Effects of nitrite on ammonia-oxidizing activity and gene regulation in three ammonia-oxidizing bacteria

Nitrite is the highly toxic end product of ammonia oxidation that accumulates in the absence of a nitrite-consuming process and is inhibitory to nitrifying and other bacteria. The effects of nitrite on ammonia oxidation rates and regulation of a common gene set were compared in three ammonia-oxidizing bacteria (AOB) to determine whether responses to this toxic metabolite were uniform. Mid-exponential-phase cells of Nitrosomonas europaea ATCC 19718, Nitrosospira multiformis ATCC 25196, and Nitrosomonas eutropha C-91 were incubated for 6 h in mineral medium supplemented with 0, 10, or 20 mM NaNO(2) . The rates of ammonia oxidation (nitrite production) decreased significantly only in NaNO(2) -supplemented incubations of N. eutropha; no significant effect on the rates was observed for N. europaea or N. multiformis. The levels of norB (nitric oxide reductases), cytL (cytochrome P460), and cytS (cytochrome c'-β) mRNA were unaffected by nitrite in all strains. The levels of nirK (nitrite reductase) mRNA increased only in N. europaea in response to nitrite (10 and 20 mM). Nitrite (20 mM) significantly reduced the mRNA levels of amoA (ammonia monooxygenase) in N. multiformis and norS (nitric oxide reductase) in the two Nitrosomonas spp. Differences in response to nitrite indicated nonuniform adaptive and regulatory strategies of AOB, even between closely related species.     

Quantitative proteomic analysis of the chemolithoautotrophic bacterium Nitrosomonas europaea: comparison of growing- and energy-starved cells

Obligately aerobic ammonia-oxidizing bacteria (AOB) like Nitrosomonas europaea play a pivotal role in the global nitrogen cycle. Although starvation tolerance is a key environmental adaptation, little is known about this response in AOB. The goal of these studies was to compare the composition of the N. europaea proteome in growing- and energy-starved cells using 1?N labeling and HPLC-ESI-MS/MS. More than 6500 peptides were sequenced with high confidence, and matched to 876 proteins (34% of the protein coding genes). Of these, 126 proteins had two or more peptide forms identified by 10 or more scans, and were used in quantitative analysis and 27 were found to be significantly different in abundance between growing and starved cells. Proteins showing greater abundance in growing cells are geared toward biosynthesis, particularly DNA replication. Energy-starved cells were shifted away from biosynthesis and toward survival functions that included: cell envelope modification, protein protection/degradation, detoxification, and implementation of alternative energy generation mechanisms. Most of these activities have not previously been reported as associated with energy-starvation stress in N. europaea. This study provides insights into the potential effects of fluctuating environmental conditions on the regulation of physiological networks in N. europaea.     

Factors Limiting Aliphatic Chlorocarbon Degradation by Nitrosomonas europaea: Cometabolic Inactivation of Ammonia Monooxygenase and Substrate Specificity

The soil nitrifying bacterium Nitrosomonas europaea is capable of degrading trichloroethylene (TCE) and other halogenated hydrocarbons. TCE cometabolism by N. europaea resulted in an irreversible loss of TCE biodegradative capacity, ammonia-oxidizing activity, and ammonia-dependent O(2) uptake by the cells. Inactivation was not observed in the presence of allylthiourea, a specific inhibitor of the enzyme ammonia monooxygenase, or under anaerobic conditions, indicating that the TCE-mediated inactivation required ammonia monooxygenase activity. When N. europaea cells were incubated with [C]TCE under conditions which allowed turnover of ammonia monooxygenase, a number of cellular proteins were covalently labeled with C. Treatment of cells with allylthiourea or acetylene prior to incubation with [C]TCE prevented incorporation of C into proteins. The ammonia-oxidizing activity of cells inactivated in the presence of TCE could be recovered through a process requiring de novo protein synthesis. In addition to TCE, a series of chlorinated methanes, ethanes, and other ethylenes were screened as substrates for ammonia monooxygenase and for their ability to inactivate the ammonia-oxidizing system of N. europaea. The chlorocarbons could be divided into three classes depending on their biodegradability and inactivating potential: (i) compounds which were not biodegradable by N. europaea and which had no toxic effect on the cells; (ii) compounds which were cooxidized by N. europaea and had little or no toxic effect on the cells; and (iii) compounds which were cooxidized and produced a turnover-dependent inactivation of ammonia oxidation by N. europaea.     

