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A betaproteobacterial chemolithotrophic ammonia-oxidizing bacterium designated APG5

In this study, the effect of the organic micropollutant and known inhibitor of nitrification, p-cresol, was investigated on the metabolism of the ammonia oxidizing bacteria (AOB) Nitrosomonas eutropha C91 using MS-based quantitative proteomics. Several studies have demonstrated that AOB are capable of biotransforming a wide variety of aromatic compounds making them suitable candidates for bioremediation, yet the underlying molecular mechanisms are poorly described. The effect of two different concentrations of the aromatic micropollutant p-cresol (1 and 10 mg L(-1)) on the metabolism of N. eutropha C91, relative to a p-cresol absent control, was investigated. Though the rate of nitrification in N. eutropha C91 appeared essentially unaffected at both concentrations of p-cresol relative to the control, the expressional pattern of the proteins of N. eutropha C91 changed significantly. The presence of p-cresol resulted in the repressed expression of several key proteins related to N-metabolism, seemingly impairing energy production in N. eutropha C91, contradicting the observed unaltered rates of nitrification. However, the expression of proteins of the TCA cycle and proteins related to xenobiotic degradation, including a p-cresol dehydrogenase, was found to be stimulated by the presence of p-cresol. This indicates that N. eutropha C91 is capable of degrading p-cresol and that it assimilates degradation intermediates into the TCA cycle. The results reveal a pathway for p-cresol degradation and subsequent entry point in the TCA cycle in N. eutropha C91. The obtained data indicate that mixotrophy, rather than cometabolism, is the major mechanism behind p-cresol degradation in N. eutropha C91.

Coexistence of an autotrophic ammonia-oxidizing bacterium (Nitrosomonas sp. RA) and heterotrophic bacteria was consistently observed when cultured in an inorganic medium without any external supply of organic carbon. The present study was undertaken to understand the association between autotrophs and the associated heterotrophs for which a system containing active autotrophs and heterotrophs controlled by Hg(2+) addition was developed. The study revealed interdependence of heterotrophs and Nitrosomonas sp. RA for growth under iron-limited condition. Growth of Nitrosomonas sp. RA was supported by siderophores produced by the associated heterotroph, Pusillimonas sp., thereby complementing its high iron requirement while the organics (such as pyruvate) excreted by Nitrosomonas sp. RA during its autotrophic growth supported the survival of heterotrophs in the inorganic medium. The study thus sheds light on the nature of the mutual interactions between heterotrophs and autotrophs that play a role in the ammonia-oxidizing system involved in wastewater treatment.

The intracellular location of the membrane-bound ammonia monooxygenase (AMO) in all genera of ammonia oxidizing bacteria (Nitrosomonas, Nitrosococcus and Nitrosospira) was determined by electron microscopic immunocytochemistry. Polyclonal antibodies recognizing the two subunits, AmoA- and AmoB-proteins, were used for post-embedding labeling. Ultrathin sections revealed that the AmoB-protein was located in all genera on the cytoplasmic membrane. In cells of Nitrosomonas and Nitrosococus additional but less AmoB-labeling was found on the intracytoplasmic membrane (ICM). In contrast to the detection of AmoB-protein, the AmoA-antibodies failed to detect the AmoA-protein. Based on quantitative immunoblots the extent of ICM in Nitrosomonas eutropha was correlated with the amount of AmoA in the cells. The highest extent of ICM and amount of AmoA was found at low ammonium substrate concentrations.

A pure culture of the obligately lithoautotrophic ammonia-oxidizer Nitrosomonas eutropha was grown in a laboratory-scale bioreactor with complete biomass retention. The air supply was supplemented with nitrogen dioxide (NO2; 25 or 50 ppm) or nitric oxide (NO; 25 or 50 ppm). Compared to cultures grown without these nitrogenous oxides, the addition of NO2 or NO to the culture resulted in a significant increase of the nitrification rate, specific activity of ammonia oxidation, growth rate, and maximum cell densities. In contrast, the growth yield slightly decreased in the presence of NO or NO2. Maximum cell densities of about 2 x 10(10) cells ml-1 and a maximum nitrification rate of about 221 mmol NH4+ l-1 day-1 were obtained after 3 weeks in the presence of 50 ppm NO2. Furthermore, in the stationary phase about 50% of the nitrite produced was aerobically denitrified to dinitrogen (N2) and traces of nitrous oxide (N2O). When cells were supplemented with NO, a high rate of aerobic denitrification occurred only during the first days of the exponential growth phase.

