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PubMed2003

Real-time PCR quantification of nitrifying bacteria in a municipal wastewater treatment plant.

Real-time PCR assays using TaqMan or Molecular Beacon probes were developed and optimized for the quantification of total bacteria, the nitrite-oxidizing bacteria Nitrospira, and Nitrosomonas oligotropha-like ammonia oxidizing bacteria (AOB) in mixed liquor suspended solids (MLSS) from a municipal wastewater treatment plant (WWTP) using a single-sludge nitrification process. The targets for the real-time PCR assays were the 16S rRNA genes (16S rDNA) for bacteria and Nitrospira spp. and the amoA gene for N. oligotropha. A previously reported assay for AOB 16S rDNA was also tested for its application to activated sludge. The Nitrospira 16S rDNA, AOB 16S rDNA, and N. oligotropha-like amoA assays were log-linear over 6 orders of magnitude and the bacterial 16S rDNA real-time PCR assay was log-linear over 4 orders of magnitude with DNA standards. When these real-time PCR assays were applied to DNA extracted from MLSS, dilution of the DNA extracts was necessary to prevent PCR inhibition. The optimal DNA dilution range was broad for the bacterial 16S rDNA (1000-fold) and Nitrospira 16S rDNA assays (2500-fold) but narrow for the AOB 16S rDNA assay (10-fold) and N. oligotropha-like amoA real-time PCR assay (5-fold). In twelve MLSS samples collected over one year, mean cell per L values were 4.3 +/- 2.0 x 10(11) for bacteria, 3.7 +/- 3.2 x 10(10) for Nitrospira, 1.2 +/- 0.9 x 10(10) for all AOB, and 7.5 +/- 6.0 x 10(9) for N. oligotropha-like AOB. The percent of the nitrifying population was 1.7% N. oligotropha-like AOB based on the N. oligotropha amoA assay, 2.9% total AOB based on the AOB 16S rDNA assay, and 8.6% nitrite-oxidizing bacteria based on the Nitrospira 16S rDNA assay. Ammonia-oxidizing bacteria in the wastewater treatment plant were estimated to oxidize 7.7 +/- 6.8 fmol/hr/cell based on the AOB 16S rDNA assay and 12.4 +/- 7.3 fmol/hr/cell based on the N. oligotropha amoA assay.

Biological Assay↗

PubMed2004

Folding, conformational changes, and dynamics of cytochromes C probed by NMR spectroscopy.

NMR spectroscopy has become a vital tool for studies of protein conformational changes and dynamics. Oxidized Fe(III)cytochromes c are a particularly attractive target for NMR analysis because their paramagnetism (S = (1)/(2)) leads to high (1)H chemical shift dispersion, even for unfolded or otherwise disordered states. In addition, analysis of shifts induced by the hyperfine interaction reveals details of the structure of the heme and its ligands for native and nonnative protein conformational states. The use of NMR spectroscopy to investigate the folding and dynamics of paramagnetic cytochromes c is reviewed here. Studies of nonnative conformations formed by denaturation and by anomalous in vivo maturation (heme attachment) are facilitated by the paramagnetic, low-spin nature of native and nonnative forms of cytochromes c. Investigation of the dynamics of folded cytochromes c also are aided by their paramagnetism. As an example of this analysis, the expression in Escherichia coli of cytochrome c(552) from Nitrosomonas europaea is reported here, along with analysis of its unusual heme hyperfine shifts. The results are suggestive of heme axial methionine fluxion in N. europaea ferricytochrome c(552). The application of NMR spectroscopy to investigate paramagnetic cytochrome c folding and dynamics has advanced our understanding of the structure and dynamics of both native and nonnative states of heme proteins.

Algorithms↗

PubMed2005

Redox properties of wild-type and heme-binding loop mutants of bacterial cytochromes C measured by direct electrochemistry.

We have used protein film voltammetry (PFV) to determine the midpoint potentials of the Pseudomonas aeruginosa, Hydrogenobacter thermophilus, and Nitrosomonas europaea wild-type monoheme cytochromes c (cyts c; PA, HT, and NE, respectively), as well as PA N64Q, HT Q64N, and NE V65delta mutants, as a function of pH, and buffer conditions. Recent studies have suggested that the identity of the 64 position of the heme-binding loop (either Asn or Gln) strongly influences the conformation of the Met ligand that binds the heme iron. The PFV studies reveal that HT and NE possess significantly lower potentials (wild-type cyts c having E(m) values of +227 and +250 mV vs SHE) than PA (+290 mV) in 50 mM phosphate buffer, pH 7 at 3 degrees C. The HT Q64N mutant rises in potential compared to wild-type, and the PA N64Q mutant has a lower potential, indicating relationships between Met ligand fluxion, hydrogen bonding to the Met ligand, and redox chemistry. Surprisingly, NE V65delta, possessing a heme binding loop nearly identical to that of the PA protein, displayed an E(m) of +232 mV, even lower than wild-type NE. These data are discussed in terms of models of Met ligand properties and proton dependence.