Protocol for mutagenesis of alkene monooxygenase and screening for modified enantiocomposition of the epoxypropane product

Alkene monooxygenase (AMO) from Rhodococcus rhodochrous B-276 is a 3-component enzyme system encoded by the 4-gene operon amoABCD, which catalyzes the stereoselective epoxidation of aliphatic alkenes yielding primarily the R enantiomer. With propene as the substrate, wild-type AMO yields R-epoxypropane with an enantiomeric excess (e.e.) of 83%. The presumed site of alkene oxidation is a dinuclear iron center situated within the large subunit of the epoxygenase component, AmoC. Substantial problems with the expression of recombinant AMO were previously overcome. In this study, the authors have further developed this expression system to allow amoC to be subjected to mutagenesis by means of error-prone PCR, with the aim of developing a system that could be used to manipulate the enantioselectivity of the enzyme. The mutants were screened for altered stereoselectivity in the propene/epoxypropane reaction by a whole-cell assay, solvent extraction, and chiral gas chromatography analysis protocol that is suitable for scale up to several thousand mutants and that is estimated to detect differences in e.e. of as little as 5%.     

Inhibition of Ammonia Oxidation in Nitrosomonas europaea by Sulfur Compounds: Thioethers Are Oxidized to Sulfoxides by Ammonia Monooxygenase

Organic sulfur compounds are well-known nitrification inhibitors. The inhibitory effects of dimethylsulfide, dimethyldisulfide, and ethanethiol on ammonia oxidation by Nitrosomonas europaea were examined. Both dimethylsulfide and dimethyldisulfide were weak inhibitors of ammonia oxidation and exhibited inhibitory characteristics typical of substrates for ammonia monooxygenase (AMO). Depletion of dimethylsulfide required O(2) and was prevented with either acetylene or allylthiourea, two inhibitors of AMO. The inhibition of ammonia oxidation by dimethylsulfide was examined in detail. Cell suspensions incubated in the presence of ammonia oxidized dimethylsulfide to dimethyl sulfoxide. Depletion of six other thioethers was also prevented by treating cell suspensions with either allylthiourea or acetylene. The oxidative products of three thioethers were identified as the corresponding sulfoxides. The amount of sulfoxide formed accounted for a majority of the amount of sulfide depleted. By using gas chromatography coupled with mass spectrometry, allylmethylsulfide was shown to be oxidized to allylmethylsulfoxide by N. europaea with the incorporation of a single atom of O derived from O(2) into the sulfide. This result supported our conclusion that a monooxygenase was involved in the oxidation of allylmethylsulfide. The thioethers are concluded to be a new class of substrates for AMO. This is the first report of the oxidation of the sulfur atom by AMO in whole cells of N. europaea. The ability of N. europaea to oxidize dimethylsulfide is not unique among the ammonia-oxidizing bacteria. Nitrosococcus oceanus, a marine nitrifier, was also demonstrated to oxidize dimethylsulfide to dimethyl sulfoxide.     

Molecular biology and biochemistry of ammonia oxidation by Nitrosomonas europaea

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.     

