Amplification of the amoA gene from diverse species of ammonium-oxidizing bacteria and from an indigenous bacterial population from seawater.

Because the chemolithotrophic ammonium-oxidizing bacteria are an integral component of nitrogen biogeochemistry, a sensitive and accurate method to detect this ecologically important group of microorganisms is needed. The amoA gene of these organisms encodes the active site of ammonia monooxygenase, an enzyme unique to this group of nitrifying bacteria. We report here the use of the PCR technique to detect the amoA gene from pure cultures of chemolithotrophic ammonium-oxidizing bacteria, ammonium oxidizers introduced into filtered seawater, and the natural bacterial population of an unfiltered seawater sample. Oligonucleotide primers, based on the published amoA sequence from Nitrosomonas europaea, were used to amplify DNA from pure cultures of Nitrosomonas europaea, Nitrosomonas cryotolerans, and Nitrosococcus oceanus and from bacteria in seawater collected offshore near the Florida Keys. Partial sequencing of the amplification products verified that they were amoA. These primers, used in conjunction with a radiolabeled amoA gene probe from Nitrosomonas europaea, could detect Nitrosococcus oceanus inoculated into filter-sterilized seawater at 10(4) cells liter-1. Native marine bacteria containing amoA could also be detected at their naturally occurring titer in oligotrophic seawater. Amplification of the gene for ammonia monooxygenase may provide a method to estimate the distribution and relative abundance of chemolithotrophic ammonium-oxidizing bacteria in the environment.     

Genera-Specific Immunofluorescence Labeling of Ammonia Oxidizers with Polyclonal Antibodies Recognizing Both Subunits of the Ammonia Monooxygenase

Polyclonal antibodies that recognize the two subunits AmoA and AmoB of the ammonia monooxygenase (AMO) were applied to identify ammonia-oxidizing bacteria by immunofluorescence (IF) labeling in pure, mixed, and enriched cultures. The antibodies against the AmoA were produced using a synthetic peptide of the AmoA of Nitrosomonas eutropha, whereas the antibodies against the AmoB had been developed previously is against the whole B-subunit of the AMO [Pinck et al. (2001) Appl Environ Microbiol 67:118–124]. Using IF labeling, the AmoA antibodies were specific for the detection of all species of the genus Nitrosomonas. In contrast, the antiserum against AmoB labeled all genera of ammonia oxidizers of the -subclass of Proteobacteria (Nitrosomonas, Nitrosospira, Nitrosolobus, and Nitrosovibrio). The fluorescence signals of the AmoA antibodies were spread all over the cells, whereas the signals of the AmoB antibodies were associated with the cytoplasmic membranes. The specificity of the reactions of the antisera with ammonia oxidizers were proven in pure and mixed cultures, and the characteristic IF labeling and the morphology of the cells enabled their identification at the genus level. The genus-specific IF labeling could be used to identify ammonia oxidizers enriched from various habitats. In enrichment cultures of natural sandstone, cells of the genera Nitrosomonas, Nitrosovibrio, and Nitrosospira were detected. Members of the genus Nitrosovibrio and Nitrosolobus were most prominent in enriched garden soil samples, whereas members of the genus Nitrosomonas dominated in enriched activated sludge. The antibodies caused only slight background fluorescence on sandstone and soil particles compared to oligonucleotide probes, which could not be used to detect ammonia oxidizers on these materials because of strong nonspecific fluorescence.     

Pacific Northwest marine sediments contain ammonia-oxidizing bacteria in the beta subdivision of the Proteobacteria

The diversity of ammonia-oxidizing bacteria in aquatic sediments was studied by retrieving ammonia monooxygenase and methane monooxygenase gene sequences. Methanotrophs dominated freshwater sediments, while beta-proteobacterial ammonia oxidizers dominated marine sediments. These results suggest that gamma-proteobacteria such as Nitrosococcus oceani are minor members of marine sediment ammonia-oxidizing communities.     