Bacterial Proteins↗

PubMed2001

Correlations of structure and electronic properties from EPR spectroscopy of hydroxylamine oxidoreductase.

Hydroxylamine oxidoreductase (HAO) from the autotrophic nitrifying bacterium Nitrosomonas europaea catalyzes the oxidation of NH(2)OH to HNO(2). The enzyme contains eight hemes per subunit which participate in catalytic function and electron transport. The structure of the enzyme shows a unique spatial arrangement of the eight hemes, subsets of which are now observed in four other proteins. The spatial arrangement displays three types of diheme pairing motifs. At least four of the eight hemes are electronically coupled in two distinguishable pairs and one of these pairs is at the active site of the enzyme. Here, the use of quantitative simulation of the EPR signals allows determination of exchange couplings, and assignments of signals and reduction potentials to hemes of the crystal structure. The absence of any obvious heme-to-heme bonding pathway in the crystal structure suggests that the observed exchange interactions are derived from direct electronic overlap of porphyrin orbitals. This provides evidence for heme pairs which function as biological two-electron redox centers in electron-transfer processes.

Catalysis↗

PubMed2000

Nitrification inhibitors from the roots of Leucaena leucocephala.

The nitrification inhibition (NI) bioassay guided fractionation of the methanol extract of lyophilized and milled roots of Leuceana leucocephala resulted in the isolation of four compounds, 1-4, as confirmed from their 1H and 13C NMR spectral data. Compound 1, gallocatechin, was the most active NI inhibitor at 12 microg/mL. Epigallocatechin, 2, and epicatechin, 4, isolated as mixtures, were not assayed individually for their NI inhibitory activities against the nitrification bacterium Nitrosomonas europaea.

Catechin↗

PubMed2006

Classification of proteins based on minimal modular repeats: lessons from nature in protein design.

Proteins containing internal repeats within their primary sequence have received increased attention recently, as the extent of their presence in various organisms is recognized more fully, and their role in evolution is more thoroughly studied. Presented here is a technique used to detect and classify proteins based on a modular evolutionary phenomenon that results in a series of small internal repeats. The parameters chosen are based on a minimum segment of seven residues that result in simple functional scaffolds. The genomes and corresponding proteomes of a variety of eubacteria and archaea have been analyzed using an algorithm that searches prokaryotic genomes for proteins containing small conserved repeats assembled in a modular fashion similar to a recently characterized protein from the organism Nitrosomonas europaea. This analysis has revealed additional proteins present in N. europaea with similar modular characteristics. A further survey of a variety of organisms demonstrates that this evolutionary pathway has been utilized in other organisms as well, to yield a broad assortment of small modular proteins. A thorough description of the sequential characteristics of these modular proteins follows, along with a selection and discussion of the various proteins uncovered through this expanded search and analysis. Several databases of the proteins uncovered from this work and the program used to perform the search are available.

Algorithms↗

PubMed2001

Activity monitoring for nitrifying bacteria by fluorescence in situ hybridization and respirometry.

The activities of two families of nitrifying bacteria were measured by applied respirometer and fluorescence in situ hybridization (FISH) for the purpose of monitoring nitrogen removal. Oxygen uptake rates (OUR) for Nitrosomonas and Nitrobacter were separately measured with the dosing of selective inhibitors during respirometric monitoring. The FISH method was applied to analyze microorganism communities. In 5 mg NH4+-N/L solution, the total nitrogen oxygen demand (NOD) was found to be 6,672 mg DO/L-day with ammonia and nitrite oxidation rate of 4,512 and 2,160 mg DO/L-day, respectively. The oxygen consumption by endogenous oxidation was 288 mg DO/L-day and the biological oxygen demand 936 mg DO/L-day. Respirometric measurements were well matched by the FISH result, which described the presence and quantity of each nitrifying bacteria as nitrification proceeded. It was found that when coupled with OUR data, FISH could provide valuable information about the nitrogen removal process in many practical wastewater treatment systems.

Ammonia↗

PubMed1985

Spectroscopic evidence for a photosensitive oxygenated state of ammonia mono-oxygenase.