Whole-genome analysis of the ammonia-oxidizing bacterium, Nitrosomonas eutropha C91: implications for niche adaptation

Analysis of the structure and inventory of the genome of Nitrosomonas eutropha C91 revealed distinctive features that may explain the adaptation of N. eutropha-like bacteria to N-saturated ecosystems. Multiple gene-shuffling events are apparent, including mobilized and replicated transposition, as well as plasmid or phage integration events into the 2.66 Mbp chromosome and two plasmids (65 and 56 kbp) of N. eutropha C91. A 117 kbp genomic island encodes multiple genes for heavy metal resistance, including clusters for copper and mercury transport, which are absent from the genomes of other ammonia-oxidizing bacteria (AOB). Whereas the sequences of the two ammonia monooxygenase and three hydroxylamine oxidoreductase gene clusters in N. eutropha C91 are highly similar to those of Nitrosomonas europaea ATCC 19718, a break of synteny in the regions flanking these clusters in each genome is evident. Nitrosomonas eutropha C91 encodes four gene clusters for distinct classes of haem-copper oxidases, two of which are not found in other aerobic AOB. This diversity of terminal oxidases may explain the adaptation of N. eutropha to environments with variable O(2) concentrations and/or high concentrations of nitrogen oxides. As with N. europaea, the N. eutropha genome lacks genes for urease metabolism, likely disadvantaging nitrosomonads in low-nitrogen or acidic ecosystems. Taken together, this analysis revealed significant genomic variation between N. eutropha C91 and other AOB, even the closely related N. europaea, and several distinctive properties of the N. eutropha genome that are supportive of niche specialization.     

A rapid and simple respirometric biosensor with immobilized cells of Nitrosomonas europaea for detecting inhibitors of ammonia oxidation

As obligate chemolithotrophs, ammonia-oxidizing bacteria (AOB) grow very slowly and are known to be extremely sensitive to a wide variety of inhibitors. Since it is generally accepted that inhibition of ammonia oxidation by AOB results in a total failure of nitrogen removal, it is necessary to develop a method to detect inhibitors of ammonia oxidation in wastewater. Since ammonia oxidation accompanies oxygen consumption, ammonia oxidation can be easily evaluated by measuring oxygen consumption rate using a dissolved oxygen (DO) probe. In this study, a rapid and simple respirometric biosensor using the pure culture of Nitrosomonas europaea was developed. N. europaea was cultivated in a continuous fermentor operating at the dilution rate of 0.008 h(-1) to obtain physiologically constant cells and was immobilized onto the dialysis membrane through filtration. DO, determined by the biosensor, started to increase 30 s later after ammonia oxidation inhibitor was fed, and a new steady-state DO was obtained in 10-30 min. For this DO profile, steady-state kinetics was applied to evaluate ammonia oxidation efficiency. The concentration of a toxic compound causing 50% decrease of oxygen-consumption activity (EC50) was determined for different chemicals. The EC50 values obtained with the biosensor (0.018 mg l(-1) for allylthiourea, 0.027 mg l(-1) for thioacetamide, 1.10 mg l(-1) for phenol and 0.0 1mg l(-1) for thiourea) indicated that the developed biosensor was highly sensitive to a variety of the inhibitors. It was also shown that the biosensor is applicable for on-line real time monitoring.     

Habitat-Associated Phylogenetic Community Patterns of Microbial Ammonia Oxidizers

Microorganisms mediating ammonia oxidation play a fundamental role in the connection between biological nitrogen fixation and anaerobic nitrogen losses. Bacteria and Archaea ammonia oxidizers (AOB and AOA, respectively) have colonized similar habitats worldwide. Ammonia oxidation is the rate-limiting step in nitrification, and the ammonia monooxygenase (Amo) is the key enzyme involved. The molecular ecology of this process has been extensively explored by surveying the gene of the subunit A of the Amo (amoA gene). In the present study, we explored the phylogenetic community ecology of AOB and AOA, analyzing 5776 amoA gene sequences from >300 isolation sources, and clustering habitats by environmental ontologies. As a whole, phylogenetic richness was larger in AOA than in AOB, and sediments contained the highest phylogenetic richness whereas marine plankton the lowest. We also observed that freshwater ammonia oxidizers were phylogenetically richer than their marine counterparts. AOA communities were more dissimilar to each other than those of AOB, and consistent monophyletic lineages were observed for sediments, soils, and marine plankton in AOA but not in AOB. The diversification patterns showed a more constant cladogenesis through time for AOB whereas AOA apparently experienced two fast diversification events separated by a long steady-state episode. The diversification rate (γ statistic) for most of the habitats indicated γ_AOA > γ_AOB. Soil and sediment experienced earlier bursts of diversification whereas habitats usually eutrophic and rich in ammonium such as wastewater and sludge showed accelerated diversification rates towards the present. Overall, this work shows for the first time a global picture of the phylogenetic community structure of both AOB and AOA assemblages following the strictest analytical standards, and provides an ecological view on the differential evolutionary paths experienced by widespread ammonia-oxidizing microorganisms. The emerged picture of AOB and AOA distribution in different habitats provides a new view to understand the ecophysiology of ammonia oxidizers on Earth.     