Nitrosomonas eutropha, an obligately lithoautotrophic bacterium, was able to nitrify and denitrify simultaneously under anoxic conditions when gaseous nitrogen dioxide (NO2) was supplemented to the atmosphere. In the presence of gaseous NO2, ammonia was oxidized, nitrite and nitric oxide (NO) were formed, and hydroxylamine occurred as an intermediate. Between 40 and 60% of the produced nitrite was denitrified to dinitrogen (N2). Nitrous oxide (N2O) was shown to be an intermediate of denitrification. Under an N2 atmosphere supplemented with 25 ppm NO2 and 300 ppm CO2, the amount of cell protein increased by 0.87 mg protein per mmol ammonia oxidized, and the cell number of N. eutropha increased by 5.8 x 10(9) cells per mmol ammonia oxidized. In addition, the ATP and NADH content increased by 4.3 micromol ATP (g protein)-1 and 6.3 micromol NADH (g protein)-1 and was about the same in both anaerobically and aerobically grown cells. Without NO2, the ATP content decreased by 0.7 micromol (g protein)-1, and the NADH content decreased by 1.2 micromol (g protein)-1. NO was shown to inhibit anaerobic ammonia oxidation.

Cells of the obligately lithotrophic species Nitrosomonas europaea and Nitrosomonas eutropha were able to nitrify and denitrify at the same time when grown under oxygen limitation. In addition to oxygen, nitrite was used as an electron acceptor. The simultaneous nitrification and denitrification resulted in significant formation of the gaseous N-compounds nitrous oxide and dinitrogen, causing significant nitrogen loss. In mixed cultures of N. europaea and various chemoorganotrophic bacteria, the nitrogen loss was strongly influenced by the partners growing under oxygen limitation. Under anoxic conditions, pure cultures of N. eutropha were able to denitrify with molecular hydrogen as electron donor and nitrite as the only electron acceptor in a sulfide-reduced complex medium. The increase of cell numbers was directly coupled to nitrite reduction. Nitrous oxide and dinitrogen were the only detectable end products. In pure cultures of N. eutropha and mixed cultures of N. eutropha and Enterobacter aerogenes, ammonium and nitrite disappeared slowly at a molar ratio of about one when oxygen was absent. However, under these conditions cell growth was not measurable.

Nitrosomonas europaea and Nitrosovibrio sp. produced NO and N2O during nitrification of ammonium. Less then 15% of the produced NO was due to chemical decomposition of nitrite. Production of NO and especially of N2O increased when the bacteria were incubated under anaerobic conditions at decreasing flow rates of air, or at increasing cell densities. Low concentrations of chlorite (10 μM) inhibited the production of NO and N2, but not of nitrite indicating that NO and N2O were not produced during the oxidative conversion of ammonium to nitrite. NO and N2O were produced during reduction of nitrite with hydrazine as electron donor in almost stoichiometric quantities indicating that reduction of nitrite was the main source of NO and N2O.

