role in Fe regulation by the Fur protein encoded by N. europaea NE0616 gene. Additional studies are required to fully delineate role of this fur homolog.

Role of Nitrosomonas europaea NitABC iron transporter in the uptake of Fe3+-siderophore complexes.

Nitrosomonas europaea has a single three-gene operon (nitABC) encoding an iron ABC transporter system (NitABC). Phylogenetic analysis clustered the subunit NitB with Fe(3+)-ABC transporter permease components from other organisms. The N. europaea strain deficient in nitB (nitB::kan) grew well in either Fe-replete or Fe-limited media and in Fe-limited medium containing the catecholate-type siderophore, enterobactin or the citrate-based dihydroxamate-type siderophore, aerobactin. However, the nitB::kan mutant strain was unable to grow in Fe-limited media containing either the hydroxamate-type siderophores, ferrioxamine and ferrichrome or the mixed-chelating type siderophore, pyoverdine. Exposure of N. europaea cells to a ferrichrome analog coupled to the fluorescent moiety naphthalic diimide (Fhu-NI) led to increase in fluorescence in the wild type but not in nitB::kan mutant cells. Spheroplasts prepared from N. europaea wild type exposed to Fhu-NI analog retained the fluorescence, while spheroplasts of the nitB::kan mutant were not fluorescent. NitABC transports intact Fe(3+)-ferrichrome complex into the cytoplasm and is an atypical ABC type iron transporter for Fe(3+) bound to ferrioxamine, ferrichrome or pyoverdine siderophores into the cytoplasm. The mechanisms to transport iron in either the Fe(3+) or Fe(2+) forms or Fe(3+) associated with enterobactin or aerobactin siderophores into the cell across the cytoplasmic membrane are as yet undetermined.

Nitrification and degradation of halogenated hydrocarbons--a tenuous balance for ammonia-oxidizing bacteria.

The process of nitrification has the potential for the in situ bioremediation of halogenated compounds provided a number of challenges can be overcome. In nitrification, the microbial process where ammonia is oxidized to nitrate, ammonia-oxidizing bacteria (AOB) are key players and are capable of carrying out the biodegradation of recalcitrant halogenated compounds. Through industrial uses, halogenated compounds often find their way into wastewater, contaminating the environment and bodies of water that supply drinking water. In the reclamation of wastewater, halogenated compounds can be degraded by AOB but can also be detrimental to the process of nitrification. This minireview considers the ability of AOB to carry out cometabolism of halogenated compounds and the consequent inhibition of nitrification. Possible cometabolism monitoring methods that were derived from current information about AOB genomes are also discussed. AOB expression microarrays have detected mRNA of genes that are expressed at higher levels during stress and are deemed "sentinel" genes. Promoters of selected "sentinel" genes have been cloned and used to drive the expression of gene-reporter constructs. The latter are being tested as early warning biosensors of cometabolism-induced damage in Nitrosomonas europaea with promising results. These and other biosensors may help to preserve the tenuous balance that exists when nitrification occurs in waste streams containing alternative AOB substrates such as halogenated hydrocarbons.

Transcriptomic Response of Nitrosomonas europaea Transitioned from Ammonia- to Oxygen-Limited Steady-State Growth.