Analysis of ammonia-oxidizing bacteria from hypersaline Mono Lake, California, on the basis of 16S rRNA sequences

Ammonia-oxidizing bacteria were detected by PCR amplification of DNA extracted from filtered water samples throughout the water column of Mono Lake, California. Ammonia-oxidizing members of the beta subdivision of the division Proteobacteria (beta-subdivision Proteobacteria) were detected using previously characterized PCR primers; target sequences were detected by direct amplification in both surface water and below the chemocline. Denaturing gradient gel electrophoresis analysis indicated the presence of at least four different beta-subdivision ammonia oxidizers in some samples. Subsequent sequencing of amplified 16S rDNA fragments verified the presence of sequences very similar to those of cultured Nitrosomonas strains. Two separate analyses, carried out under different conditions (different reagents, locations, PCR machines, sequencers, etc.), 2 years apart, detected similar ranges of sequence diversity in these samples. It seems likely that the physiological diversity of nitrifiers exceeds the diversity of their ribosomal sequences and that these sequences represent members of the Nitrosomonas europaea group that are acclimated to alkaline, high-salinity environments. Primers specific for Nitrosococcus oceanus, a marine ammonia-oxidizing bacterium in the gamma subdivision of the Proteobacteria, did not amplify target from any samples.     

Pacific Northwest Marine Sediments Contain Ammonia-Oxidizing Bacteria in the β Subdivision of the Proteobacteria

The diversity of ammonia-oxidizing bacteria in aquatic sediments was studied by retrieving ammonia monooxygenase and methane monooxygenase gene sequences. Methanotrophs dominated freshwater sediments, while β-proteobacterial ammonia oxidizers dominated marine sediments. These results suggest that γ-proteobacteria such as Nitrosococcus oceani are minor members of marine sediment ammonia-oxidizing communities.     

Diversity of ammonia monooxygenase operon in autotrophic ammonia-oxidizing bacteria

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

Linking Diversity and Stable Isotope Fractionation in Ammonia-Oxidizing Bacteria

The link between similarity in amino acid sequence for ammonia monooxygenase (AMO) and isotopic discrimination for ammonia oxidation ( l AMO ) was investigated in g -subdivision ammonia-oxidizing bacteria. The isotope effects for ammonia oxidation in pure cultures of the nitrifying strains Nitrosomonas marina , Nitrosomonas C-113a, Nitrosospira tenuis , Nitrosomonas europaea , and Nitrosomonas eutropha ranged from 14.2 to 38.2. The differences in isotope effects could not be readily explained by differential rates of ammonia oxidation, transport of NH 4 + , or accumulation of NH 2 OH or N 2 O among the strains. The major similarities and differences observed in l AMO are, however, paralleled by similarities and differences in amino acid sequences for the f -subunit of AMO (AmoA). Robust differences in l AMO among nitrifying bacteria may be expected to influence the stable isotopic signatures of nitrous oxide (N 2 O) produced in various environments.     

Methane Oxidation by Nitrosococcus oceanus and Nitrosomonas europaea

Chemolithotrophic ammonium-oxidizing and nitrite-oxidizing bacteria including Nitrosomonas europaea, Nitrosococcus oceanus, Nitrobacter sp., Nitiospina gracilis, and Nitrococcus mobilis were examined as to their ability to oxidize methane in the absence of ammonium or nitrite. All ammonium oxidizers tested had the ability to oxidize significant amounts of methane to CO₂ and incorporate various amounts into cellular components. None of the nitrite-oxidizing bacteria were capable of methane oxidation. The methane-oxidizing capabilities of Nitrosococcus oceanus and Nitrosomonas europaea were examined with respect to ammonium and methane concentrations, nitrogen source, and pH. The addition of ammonium stimulated both CO₂ production and cellular incorporation of methane-carbon by both organisms. Less than 0.1 mM CH₄ in solution inhibited the oxidation of ammonium by Nitrosococcus oceanus by 87%. Methane concentrations up to 1.0 mM had no inhibitory effects on ammonium oxidation by Nitrosomonas europaea. In the absence of NH₄-N, Nitrosococcus oceanus achieved a maximum methane oxidation rate of 2.20 × 10⁻² μmol of CH₄ h⁻¹ mg (dry weight) of cells⁻¹, which remained constant as the methane concentration was increased. In the presence of NH₄-N (10 ppm [10 μg/ml]), its maximum rate was 26.4 × 10⁻² μmol of CH₄ h⁻¹ mg (dry weight) of cells⁻¹ at a methane concentration of 1.19 × 10⁻² mM. Increasing the methane concentration above this level decreased CO₂ production, whereas cellular incorporation of methane-carbon continued to increase. Nitrosomonas europaea showed a linear response throughout the test range, with an activity of 196.0 × 10⁻² μmol of CH₄ h⁻¹ mg (dry weight) of cells ⁻¹ at a methane concentration of 1.38 × 10⁻¹ mM. Both nitrite and nitrate stimulated the oxidation of methane. The pH range was similar to that for ammonium oxidation, but the points of maximum activity were at lower values for the oxidation of methane.     