Photoinactivation of ammonia oxidation by Nitrosomonas europaea cells by near-u.v. light was confirmed and further shown to occur with the same rate constant as loss of bromoethane-oxidation activity. Hydroxylamine oxidation was much less photosensitive. Protection against inactivation was afforded by anaerobiosis, organic substrates of ammonia mono-oxygenase such as bromoethane, or metal-ion-chelating agents such as thiourea. The presence of 10 mM-NH4+ or 1 mM-hydroxylamine made little difference, whereas hydrazine had a potentiating effect. Illumination of cells also caused a bleaching in the absorption spectrum around 380 nm, along with changes in the cytochrome gamma-band region. Similar effects below 400 nm were obtained when organic substrates and inhibitors of the mono-oxygenase were added to cells in the dark. The copper proteins haemocyanin and tyrosinase have a photosensitive oxygenated state with a near-u.v. absorption band of similar half-width. They also have a sensitivity to chelating agents similar to that of ammonia mono-oxygenase. The experimental results are explained in terms of a three-stage catalytic cycle analogous to that for tyrosinase. In resting cells most of the enzyme is believed to be in an oxygenated (Oxy) form, which absorbs maximally at 378 nm and is photosensitive. In the presence of a substrate, one O atom is inserted into the substrate and the other is reduced to water, leaving the enzyme in an oxidized (Met) state. This is followed by a two-electron reduction of the proposed binuclear copper site to give a reduced (Deoxy) state, which can bind O2 to complete the cycle.

Ammonia↗

PubMed1985

Suicidal inactivation and labelling of ammonia mono-oxygenase by acetylene.

Acetylene brings about a progressive inactivation of ammonia mono-oxygenase, the ammonia-oxidizing enzyme in Nitrosomonas europaea. High NH4+ ion concentrations were protective. The inactivation followed first-order kinetics, with a rate constant of 1.5 min-1 at saturating concentrations of acetylene. If acetylene was added in the absence of O2, the cells remained active until O2 was re-introduced. A protective effect was also demonstrated with thiourea, a reversible non-competitive inhibitor of ammonia oxidation. Incubation of cells with [14C]acetylene was found to cause labelling of a single membrane polypeptide. This ran on dodecyl sulphate/polyacrylamide-gel electrophoresis with an Mr value of 28 000. It is concluded that acetylene is a suicide substrate for the mono-oxygenase. The labelling experiment provides the first identification of a constituent polypeptide of ammonia mono-oxygenase.

Acetylene↗

PubMed1996

The biochemical characterization of a novel non-haem-iron hydroxylamine oxidase from Paracoccus denitrificans GB17.

The characterization of the hydroxylamine oxidase from the heterotrophic nitrifier Paracoccus denitrificans GB17 indicates the enzyme to be entirely distinct from the hydroxylamine oxidase from the autotrophic nitrifier Nitrosomonas europaea. Hydroxylamine oxidase from P. denitrificans contains three to five non-haem, non-iron-sulphur iron atoms as prosthetic groups, predominantly co-ordinated by carboxylate ligands. The interaction of the enzyme with the electron-accepting proteins cytochrome C556 and pseudoazurin is mainly hydrophobic. The catalytic mechanism of hydroxylamine oxidase from P. denitrificans is different from the enzyme from N. europaea because the production of nitrite by the former requires molecular oxygen. Under anaerobic conditions the enzyme makes nitrous oxide as a sole product.

Edetic Acid↗

PubMed2006

Role of nitrogen oxides in the metabolism of ammonia-oxidizing bacteria.

Ammonia-oxidizing bacteria (AOB) can use oxygen and nitrite as electron acceptors. Nitrite reduction by Nitrosomonas is observed under three conditions: (i) hydrogen-dependent denitrification, (ii) anoxic ammonia oxidation with nitrogen dioxide (NO(2)) and (iii) NO(x)-induced aerobic ammonia oxidation. NO(x) molecules play an important role in the conversion of ammonia and nitrite by AOB. Absence of nitric oxide (NO), which is generally detectable during ammonia oxidation, severely impairs ammonia oxidation by AOB. The lag phase of recovery of aerobic ammonia oxidation was significantly reduced by NO(2) addition. Acetylene inhibition tests showed that NO(2)-dependent and oxygen-dependent ammonia oxidation can be distinguished. Addition of NO(x) increased specific activity of ammonia oxidation, growth rate and denitrification capacity. Together, these findings resulted in a hypothetical model on the role of NO(x) in ammonia oxidation: the NO(x) cycle.

Ammonia↗

PubMed2000

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.

Acetylene↗

PubMed2000

Analysis of ammonia-oxidizing bacteria populations in acid forest soil during conditions of moisture limitation.