The nitrifying bacterium Nitrosomonas europaea contains three copies of the gene (hao) encoding hydroxylamine oxidoreductase (HAO), the second enzyme in the nitrification pathway which oxidizes NH(2)OH to NO(2)(-). The nucleotide sequences of the hao genes differ by only one nucleotide. Two of the three gene copies have identical promoter sequences, while the third promoter has a different nucleotide sequence. Mutant strains with two of the three copies of hao inactivated were created by insertional inactivation, using DNA cassettes containing kanamycin- and gentamycin-resistance genes. All three double-mutant combinations were obtained. These double mutants were phenotypically identical under the conditions tested. Two of these double mutants were similar to wild-type cells or cells having a single hao copy inactivated regarding growth rates or hydroxylamine-dependent O(2) uptake activity, but had only about 50% of the wild-type level of in vitro HAO activity and hao mRNA. The third hao double mutant had an unstable genotype, resulting in recombination of the gentamycin marker into another copy of hao. The N. europaea genomic sequence was recently completed, revealing the locations of the copies of hao and other nitrification genes. Comparison with the arrangement of hao genes in the closely related strain, Nitrosomonas sp. strain ENI-11, showed a similar organization.

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.

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.

Five mixed samples prepared from the surface sediments of 20 north-east Mongolian soda lakes with total salt contents from 5 to 360 g/l and pH values from 9.7 to 10.5 were used to enrich for alkaliphilic ammonia-oxidizing bacteria. Successful enrichments at pH 10 were achieved on carbonate mineral medium containing 0.6 M total Na(+) and < or =4 mM NH(4)Cl. Five isolates (ANs1-ANs5) of ammonia-oxidizing bacteria capable of growth at pH 10 were obtained from the colonies developed on bilayered gradient plates. The cells were motile and coccoid, with well-developed intracytoplasmic membranes (ICPM) and carboxysomes. At pH 10.0, ammonia was toxic for growth at concentrations higher than 5 mM NH(4)Cl. The bacteria were able to grow within the salinity range of 0.1-1.0 M of total Na+ (optimum 0.3 M). In media containing 0.3-0.6 M total Na(+), optimal growth in batch cultures occurred in the presence of a bicarbonate/carbonate buffer system within the pH range 8.5-9.5, with the highest pH limit at pH 10.5. At pH lower than 8.0, growth was slower, most probably due to decreasing free ammonia. The pH profile of the respiratory activity was broader, with limits at 6.5-7.0 and 11.0 and an optimum at 9.5-10.0. In pH-controlled, NH(3)-limited continuous culture, isolate ANs5 grew up to pH 11.3, which is the highest pH limit known for ammonia-oxidizing bacteria so far. This showed the existence of extremely alkali-tolerant ammonia-oxidizing bacteria in the soda lakes. Comparative 16S rDNA sequence analysis of the five isolates demonstrated that they possess identical 16S rDNA genes and that they are closely related to Nitrosomonas halophila (sequence similarity 99.3%), a member of the beta-subclass of the Proteobacteria. This affiliation was confirmed by comparative sequence analysis of the amoA gene, encoding the active-site subunit of the ammonia-monoxygenase, of one of the isolates. DNA-DNA hybridization data further supported that the soda lake isolates are very similar to each other and represent an alkali-tolerant subpopulation of N. halophila whose species description is herewith amended.

Cloned luciferase-encoding operons were transferred by conjugation to a natural isolate of the ammonia-oxidizing bacterial strain Nitrosomonas sp. RST41-3, thereby establishing conjugation as a tool for gene transfer into Nitrosomonas strains. Luminescence was dependent on the pH of the medium and the concentration of the substrate ammonium chloride. Moreover, the luminescence of the transconjugants was reduced immediately by micromolar concentrations of nitrapyrin and allylthiourea, which are specific inhibitors of nitrification. Our results indicate that luminescent Nitrosomonas strains may be useful as a probe to detect nitrification conditions in the natural environment as well as in sewage plants.