Physiology, biochemistry, and specific inhibitors of CH4, NH4+, and CO oxidation by methanotrophs and nitrifiers.

Ammonia oxidizers (family Nitrobacteraceae) and methanotrophs (family Methylococcaceae) oxidize CO and CH4 to CO2 and NH4+ to NO2-. However, the relative contributions of the two groups of organisms to the metabolism of CO, CH4, and NH4+ in various environments are not known. In the ammonia oxidizers, ammonia monooxygenase, the enzyme responsible for the conversion of NH4+ to NH2OH, also catalyzes the oxidation of CH4 to CH3OH. Ammonia monooxygenase also mediates the transformation of CH3OH to CO2 and cell carbon, but the pathway by which this is done is not known. At least one species of ammonia oxidizer, Nitrosococcus oceanus, exhibits a Km for CH4 oxidation similar to that of methanotrophs. However, the highest rate of CH4 oxidation recorded in an ammonia oxidizer is still five times lower than rates in methanotrophs, and ammonia oxidizers are apparently unable to grow on CH4. Methanotrophs oxidize NH4+ to NH2OH via methane monooxygenase and NH4+ to NH2OH via methane monooxygenase and NH2OH to NO2- via an NH2OH oxidase which may resemble the enzyme found in ammonia oxidizers. Maximum rates of NH4+ oxidation are considerably lower than in ammonia oxidizers, and the affinity for NH4+ is generally lower than in ammonia oxidizers. NH4+ does not apparently support growth in methanotrophs. Both ammonia monooxygenase and methane monooxygenase oxidize CO to CO2, but CO cannot support growth in either ammonia oxidizers or methanotrophs. These organisms have affinities for CO which are comparable to those for their growth substrates and often higher than those in carboxydobacteria. The methane monooxygenases of methanotrophs exist in two forms: a soluble form and a particulate form. The soluble form is well characterized and appears unrelated to the particulate. Ammonia monooxygenase and the particulate methane monooxygenase share a number of similarities. Both enzymes contain copper and are membrane bound. They oxidize a variety of inorganic and organic compounds, and their inhibitor profiles are similar. Inhibitors thought to be specific to ammonia oxidizers have been used in environmental studies of nitrification. However, almost all of the numerous compounds found to inhibit ammonia oxidizers also inhibit methanotrophs, and most of the inhibitors act upon the monooxygenases. Many probably exert their effect by chelating copper, which is essential to the proper functioning of some monooxygenases. The lack of inhibitors specific for one or the other of the two groups of bacteria hampers the determination of their relative roles in nature.     

Application of Molecular Biological Techniques to a Seasonal Study of Ammonia Oxidation in a Eutrophic Freshwater Lake

