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At least 145 records · Page 8Linked to original sources
PubMed2005

Cometabolism of trihalomethanes by Nitrosomonas europaea.

The ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718) was shown to degrade low concentrations (50 to 800 mug/liter) of the four trihalomethanes (trichloromethane [TCM], or chloroform; bromodichloromethane [BDCM]; dibromochloromethane [DBCM]; and tribromomethane [TBM], or bromoform) commonly found in treated drinking water. Individual trihalomethane (THM) rate constants (k1THM) increased with increasing THM bromine substitution, with TBM > DBCM > BDCM > TCM (0.23, 0.20, 0.15, and 0.10 liters/mg/day, respectively). Degradation kinetics were best described by a reductant model that accounted for two limiting reactants, THMs and ammonia-nitrogen (NH3-N). A decrease in the temperature resulted in a decrease in both ammonia and THM degradation rates with ammonia rates affected to a greater extent than THM degradation rates. Similarly to the THM degradation rates, product toxicity, measured by transformation capacity (Tc), increased with increasing THM bromine substitution. Because both the rate constants and product toxicities increase with increasing THM bromine substitution, a water's THM speciation will be an important consideration for process implementation during drinking water treatment. Even though a given water sample may be kinetically favored based on THM speciation, the resulting THM product toxicity may not allow stable treatment process performance.

Ammonia↗
PubMed2005

Identification of acyl-homoserine lactone signal molecules produced by Nitrosomonas europaea strain Schmidt.

Nitrosomonas europaea strain Schmidt produces at least three acyl homoserine lactone (AHL) signal molecules: C(6)-homoserine lactone (HSL), C(8)-HSL, and C(10)-HSL. These compounds were identified in extracts of chemostat culture effluent by three independent methods. The concentrations of AHL in effluent were low (0.4 to 2.2 nM) but within the range known to induce AHL-responsive systems. The absence of LuxI and LuxM homologs from the genome of N. europaea strain Schmidt suggested that AHL synthesis occurs by an alternate pathway, possibly mediated by an HdtS homolog. To the best of our knowledge, the present report is the first to document the types and levels of AHLs produced by N. europaea.

4-Butyrolactone↗
PubMed1973

Specific inhibitors of ammonia oxidation in Nitrosomonas.

The following compounds or treatments have been shown to inhibit the oxidation of ammonia, but not the oxidation of hydroxylamine in cells of Nitrosomonas: (i) metal-binding agents such as allylthiourea or potassium cyanide; (ii) compounds such as SKF 525 which interact with cytochrome P-450 of mammalian microsomes; (iii) carbon monoxide; (iv) inhibitors of catalase, peroxidase, and amine oxidases such as thiosemicarbazide, ethylxanthate, and iproniazid, respectively; (v) uncouplers of oxidative phosphorylation such as m-chlorocarbonylcyanidephenylhydrazone; (vi) electron acceptors such as phenazine methosulfate; (vii) compounds such as methanol or N(2)O which react with free radicals; and (viii) illumination with 420 lux (5,000 foot candles) of light.

Alcohols↗
PubMed1974

Photoinactivation of ammonia oxidation in Nitrosomonas.

Photoinactivation of ammonia oxidation in cells of Nitrosomonas was shown to follow first-order kinetics with a rate constant proportional to incident light intensity. The action spectrum for photoinactivation consisted of a broad peak in the ultraviolet range, where both hydroxylamine and ammonia oxidation were affected, and a shoulder at approximately 410 nm where only ammonia oxidation was affected. In photoinactivated cells, hydroxylamine but not ammonia was oxidized to nitrite and hydroxylamine but not ammonia caused reduction of cytochromes in vivo. The amount per cell of the following constituents was not measurably altered by photoinactivation: cytochromes b, c, a, and P460; ubiquinone; phospholipid; free amino acids; hydroxylamine-dependent nitrite synthetase; nitrite reductase; p-phenylenediamine oxidase; and cytochrome c oxidase. Malonaldehyde or lipid peroxides were not detected in photoinactivated cells. Photoinactivation was prevented (i) under anaerobic conditions, (ii) in the presence of methanol, allylthiourea, thiosemicarbazide, hydroxylamine, ethylxanthate, or CO at concentrations wich caused 100% inhibition of ammonia oxidation, and (iii) at concentrations of ammonia or hydroxylamine which gave a rapid rate of nitrite production. Recovery of ammonia oxidation activity in 90% inactivated cells took place in 6 h, required an energy and/or nitrogen source, and was inhibited by 400 mug of chloramphenicol per ml.

Amino Acids↗
PubMed1990

Inhibition of ammonia monooxygenase in Nitrosomonas europaea by carbon disulfide.

Carbon disulfide has long been recognized as a potent inhibitor of nitrification, and it is the likely active component in several nitrification inhibitors suitable for field use. The effects of this compound on Nitrosomonas europaea have been investigated, and the site of action has been determined. Low concentrations of CS2 (less than 400 microM) produced a time-dependent inhibition of ammonia-dependent O2 uptake but did not inhibit hydrazine-oxidizing activity. CS2 also produced distinct changes in difference spectra of whole cells. These results suggest that ammonia monooxygenase (AMO) is the site of action of CS2. Unlike the case for thiourea and acetylene, saturating concentrations of CS2 did not fully inhibit AMO, and the inhibition resulted in a low but significant rate of ammonia-dependent O2 uptake. The effects of CS2 were not competitive with respect to ammonia concentration, and the inhibition by CS2 did not require the turnover of AMO to take effect. The ability of CS2-treated cells to incorporate [14C]acetylene into the 28-kilodalton polypeptide of AMO was used to demonstrate that the effects of CS2 are compatible with a mode of action which involves a reduction of the rate of turnover of AMO without effects on the catalytic mechanism. It is proposed that CS2 may act on AMO by reversibly reacting with a suitable nucleophilic amino acid in close proximity to the active site copper.