Ammonia-oxidizing microorganisms perform the first step of nitrification, the oxidation of ammonia to nitrite. The bacterium Nitrosomonas europaea is the best-characterized ammonia oxidizer to date. Exposure to hypoxic conditions has a profound effect on the physiology of N. europaea, e.g., by inducing nitrifier denitrification, resulting in increased nitric and nitrous oxide production. This metabolic shift is of major significance in agricultural soils, as it contributes to fertilizer loss and global climate change. Previous studies investigating the effect of oxygen limitation on N. europaea have focused on the transcriptional regulation of genes involved in nitrification and nitrifier denitrification. Here, we combine steady-state cultivation with whole-genome transcriptomics to investigate the overall effect of oxygen limitation on N. europaea Under oxygen-limited conditions, growth yield was reduced and ammonia-to-nitrite conversion was not stoichiometric, suggesting the production of nitrogenous gases. However, the transcription of the principal nitric oxide reductase (cNOR) did not change significantly during oxygen-limited growth, while the transcription of the nitrite reductase-encoding gene (nirK) was significantly lower. In contrast, both heme-copper-containing cytochrome c oxidases encoded by N. europaea were upregulated during oxygen-limited growth. Particularly striking was the significant increase in transcription of the B-type heme-copper oxidase, proposed to function as a nitric oxide reductase (sNOR) in ammonia-oxidizing bacteria. In the context of previous physiological studies, as well as the evolutionary placement of N. europaea's sNOR with regard to other heme-copper oxidases, these results suggest sNOR may function as a high-affinity terminal oxidase in N. europaea and other ammonia-oxidizing bacteria.IMPORTANCE Nitrification is a ubiquitous microbially mediated process in the environment and an essential process in engineered systems such as wastewater and drinking water treatment plants. However, nitrification also contributes to fertilizer loss from agricultural environments, increasing the eutrophication of downstream aquatic ecosystems, and produces the greenhouse gas nitrous oxide. As ammonia-oxidizing bacteria are the most dominant ammonia-oxidizing microbes in fertilized agricultural soils, understanding their responses to a variety of environmental conditions is essential for curbing the negative environmental effects of nitrification. Notably, oxygen limitation has been reported to significantly increase nitric oxide and nitrous oxide production during nitrification. Here, we investigate the physiology of the best-characterized ammonia-oxidizing bacterium, Nitrosomonas europaea, growing under oxygen-limited conditions.

Construction of recombinant Nitrosomonas europaea expressing green fluorescent protein in response to co-oxidation of chloroform.

Transcriptional fusions with gfp driven by the promoter region of mbla (NE2571) in pPRO/mbla4 and clpB (NE2402) in pPRO/clpb7 were used to transform the ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718). The two genes were chosen because their transcript levels were found at much higher levels in N. europaea in response to oxidation of chloroform and chloromethane. In N. europaea transformed with pPRO/mbla4, green fluorescent protein (GFP)-dependent fluorescence increased from 3- to 18-fold above control levels in response to increasing chloroform concentrations (7 to 28 microM), and from 8- to 10-fold in response to increasing hydrogen peroxide concentrations (2.5-7.5 mM). The GFP-dependent fluorescence of N. europaea transformed with pPRO/clpb7 also showed an increase of 6- to 10-fold in response to chloroform (28-100 microM) but did not respond to H(2)O(2). Our data provide proof of concept that biosensors can be fabricated in ammonia-oxidizing bacteria using "sentinel" genes that up-regulate in response to stress caused either by co-oxidation of chlorinated solvents or by the presence of H(2)O(2). The fabricated biosensors had a consistent concentration-dependent response to chloroform; however, these did not respond to other chlorinated compounds that cause similar cellular stress.

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

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 approximately 21 kb of a approximately 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 b(561), 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.

Nitrite-oxidizing activity responds to nitrite accumulation in soil.

The factors influencing how soil nitrite (NO2-)- and ammonia (NH3)-oxidizing activities remain coupled are unknown. A short-term study (<48 h) was conducted to examine the dynamics of NO2--oxidizing activity and the accumulation of NO2- in three Oregon soils stimulated by the addition of 1 mM NH4+ in soil slurry. Nitrite initially accumulated in all three soils; its subsequent decline or slowing of the accumulation of the NO2- pool by 24 h was accompanied by an increase in the size of the nitrate (NO3-) pool, indicating a change in NO2- oxidation kinetics. Bacterial protein synthesis inhibitors prevented the NO2- pool decline, resulting in a larger accumulation in all three soils. Although no significant increases in NO2--oxidizing bacteria nxrA (Nitrobacter) and nxrB (Nitrospira) gene abundances were detected over the time course, maximum NO2- consumption rates increased 2-fold in the treatment without antibiotics compared to no change with antibiotics. No changes were observed in the apparent half saturation constant (Km) values for NO2- consumption. This study demonstrates phenotypic flexibility among soil NO2- oxidizers, which can undergo protein synthesis-dependent increases in NO2- consumption rates to match NH3 oxidation rates and recouple nitrification.