The autotrophic ammonia-oxidizing bacteria in a eutrophic freshwater lake were studied over a 12-month period. Numbers of ammonia oxidisers in the lakewater were small throughout the year, and tangential-flow concentration was required to obtain meaningful estimates of most probable numbers. Sediments from littoral and profundal sites supported comparatively large populations of these bacteria, and the nitrification potential was high, particularly in summer samples from the littoral sediment surface. In enrichment cultures, lakewater samples nitrified at low (0.67 mM) ammonium concentrations only whereas sediment samples exhibited nitrification at high (12.5 mM) ammonium concentrations also. Enrichments at low ammonium concentration did not nitrify when inoculated into high-ammonium medium, but the converse was not true. This suggests that the water column contains a population of ammonia oxidizers that is sensitive to high ammonium concentrations. The observation of nitrification at high ammonium concentration by isolates from some winter lakewater samples, identified as nitrosospiras by 16S rRNA probing, is consistent with the hypothesis that sediment ammonia oxidizers enter the water column at overturn. With only one exception, nested PCR amplification enabled the detection of Nitrosospira 16S rDNA in all samples, but Nitrosomonas (N. europaea-eutropha lineage) 16S rDNA was never obtained. However, the latter were part of the sediment and water column communities, because their 16S rRNA could be detected by specific oligonucleotide probing of enrichment cultures. Furthermore, a specific PCR amplification regime for the Nitrosomonas europaea ammonia monooxygenase gene (amoA) yielded positive results when applied directly to sediment and lakewater samples. Patterns of Nitrosospira and Nitrosomonas detection by 16S rRNA oligonucleotide probing of sediment enrichment cultures were complex, but lakewater enrichments at low ammonium concentration were positive for nitrosomonads and not nitrosospiras. Analysis of enrichment cultures has therefore provided evidence for the existence of subpopulations within the lake ammonia-oxidizing community distinguishable on the basis of ammonium tolerance and possibly showing a seasonal distribution between the sediment and water column.     

Community analysis of ammonia and nitrite oxidizers during start-up of nitritation reactors

Partial nitrification of ammonium to nitrite under oxic conditions (nitritation) is a critical process for the effective use of alternative nitrogen removal technologies from wastewater. Here we investigated the conditions which promote establishment of a suitable microbial community for performing nitritation when starting from regular sewage sludge. Reactors were operated in duplicate under different conditions (pH, temperature, and dilution rate) and were fed with 50 mM ammonium either as synthetic medium or as sludge digester supernatant. In all cases, stable nitritation could be achieved within 10 to 20 days after inoculation. Quantitative in situ hybridization analysis with group-specific fluorescent rRNA-targeted oligonucleotides (FISH) in the different reactors showed that nitrite-oxidizing bacteria of the genus Nitrospira were only active directly after inoculation with sewage sludge (up to 4 days and detectable up to 10 days). As demonstrated by quantitative FISH and restriction fragment length polymorphism (RFLP) analyses of the amoA gene (encoding the active-site subunit of the ammonium monooxygenase), the community of ammonia-oxidizing bacteria changed within the first 15 to 20 days from a more diverse set of populations consisting of members of the Nitrosomonas communis and Nitrosomonas oligotropha sublineages and the Nitrosomonas europaea-Nitrosomonas eutropha subgroup in the inoculated sludge to a smaller subset in the reactors. Reactors operated at 30 degrees C and pH 7.5 contained reproducibly homogeneous communities dominated by one amoA RFLP type from the N. europaea-N. eutropha group. Duplicate reactors at pH 7.0 developed into diverse communities and showed transient population changes even within the ammonia oxidizer community. Reactors at pH 7.5 and 25 degrees C formed communities that were indistinguishable by the applied FISH probes but differing in amoA RFLP types. Communities in reactors fed with sludge digester supernatant exhibited a higher diversity and were constantly reinoculated with ammonium oxidizers from the supernatant. Therefore, such systems could be maintained at a higher dilution rate (0.75 day(-1) compared to 0.2 day(-1) for the synthetic wastewater reactors). Despite similar reactor performance with respect to chemical parameters, the underlying community structures were different, which may have an influence on stability during perturbations.     

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.     

Energy coupling and respiration in Nitrosomonas europaea.

Intact cells of Nitrosomonas europaea grown in an ammonium salts medium will oxidise ammonium ions, hydroxylamine and ascorbate-TMPD; there is no oxidation of carbon monoxide, methane or methanol. The Km value for ammonia oxidation is highly pH dependent with a minimum value of 0.5 mM above pH 8.0. This suggests that free ammonia is the species crossing the cytoplasmic membrane(s). The measurement of respiration driven proton translocation indicates that there is probably only one proton translocating loop (loop 3) association with hydroxylamine oxidation. The oxidation of "endogenous" substrates is sometimes associated with more than one proton-translocating loop. These results indicate that during growth hydroxylamine oxidation is probably associated with a maximum P/O ratio of 1.     