Chemolithoautotrophically growing cells of Nitrosomonas europaea quantitatively oxidized ammonia to nitrite under aerobic conditions with no loss of inorganic nitrogen. Significant inorganic nitrogen losses occurred when cells were growing mixotrophically with ammonium, pyruvate, yeast extract and peptone. Under oxygen limitation the nitrogen losses were even higher. In the absence of oxygen pyruvate was metabolized slowly while nitrite was consumed concomitantly. Nitrogen losses were due to the production of nitric oxide and nitrous oxide. In mixed cultures of Nitrosomonas and Nitrobacter, strong inhibition of nitrite oxidation was reproducibly measured. NO and ammonium were not inhibitory to Nitrobacter. First evidence is given that hydroxylamine, the intermediate of the Nitrosomonas monooxygenase-reaction, is formed. 0.2 to 1.7 μM NH2OH were produced by mixotrophically growing cells of Nitrosomonas and Nitrosovibrio. Hydroxylamine was both a selective inhibitory agent to Nitrobacter cells and a strong reductant which reduced nitrite to NO and N2O. It is discussed whether chemodenitrification or denitrification is the most abundant process for NO and N2O production of Nitrosomonas.

The adhesion of Nitrosomonas sp. and Nitrobacter sp. cells isolated from fishpond sediment to different solid particles was studied. Nitrosomonas and Nitrobacter cells rapidly attached to particles of bentonite, calcium carbonate, amberlite, and fishpond sediment, however they did not adhere to phenyl-sepharose beads. The nitrifying activity of attached bacteria was greater than the activity of freely suspended cells or the activity of cells which have been detached from CaCO3 particles. The enhancement in the nitrifying activity was rapid and was already observed within the first hour after attachment (which equals only 1/24 to 1/50 of the generation time of Nitrosomonas sp. or Nitrobacter sp. In addition, the survival of the attached bacteria under both anaerobic and under aerobic incubation was extended to weeks, compared to only a few days for the free cells. The presence of substrate (ammonia or nitrite) during the anaerobic incubation period was found not to affect the survival time of the bacteria. Finally, it was found that the attachment of Nitrosomonas and Nitrobacter cells to CaCO3 particles affected the dispersal and sinking rate of these particles.

Rates of nitrification and organic C production were determined in batch and chemostat cultures of marine nitrifying bacteria; two NH 4 + -oxidizing species and one NO 2 − -oxidizing spezies. With increasing age in batch cultures and with decreasing flow rates in chemostats, cellular organic C and N concentrations declined while the intracellular ratio of C:N remained constant. With decreasing flow rates in chemostats, there was a reduction in (a) carboxylating enzyme activity per unit of cellular organic C (the potential for chemoautotrophic CO2 fixation), and (b) the yield of organic C. For both NH 4 + and NO 2 − oxidizers, rates of nitrification and C yield were lowest at very slow chemostat growth rates, when compared with optimal growth rates in batch cultures. For both NH 4 + and NO 2 − -oxidizing species, the stoichiometric relationship between nitrification and organic C production did not remain constant and appeared to be dependent on the availability of the inorganic N substrate. The organic C yield from NH 4 + oxidation and hence the free energy efficiency declined with increasing age in batch cultures and with decreasing flow rates in chemostats. The C yield from NO 2 − oxidation and the free energy efficiency at slow chemostat growth rates was also lower than that at the optimal growth rate in batch culture.

DNA of 96 strains of the genera Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosolobus, and Nitrosovibrio was isolated and analysed spectrophotometrically. Percentages of guanine plus cytosine (G + C) content, genome sizes, and DNA-DNA homologies were determined. The results indicated the presence of eight Nitrosomonas species, three or four Nitrosococcus species, five Nitrosospira species, and two species of both Nitrosolobus and Nitrosovibrio. DNA homologies between strains of a separate species ranged from 56-100%. Average homologies between strains of different species were 33% in Nitrosococcus, 36% in Nitrosomonas, 37% in Nitrosolobus, 40% in Nitrosospira, and 42% in Nitrosovibrio. Average homologies between species of different genera were 33% and thus not significantly above the background value of 30% detected between DNA of ammonia-oxidizing bacteria and Escherichia coli. Genome sizes ranged from 1.90 - 2.74 X 10(9) dalton in Nitrosomonas, 2.09 - 2.37 X 10(9) dalton in Nitrosococcus, 1.87 - 2.15 X 10(9) dalton in Nitrosospira, 1.92 - 2.10 X 10(9) dalton in Nitrosolobus, and 1.91 - 2.15 X 10(9) dalton in Nitrosovibrio. Differences in genome sizes were in accordance with DNA homologies.