Ammonia-oxidizer numbers decreased under conditions of moisture limitation in litter, fermentation and humus layers of forest soil in the field, but the extent of regrowth after rehydration varied between layers. Nitrosospira 16S rRNA genes were amplified from all layers, regardless of moisture content or soil pH which varied between 4.1 and 5.2. Nitrosomonas spp. were detected less often, but appeared to exhibit more rapid recovery than the Nitrosospira spp. when drought conditions were relieved by rainfall.

Ammonia↗

PubMed2001

The CANON system (Completely Autotrophic Nitrogen-removal Over Nitrite) under ammonium limitation: interaction and competition between three groups of bacteria.

The CANON system (Completely Autotrophic Nitrogen Removal Over Nitrite) can potentially remove ammonium from wastewater in a single, oxygen-limited treatment step. The usefulness of CANON as an industrial process will be determined by the ability of the system to recover from major disturbances in feed composition. The CANON process relies on the stable interaction between only two bacterial populations: Nitrosomonas-like aerobic and Planctomycete-like anaerobic ammonium oxidising bacteria. The effect of extended periods of ammonium limitation was investigated at the laboratory scale in two different reactor types (sequencing batch reactor and chemostat). The lower limit of effective and stable nitrogen removal to dinitrogen gas in the CANON system was 0.1 kg N m(-3) day(-1). At this loading rate, 92% of the total nitrogen was removed. After prolonged exposure (> 1 month) to influxes lower than this critical NH4+-influx, a third population of bacteria developed in the system and affected the CANON reaction stoichiometry, resulting in a temporary decrease in nitrogen removal from 92% to 57%. The third group of bacteria were identified by activity tests and qualititative FISH (Fluorescence In Situ Hybridisation) analysis to be nitrite-oxidising Nitrobacter and Nitrospira species. The changes caused by the NH4+-limitation were completely reversible, and the system re-established itself as soon as the ammonium limitation was removed. This study showed that CANON is a robust system for ammonium removal, enduring periods of up to one month of ammonium limitation without irreversible damage.

Bacteria, Anaerobic↗

PubMed2004

A two-stage SBR process for removal of organic substrate and nitrogen via nitrite-type nitrification-denitrification.

A two-stage SBR process (TSSBR) was applied for the treatment of wastewater with high strength COD and nitrogen. Most of organic substrate was removed in the first stage SBR reactor (SBR1) under the aerobic condition. Subsequently the second stage SBR reactor (SBR2) firstly was operated under the aerobic condition for simultaneous nitrite-type nitrification and removal of a small amount of residual organic substrate. Nitrification was controlled to the nitrite-type nitrification. Then denitrification in which the wastewater was used as external carbon sources occurred in SBR2 under the anoxic condition. The experimental results indicated that two kinds of biomass with the different function were cultured in a TSSBR system, which was beneficial to increase the proportion of Nitrosomonas in the microorganism of SBR2. It avoided the inhibition of the high organic loading to nitrification and C/N was not the limiting factor affecting the nitrogen removal efficiency. The rate of COD degradation and nitrification was improved, 8 kg COD/kg MLSS/d and 0.25 kg NH4+-N/kg MLSS/d, respectively. The final effluent COD concentration further reduced because the organic substrate that was left in SBR1 effluent was removed in SBR2. Consequently, when compared to a single SBR process, a two-stage SBR (TSSBR) not only improved the treatment efficiency, but also saved the operational cost.

Bioreactors↗

PubMed2001

Detailed phylogeny of ammonia-oxidizing bacteria determined by rDNA sequences and DNA homology values.

A comparison of the phylogeny of 38 isolates of chemolithoautotrophic ammonia-oxidizing bacteria (AOB) based on 16S rRNA gene sequences, 16S-235 rDNA intergenic spacer region (ISR) sequences and species affiliations based on DNA homology values was performed. The organisms studied all belong to the beta-subclass of the Proteobacteria and included representatives of Nitrosomonas, Nitrosococcus and Nitrosospira. The similarity values of the 16S rDNA sequences were high, particularly within the Nitrosospira genus, and based on these sequences it is difficult to determine the phylogenetic position of some AOB. As an alternative and supplement to 16S rRNA gene sequencing, the ISR was sequenced and analysed phylogenetically. Due to considerably lower similarity values, the ISR-based phylogeny gives a better resolution than the phylogeny based on the functional 16S rRNA gene. Since the ISR-based phylogeny of AOB is highly consistent with the 16S rDNA based phylogeny, ISR sequencing appears as a suitable tool for resolving the detailed phylogeny of AOB. The phylogenetic position of two isolates of the former genus 'Nitrosolobus' (now included in the Nitrosospira genus) is not clear. These organisms are close relatives of the former Nitrosospira spp. and 'Nitrosovibrio' spp. (now Nitrosospira), but based on their marginal positions in the phylogenetic trees, DNA-DNA hybridization data and phenotypic characteristics, it is suggested that 'Nitrosolobus' should be a separate genus. DNA homology determination of 11 Nitrosospira isolates revealed two new species of Nitrosospira. The phylogeny of AOB reflected in the trees based on the rDNA sequences is consistent with the species affiliations of AOB by DNA homology values. This observation will probably be important for the interpretation of results from studies of natural diversity of AOB.