Bacteria dominate the ammonia-oxidizing community in a hydrothermal vent site at the Mid-Atlantic Ridge of the South Atlantic Ocean

Ammonia oxidation is the first and rate-limiting step of nitrification, which is carried out by two groups of microorganisms: ammonia-oxidizing bacteria (AOB) and the recently discovered ammonia-oxidizing archaea (AOA). In this study, diversity and abundance of AOB and AOA were investigated in five rock samples from a deep-sea hydrothermal vent site at the Mid-Atlantic Ridge (MAR) of the South Atlantic Ocean. Both bacterial and archaeal ammonia monooxygenase subunit A (amoA) gene sequences obtained in this study were closely related to the sequences retrieved from deep-sea environments, indicating that AOB and AOA in this hydrothermal vent site showed typical deep ocean features. AOA were more diverse but less abundant than AOB. The ratios of AOA/AOB amoA gene abundance ranged from 1/3893 to 1/242 in all investigate samples, indicating that bacteria may be the major members responding to the aerobic ammonia oxidation in this hydrothermal vent site. Furthermore, diversity and abundance of AOA and AOB were significantly correlated with the contents of total nitrogen and total sulfur in investigated samples, suggesting that these two environmental factors exert strong influences on distribution of ammonia oxidizers in deep-sea hydrothermal vent environment.     

好氧氨氧化菌的种群生态学研究进展

好氧氨氧化菌是一类能够在好氧条件下将NH4^＋转化为NO2^-的化能无机自养型细菌,其活动将直接或间接影响土壤养分循环、水体富营养化、温室气体（N2O）和生态系统的功能。现代分子生物学技术的发展促进了人们对好氧氨氧化菌种群生态学的研究。介绍了近年来基于16SrRNA和氨单加氧酶amoA基因序列分析的好氧氨氧化菌的系统发育研究,比较了两种基因序列分析在好氧氨氧化菌遗传多样性研究中存在的差异;概述了环境条件诸如铵浓度、酸度、氧的可利用性、温度、盐度等对好氧氨氧化菌种类、数量及其种群生态分布的影响;阐述了好氧氨氧化菌对铵、氧饥饿的响应特征及其在酸性环境中的生存机制;并对今后好氧氨氧化菌的应用生态学研究及其主要方向进行了展望。     

氨氧化古菌及其在氮循环中的重要作用

氨氧化作用作为硝化过程的第一步,是氮素生物地球化学循环的关键步骤.长期以来,变形菌纲卢和',亚群中的氨氧化细菌一直被认为是氨氧化作用的主要承担者.然而,近年来研究发现氨氧化古菌广泛存在于各种生态系统中,并且在数量上占明显优势,在氮素生物地球化学循环中起着非常重要的作用.本文概述了氨氧化古菌的形态、生理生态特性及其系统发育特征,对比分析了氨氧化古菌和氨氧化细菌的氨单加氧酶及其编码基因的异同,综述了氨氧化古菌在水生和陆地等生态系统氮素循环中的作用,同时就氨氧化古菌在应用生态和环境保护领域今后的研究重点进行了展望.     