The viable but non-culturable state in the human pathogen Vibrio vulnificus

Abstract Genes encoding paniculate methane monooxygenase and ammonia monooxygenase share high sequence identity. Degenerate oligonucleotide primers were designed, based on regions of shared amino acid sequence between the 27-kDa polypeptides, which are believed to contain the active sites, of particulate methane monooxygenase and ammonia monooxygenase. A 525-bp internal DNA fragment of the genes encoding these polypeptides ( pmoA and amoA ) from a variety of methanotrophic and nitrifying bacteria was amplified by PCR, cloned and sequenced. Representatives of each of the phylogenetic groups of both methanotrophs (α- and γ-Proteobacteria) and ammonia-oxidizing nitrifying bacteria (β-and y-Proteobacteria) were included. Analysis of the predicted amino acid sequences of these genes revealed strong conservation of both primary and secondary structure. Nitrosococcus oceanus AmoA showed higher identity to PmoA sequences from other members of the γ-Proteobacteria than to AmoA sequences. These results suggest that the particulate methane monooxygenase and ammonia monooxygenase are evolutionarily related enzymes despite their different physiological roles in these bacteria.     

Physiological and phylogenetic study of an ammonium-oxidizing culture at high nitrite concentrations

Oxidation of high-strength ammonium wastewater can lead to exceptionally high nitrite concentrations; therefore, the effect of high nitrite concentration (> 400 mM) was studied using an ammonium-oxidizing enrichment culture in a batch reactor. Ammonium was fed to the reactor in portions of 40-150 mM until ammonium oxidation rates decreased and finally stopped. Activity was restored by replacing half of the medium, while biomass was retained by a membrane. The ammonium-oxidizing population obtained was able to oxidize ammonium at nitrite concentrations of up to 500 mM. The maximum specific oxidation activity of the culture in batch test was about 0.040 mmol O(2)g(-1)proteinmin(-1) and the K(s) value was 1.5 mM ammonium. In these tests, half of the maximum oxidation activity was still present at a concentration of 600 mM nitrite and approximately 10% residual activity could still be measured at 1200 mM nitrite (pH 7.4), or as a free nitrous acid (FNA) concentration of 6.6 mg l(-1). Additional experiments showed that the inhibition was caused by nitrite and not by the high sodium chloride concentration of the medium. The added ammonium was mainly converted into nitrite and no nitrite oxidation was observed. In addition, gaseous nitrogen compounds were detected and mass balance calculations revealed a nitrogen loss of approximately 20% using this system. Phylogenetic analyses of 16S rRNA and ammonium monooxygenase (amoA) genes of the obtained enrichment culture showed that ammonium-oxidizing bacteria of the Nitrosomonas europaea/Nitrosococcus mobilis cluster dominated the two clone libraries. Approximately 25% of the 16S rRNA clones showed a similarity of 92% to Deinococcus-like organisms. Specific fluorescence in situ hybridization (FISH) probes confirmed that these microbes comprised 10-20% of the microbial community in the enrichment. The Deinococcus-like organisms were located around the Nitrosomonas clusters, but their role in the community is currently unresolved.     

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.     

Ammonium Limitation Results in the Loss of Ammonia-Oxidizing Activity in Nitrosomonas europaea

The effects of limiting concentrations of ammonium on the metabolic activity of Nitrosomonas europaea, an obligate ammonia-oxidizing soil bacterium, were investigated. Cells were harvested during late logarithmic growth and were incubated for 24 h in growth medium containing 0, 15, or 50 mM ammonium. The changes in nitrite production and the rates of ammonia- and hydroxylamine-dependent oxygen consumption were monitored. In incubations without ammonium, there was little change in the ammonia oxidation activity after 24 h. With 15 mM ammonium, an amount that was completely consumed, there was an 85% loss of the ammonia oxidation activity after 24 h. In contrast, there was only a 35% loss of the ammonia oxidation activity after 24 h in the presence of 50 mM ammonium, an amount that was not consumed to completion. There was little effect on the hydroxylamine oxidation activity in any of the incubations. The loss of ammonia oxidation activity was not due to differences in steady-state levels of ammonia monooxygenase (AMO) mRNA (amoA) or to degradation of the active site-containing subunit of AMO protein. The incubations were also conducted at a range of pH values to determine whether the loss of ammonia oxidation activity was correlated to the residual ammonium concentration. The loss of ammonia oxidation activity after 24 h was less at lower pH values (where the unoxidized ammonium concentration was higher). When added in conjunction with limiting ammonium, short-chain alkanes, which are alternative substrates for AMO, prevented the loss of ammonia oxidation activity at levels corresponding to their binding affinity for AMO. These results suggest that substrates of AMO can preserve the ammonia-oxidizing activity of N. europaea in batch incubations by protecting either AMO itself or other molecules associated with ammonia oxidation.     