Ammonia↗

PubMed1994

The dynamic progression of evolved character states for aromatic amino acid biosynthesis in gram-negative bacteria.

A systematic analysis of the evolution of aromatic amino acid biosynthesis in the Proteobacteria, previously focussed mainly upon the gamma subdivision, has now been extended to the beta subdivision. Five lineages were studied, represented by Neisseria gonorrhoeae, Nitrosomonas europaea, Alcaligenes faecalis, rRNA Group-III pseudomonads/Rubrivivax gelatinosus, and rRNA Group-II pseudomonads/Rhodocyclus tenuis. Within the phenylalanine pathway, the bifunctional P-protein (chorismate mutase/prephenate dehydratase) was present in each lineage and must have evolved in a common ancestor of the beta and gamma subdivisions. Each P-protein was found to be subject to activation by L-tyrosine, and to feedback inhibition by L-phenylalanine. Phenylalanine-inhibited (DS-phe) and tyrosine-inhibited (DS-tyr) isoenzymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase probably existed in the common beta-subdivision ancestor, with DS-tyr being lost in N. gonorrhoeae and A. faecalis. The participation of DS-phe in a dissociable multienzyme complex with one or more other common-pathway enzymes is known to exist in N. gonorrhoeae. The same complex is indicated by two peaks of DS-phe seen in chromatographic profiles of Group-III pseudomonads and A. faecalis. It is concluded that the contemporary DS-phe species present in subdivision gamma and beta must have had independent origins. Tyrosine biosynthesis was found to be quite diverse within the beta subdivision. Nit. europaea possessed an arogenate dehydrogenase which was specific for NADP+. In all other lineages, a broad-specificity cyclohexadienyl dehydrogenase (CHD) was present. In N. gonorrhoeae the CDH was specific for NAD+ while the remaining CDH species could utilize either NAD+ or NADP+. Only the CDH species within the rRNA Group-II pseudomonad/R. tenuis lineage was feedback-inhibited by L-tyrosine, and this correlated with an allosteric pattern where activation of the prephenate dehydratase component of the P-protein by L-tyrosine was relatively poor. However, the CDH enzyme present in N. gonorrhoeae and A. faecalis was subject to inhibition by 4-hydroxyphenylpyruvate, this being competitive with respect to the cyclohexadienyl substrate. The monofunctional species of chorismate mutase (CM-F) and cyclohexadienyl dehydratase, widely distributed among the gamma-subdivision assemblage and recently shown to be periplasmic enzymes, were demonstrated in Pseudomonas pickettii, a member of rRNA homology Group-II.

Alcaligenes↗

PubMed2005

Failure of the ammonia oxidation process in two pharmaceutical wastewater treatment plants is linked to shifts in the bacterial communities.

AIMS: To investigate whether two different wastewater treatment plants (WWTPs) -- treating the same pharmaceutical influent -- select for a different bacterial and/or ammonia oxidizing bacterial (AOB) community. METHODS AND RESULTS: Molecular fingerprinting demonstrated that each WWTP had its own total bacterial and AOB community structure, but Nitrosomonas eutropha and N. europea were dominant in both WWTP A and B. The DNA and RNA analysis of the AOB communities revealed different patterns; so the most abundant species may not necessarily be the most active ones. Nitritation failures, monitored by chemical parameter analysis, were reflected as AOB community shifts and visualized by denaturing gradient gel electrophoresis (DGGE)-based moving window analysis. CONCLUSIONS: This research demonstrated the link between functional performance (nitritation parameters) and the presence and activity of a specific microbial ecology (AOB). Clustering and moving window analysis based on DGGE showed to be valuable to monitor community shifts in both WWTPs. SIGNIFICANCE AND IMPACT OF THE STUDY: This study of specific community shifts together with functional parameter analysis has potential as a tool for relating functional instability (such as operational failures) to specific-bacterial community shifts.

Ammonia↗