Nitrification and anammox with urea as the energy source

Urea is present in many ecosystems and can be used as an energy source by chemolithotrophic aerobic ammonia oxidizing bacteria (AOB). Thus the utilization of urea in comparison to ammonia, by AOB as well as anaerobic ammonia oxidizing (Anammox) bacteria was investigated, using enrichments cultures, inoculated with activated sludge, and molecular ecological methods. In batch enrichment cultures grown with ammonia a population established in 2 weeks, which was dominated by halophilic and halotolerant AOB as determined by fluorescence in situ hybridization (FISH) experiments, with the 16S rRNA targeting oligonucleotide probe NEU. In other batch enrichment cultures using urea, the AOB population was assessed by PCR amplification, cloning and phylogenetic analysis of amoA and ribosomal 16S rRNA genes. While only one of the 48 16S rRNA gene clones could be identified as AOB (Nitrosomonas oligotropha), the amoA approach revealed two more AOB, Nitrosomonas europaea and Nitrosomonas nitrosa to be present in the enrichment. FISH analysis of the enrichment with probe NEU and newly designed probes for a specific detection of N. oligotropha and N. nitrosa related organisms, respectively, showed that N. oligotropha-like AOB formed about 50% of the total bacterial population. Also N. nitrosa (about 15% of the total population) and N. europaea (about 5% of the total population) were relatively abundant. Additionally, continuous enrichments were performed under oxygen limitation. When ammonia was the energy source, the community in this reactor consisted of Anammox bacteria and AOB hybridizing with probe NEU. As the substrate was changed to urea, AOB related to N. oligotropha became the dominant AOB in this oxygen limited consortium. This resulted in a direct conversion of urea to dinitrogen gas, without the addition of organic carbon.     

Functionally distinct communities of ammonia-oxidizing bacteria along an estuarine salinity gradient

The relationship between ammonia-oxidizing bacteria (AOB) and potential nitrification rates was examined along a salinity gradient in a New England estuary in spring and late summer over 3 years. Ammonia-oxidizing bacteria abundance was estimated by measuring gene copies of the ammonia monooxygenase catalytic subunit (amoA) using real-time polymerase chain reaction. Ammonia-oxidizing bacteria abundance ranged from below detection to 6.0 x 10(7)amoA copies (gdw sediment)(-1). Mean potential nitrification rates ranged from 0.5 to 186.5 nmol N (gdw sediment)(-1) day(-1). Both AOB abundance and potential rates were significantly higher in spring than late summer. Correlations between rates and abundance varied significantly among sites, but showed site-specific ammonia oxidation kinetics related to AOB community structure. The effect of salinity on potential nitrification rates was evaluated by incubating sediment from each site under four salinity conditions (0, 5, 10 and 30 psu). At all sites, rates were generally highest in the intermediate salinity treatments, but rates at the upstream site were inhibited at high salinity, while rates at the two downstream sites were inhibited at the lowest salinity. Although salinity appears to be an important factor in determining AOB distribution, it may not be the primary factor as AOB exhibited a broad range of salinity tolerance in our experiments. Our results indicate that there are significant differences in abundance and community composition of AOB along the salinity gradient, and the differences are reflected in community function.     

Growth at low ammonium concentrations and starvation response as potential factors involved in niche differentiation among ammonia-oxidizing bacteria

In nature, ammonia-oxidizing bacteria have to compete with heterotrophic bacteria and plants for limiting amounts of ammonium. Previous laboratory experiments conducted with Nitrosomonas europaea suggested that ammonia-oxidizing bacteria are weak competitors for ammonium. To obtain a better insight into possible methods of niche differentiation among ammonia-oxidizing bacteria, we carried out a growth experiment at low ammonium concentrations with N. europaea and the ammonia oxidizer G5-7, a close relative of Nitrosomonas oligotropha belonging to Nitrosomonas cluster 6a, enriched from a freshwater sediment. Additionally, we compared the starvation behavior of the newly enriched ammonia oxidizer G5-7 to that of N. europaea. The growth experiment at low ammonium concentrations showed that strain G5-7 was able to outcompete N. europaea at growth-limiting substrate concentrations of about 10 micro M ammonium, suggesting better growth abilities of the ammonia oxidizer G5-7 at low ammonium concentrations. However, N. europaea displayed a more favorable starvation response. After 1 to 10 weeks of ammonium deprivation, N. europaea became almost immediately active after the addition of fresh ammonium and converted the added ammonium within 48 to 96 h. In contrast, the regeneration time of the ammonia oxidizer G5-7 increased with increasing starvation time. Taken together, these results provide insight into possible mechanisms of niche differentiation for the ammonia-oxidizing bacteria studied. The Nitrosomonas cluster 6a member, G5-7, is able to grow at ammonium concentrations at which the growth of N. europaea, belonging to Nitrosomonas cluster 7, has already ceased, providing an advantage in habitats with continuously low ammonium concentrations. On the other hand, the ability of N. europaea to become active again after longer periods of starvation for ammonium may allow better exploitation of irregular pulses of ammonium in the environment.     