Differential inhibition in vivo of ammonia monooxygenase, soluble methane monooxygenase and membrane-associated methane monoxygenase by phenylacetylene

Phenylacetylene was investigated as a differential inhibitor of ammonia monooxygenase (AMO), soluble methane monooxygenase (sMMO) and membrane-associated or particulate methane monooxygenase (pMMO) in vivo. At phenylacetylene concentrations > 1 microM, whole-cell AMO activity in Nitrosomonas europaea was completely inhibited. Phenylacetylene concentrations above 100 microM inhibited more than 90% of sMMO activity in Methylococcus capsulatus Bath and Methylosinus trichosporium OB3b. In contrast, activity of pMMO in M. trichosporium OB3b, M. capsulatus Bath, Methylomicrobium album BG8, Methylobacter marinus A45 and Methylomonas strain MN was still measurable at phenylacetylene concentrations up to 1,000 microM. AMO of Nitrosococcus oceanus has more sequence similarity to pMMO than to AMO of N. europaea. Correspondingly, AMO in N. oceanus was also measurable in the presence of 1,000 microM phenylacetylene. Measurement of oxygen uptake indicated that phenylacetylene acted as a specific and mechanistic-based inhibitor of whole-cell sMMO activity; inactivation of sMMO was irreversible, time dependent, first order and required catalytic turnover. Corresponding measurement of oxygen uptake in whole cells of methanotrophs expressing pMMO showed that pMMO activity was inhibited by phenylacetylene, but only if methane was already being oxidized, and then only at much higher concentrations of phenylacetylene and at lower rates compared with sMMO. As phenylacetylene has a high solubility and low volatility, it may prove to be useful for monitoring methanotrophic and nitrifying activity as well as identifying the form of MMO predominantly expressed in situ.     

Identification of major subgroups of ammonia-oxidizing bacteria in environmental samples by T-RFLP analysis of amoA PCR products

A cloning-independent method based on T-RFLP (terminal restriction fragment length polymorphism) analysis of amoA PCR products was developed to identify major subgroups of autotrophic ammonia oxidizers of the beta-subclass of the class Proteobacteria in total community DNA. Based on a database of 28 partial gene sequences encoding the active-site polypeptide of ammonia monooxygenase (amoA), defined lengths of terminal restriction fragments (= operational taxonomic units, OTUs) of amoA were predicted to correlate in TaqI-based T-RFLP analysis with phylogenetically defined subgroups of ammonia oxidizers. Members of the genus Nitrosospira showed a specific OTU of 283 bp in length, while a fragment size of 219 bp was indicative of Nitrosomonas-like sequence types including N. europaea, N. eutropha, and N. halophila. Two amoA sequence clusters designated previously as the lineages 'PluBsee' and 'Sch?hsee' [Rotthauwe, J.-H., Witzel, K.-P., Liesack, W., 1997. Appl. Environ. Microbiol. 63, 4704-4712] shared a TaqI-based OTU with a fragment size of 48 bp, but sequence types of these two lineages could be differentiated by AluI-based T-RFLP analysis. A survey of various environmental samples and enrichment cultures by T-RFLP analysis and by comparative analysis of cloned amoA sequences confirmed the predicted correlations between distinct OTUs and phylogenetic information. Our data suggest that amoA-based T-RFLP analysis is a reliable tool to rapidly assess the complexity of ammonia-oxidizing communities in environmental samples with respect to the presence of major subgroups, i.e. nitrosospiras versus nitrosomonads.