Genome-Scale, Constraint-Based Modeling of Nitrogen Oxide Fluxes during Coculture of Nitrosomonas europaea and Nitrobacter winogradskyi.

Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, emits nitrogen (N) oxide gases (NO, NO2, and N2O), which are potentially hazardous compounds that contribute to global warming. To better understand the dynamics of nitrification-derived N oxide production, we conducted culturing experiments and used an integrative genome-scale, constraint-based approach to model N oxide gas sources and sinks during complete nitrification in an aerobic coculture of two model nitrifying bacteria, the ammonia-oxidizing bacterium Nitrosomonas europaea and the nitrite-oxidizing bacterium Nitrobacter winogradskyi. The model includes biotic genome-scale metabolic models (iFC578 and iFC579) for each nitrifier and abiotic N oxide reactions. Modeling suggested both biotic and abiotic reactions are important sources and sinks of N oxides, particularly under microaerobic conditions predicted to occur in coculture. In particular, integrative modeling suggested that previous models might have underestimated gross NO production during nitrification due to not taking into account its rapid oxidation in both aqueous and gas phases. The integrative model may be found at https://github.com/chaplenf/microBiome-v2.1. IMPORTANCE Modern agriculture is sustained by application of inorganic nitrogen (N) fertilizer in the form of ammonium (NH4+). Up to 60% of NH4+-based fertilizer can be lost through leaching of nitrifier-derived nitrate (NO3-), and through the emission of N oxide gases (i.e., nitric oxide [NO], N dioxide [NO2], and nitrous oxide [N2O] gases), the latter being a potent greenhouse gas. Our approach to modeling of nitrification suggests that both biotic and abiotic mechanisms function as important sources and sinks of N oxides during microaerobic conditions and that previous models might have underestimated gross NO production during nitrification.

Stress response of a marine ammonia-oxidizing archaeon informs physiological status of environmental populations.

High representation by ammonia-oxidizing archaea (AOA) in marine systems is consistent with their high affinity for ammonia, efficient carbon fixation, and copper (Cu)-centric respiratory system. However, little is known about their response to nutrient stress. We therefore used global transcriptional and proteomic analyses to characterize the response of a model AOA, Nitrosopumilus maritimus SCM1, to ammonia starvation, Cu limitation and Cu excess. Most predicted protein-coding genes were transcribed in exponentially growing cells, and of ~74% detected in the proteome, ~6% were modified by N-terminal acetylation. The general response to ammonia starvation and Cu stress was downregulation of genes for energy generation and biosynthesis. Cells rapidly depleted transcripts for the A and B subunits of ammonia monooxygenase (AMO) in response to ammonia starvation, yet retained relatively high levels of transcripts for the C subunit. Thus, similar to ammonia-oxidizing bacteria, selective retention of amoC transcripts during starvation appears important for subsequent recovery, and also suggests that AMO subunit transcript ratios could be used to assess the physiological status of marine populations. Unexpectedly, cobalamin biosynthesis was upregulated in response to both ammonia starvation and Cu stress, indicating the importance of this cofactor in retaining functional integrity during times of stress.

Draft Genome Sequence of Nitrobacter vulgaris Strain Ab1, a Nitrite-Oxidizing Bacterium.

Here, we present the 3.9-Mb draft genome sequence of Nitrobacter vulgaris strain Ab1, which was isolated from a sewage system in Hamburg, Germany. The analysis of its genome sequence will contribute to our knowledge of nitrite-oxidizing bacteria and acyl-homoserine lactone quorum sensing in nitrifying bacteria.

Transcript profiles of Nitrosomonas europaea during growth and upon deprivation of ammonia and carbonate.

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.