从典型硝化细菌到全程氨氧化微生物：发现及研究进展

生物硝化过程在全球氮循环中起关键性作用,被认为由氨氮氧化成亚硝酸盐和亚硝酸盐氧化成硝酸盐两个步骤组成,分别由氨氧化微生物(Ammonia oxidizing microorganisms,AOM)和硝化细菌(Nitrite oxidizing bacteria,NOB)催化完成。AOM包括氨氧化细菌(Ammonia oxidizing bacteria,AOB)和氨氧化古菌(Ammonia oxidizing archaea,AOA),AOB与AOA分布广泛,两者的相对丰度和氨氮浓度密切相关。2015年底,3个硝化螺菌属(Nitrospira)谱系Ⅱ的NOB被证实含有AOM的特征功能酶,包括氨单加氧酶(AMO)和羟胺脱氢酶(HAO),并证明NOB同时具有氨氧化和亚硝酸盐氧化的能力,命名为全程氨氧化微生物(Complete ammonia oxidizer,Comammox)。根据AMO的α亚基基因amoA的相似性将Comammox分为两大分支clade A和clade B。它们广泛分布于自然环境和人工系统,包括土壤(稻田、森林)、淡水(湿地、河流、湖泊沉积物、蓄水层)、污水处理厂和自来水厂等。本文综述了Comammox的发现及其最新的研究进展,并展望了Comammox作为氮循环关键功能菌群的研究方向和应用前景。     

Phylogeny and functional expression of ribulose 1,5-bisphosphate carboxylase/oxygenase from the autotrophic ammonia-oxidizing bacterium Nitrosospira sp. isolate 40KI.

The autotrophic ammonia-oxidizing bacteria (AOB), which play an important role in the global nitrogen cycle, assimilate CO(2) by using ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Here we describe the first detailed study of RubisCO (cbb) genes and proteins from the AOB. The cbbLS genes from Nitrosospira sp. isolate 40KI were cloned and sequenced. Partial sequences of the RubisCO large subunit (CbbL) from 13 other AOB belonging to the beta and gamma subgroups of the class Proteobacteria are also presented. All except one of the beta-subgroup AOB possessed a red-like type I RubisCO with high sequence similarity to the Ralstonia eutropha enzyme. All of these new red-like RubisCOs had a unique six-amino-acid insert in CbbL. Two of the AOB, Nitrosococcus halophilus Nc4 and Nitrosomonas europaea Nm50, had a green-like RubisCO. With one exception, the phylogeny of the AOB CbbL was very similar to that of the 16S rRNA gene. The presence of a green-like RubisCO in N. europaea was surprising, as all of the other beta-subgroup AOB had red-like RubisCOs. The green-like enzyme of N. europaea Nm50 was probably acquired by horizontal gene transfer. Functional expression of Nitrosospira sp. isolate 40KI RubisCO in the chemoautotrophic host R. eutropha was demonstrated. Use of an expression vector harboring the R. eutropha cbb control region allowed regulated expression of Nitrosospira sp. isolate 40KI RubisCO in an R. eutropha cbb deletion strain. The Nitrosospira RubisCO supported autotrophic growth of R. eutropha with a doubling time of 4.6 h. This expression system may allow further functional analysis of AOB cbb genes.