The uptake of ammonia and O2 by washed cells of Nitrosomonas has been followed simultaneously and continuously using electrode techniques. The stoichiometry of NH4+oxidation, O2 uptake and NO2-production was 1 : 1.5 : 1.0 and for NH2OH oxidation a ratio of 1 for O2 : NO2-. A variety of inhibitors of electron transport and metals as well as uncouplers restricted ammonia uptake more markedly than O2 utilization. There is good evidence for the involvement of copper in the NH4+uptake process.

The effect of various organic compounds on the growth of ammonia-oxidizing bacteria was examined.Nitrosococcus oceanus, a strongly halophilic bacterium, had a very low tolerance to organic matter compared with other organisms tested. Organic compounds scarcely affected the growth of theNitrosomonas strains whereas nitrite formation by bothNitrosococcus mobilis strains was inhibited by nearly all of the substances tested. The growth ofNitrosospira strain Nsp1 was enhanced more than 30% by acetate and formate, but not growth was detectable in the presence of pyruvate. On the contrary,Nitrosospira strain Nsp5 was stimulated only by pyruvate. Nitrite formation by the twoNitrosovibrio tenuis strains tested was similar. The growth of both strains was enhanced considerably by formate and glucose; acetate and, to a greater extent, pyruvate inhibited these bacteria. In batch culture, the energy efficiency of autotrophically grown ammonia-oxidizing bacteria varied from strain to strain. The cell yield of mixotrophically grown cultures, per unit of ammonia oxidized, was increased in comparison with autotrophic ones. No heterotrophic growth was detected.

Rabbit antisera were prepared against living cells of six different ammonia oxidizing nitrifying bacteria. They were examined as to cross-reactivity in the agglutination test (Microtiter-system) with 24 nitrifier strains, including members of all known genera. Usually distinct cross-reactions were obtained only within the genera, but some exceptions were noticed. There was stated a clear cross-reaction between the two anti-Nitrosospira-antisera and the four tested Nitrosolobus strains. In some cases cross-reactions between cells of the Nitrosovibrio strains and the anti-Nitrosospira- as well as the anti-Nitrosococcus-antisera could be observed. The interpretation of the results obtained with the Nitrosomonas group was complicated by the fact that all strains showed positive zero titers with the control sera. In seven cases lipopolysaccharides were isolated and tested in the passive hemagglutination test to their cross-reactivity with the above mentioned antisera. Hemagglutination could only be observed in the homologous system, cross-reactivity was never expressed.

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 ammonia oxidizing bacterium Nitrosomonas europaea was grown either (a) with added bicarbonate in the absence of added CO2 (bubbled through the culture), (b) with added bicarbonate plus low added CO2 (0.03% v/v), or (c) without added bicarbonate with high added CO2 (1% v/v). Cell doubling times of 12 h were observed in 1% cultures; doubling times of 2 to 3-fold longer wre found with 0.03% CO2 and/or bicarbonate grown cultures. The specific activity of carbonic anhydrase was 40–80% lower in cultures grown on 1% CO2. These results are compared with those in heterotrophic and photosynthetic microorganisms.

Polyhedral inclusion bodies were observed in cells of a Nitrosomonas species. They were present in growing cells as well as in resting cells. In thin sections their size was about 130 nm in growing cells and about 185 nm in diameter in resting cells. The bodies were commonly located in the nucleoplasm. They appeared to be bounded by a nonunit membrane and had a granular substructure. In thin sections about 70% of the exponentially grown cells and about 20% of the resting cells of the investigated strain showed 1-7 respectively 1-3 inclusion bodies.

Polydedral inclusion bodies were isolated from exponentially grown cells of Nitrosomonas spec. The bodies contained d-ribulose, 1,5-bisphosphate carboxylase. The specific activity of the enzyme was 0.0122 μmol CO2 fixed per min per mg of protein.