Acetylene↗
PubMed1990

Oxidation of monohalogenated ethanes and n-chlorinated alkanes by whole cells of Nitrosomonas europaea.

We have investigated the substrate specificity of ammonia monooxygenase in whole cells of the nitrifying bacterium Nitrosomonas europaea for a number of aliphatic halogenated hydrocarbons. To determine the effect of the halogen substituent and carbon chain length on substrate reactivity, we measured the rates of oxidation of the monohalogenated ethanes (fluoroethane, chloroethane, bromoethane, and iodoethane) and n-chlorinated C1 to C4 alkanes by whole cells of N. europaea. For monohalogenated ethanes, acetaldehyde was the major organic product and little or none of any of the alternate predicted products (2-halogenated alcohols) were detected. The maximum rate of haloethane oxidation increased with decreasing halogen molecular weight from iodoethane to chloroethane (19 to 221 nmol/min per mg of protein). In addition, the amount of substrate required for the highest rate of haloethane oxidation increased with decreasing halogen molecular weight. For the n-chlorinated alkanes, the rate of dechlorination, as measured by the appearance of the corresponding aldehyde product, was greatest for chloroethane and decreased dramatically for chloropropane and chlorobutane (118, 4, and 8 nmol of aldehyde formed per min per mg of protein, respectively). The concentration profiles for halocarbon oxidation by ammonia monooxygenase showed apparent substrate inhibition when ammonia was used as the reductant source. When hydrazine was used as the electron donor, no substrate inhibition was observed, suggesting that the inhibition resulted from reductant limitation.

Alkanes↗
PubMed1993

In vitro activation of ammonia monooxygenase from Nitrosomonas europaea by copper.

The effect of copper on the in vivo and in vitro activity of ammonia monooxygenase (AMO) from the nitrifying bacterium Nitrosomonas europaea was investigated. The addition of CuCl2 to cell extracts resulted in 5- to 15-fold stimulation of ammonia-dependent O2 consumption, ammonia-dependent nitrite production, and hydrazine-dependent ethane oxidation. AMO activity was further stimulated in vitro by the presence of stabilizing agents, including serum albumins, spermine, or MgCl2. In contrast, the addition of CuCl2 and stabilizing agents to whole-cell suspensions did not result in any stimulation of AMO activity. The use of the AMO-specific suicide substrate acetylene revealed two populations of AMO in cell extracts. The low, copper-independent (residual) AMO activity was completely inactivated by acetylene in the absence of exogenously added copper. In contrast, the copper-dependent (activable) AMO activity was protected against acetylene inactivation in the absence of copper. However, in the presence of copper both populations of AMO were inactivated by acetylene. [14C]acetylene labelling of the 27-kDa polypeptide of AMO revealed the same extent of label incorporation in both whole cells and optimally copper-stimulated cell extracts. In the absence of copper, the label incorporation in cell extracts was proportional to the level of residual AMO activity. Other metal ions tested, including Zn2+, Co2+, Ni2+, Fe2+, Fe3+, Ca2+, Mg2+, Mn2+, Cr3+, and Ag+, were ineffective at stimulating AMO activity or facilitating the incorporation of 14C label from [14C]acetylene into the 27-kDa polypeptide. On the basis of these results, we propose that loss of AMO activity upon lysis of N. europaea results from the loss of copper from AMO, generating a catalytically inactive, yet stable and activable, form of the enzyme.

Acetylene↗
PubMed1993

Sequence of the gene coding for ammonia monooxygenase in Nitrosomonas europaea.

Nitrosomonas europaea, a chemolithotrophic bacterium, was found to contain two copies of the gene coding for the presumed active site polypeptide of ammonia monooxygenase, the 32-kDa acetylene-binding polypeptide. One copy of this gene was cloned, and its complete nucleotide sequence is presented. Immediately downstream of this gene, in the same operon, is the gene for a 40-kDa polypeptide that copurifies with the ammonia monooxygenase acetylene-binding polypeptide. The sequence of the first 692 nucleotides of this structural gene, coding for about two-thirds of the protein, is presented. These sequences are the first sequences of protein-encoding genes from an ammonia-oxidizing autotrophic nitrifying bacterium. The two protein sequences are not homologous with the sequences of any other monooxygenase. From radioactive labelling of ammonia monooxygenase with [14C]acetylene it was determined that there are 23 nmol of ammonia monooxygenase per g of cells. The kcat of ammonia monooxygenase for NH3 in vivo was calculated to be 20 s-1.

Acetylene↗
PubMed1993

Multiple copies of genes coding for electron transport proteins in the bacterium Nitrosomonas europaea.

The genome of Nitrosomonas europaea contains at least three copies each of the genes coding for hydroxylamine oxidoreductase (HAO) and cytochrome c554. A copy of an HAO gene is always located within 2.7 kb of a copy of a cytochrome c554 gene. Cytochrome P-460, a protein that shares very unusual spectral features with HAO, was found to be encoded by a gene separate from the HAO genes.

Amino Acid Sequence↗
PubMed1994

Organization of the hao gene cluster of Nitrosomonas europaea: genes for two tetraheme c cytochromes.

The organization of genes for three proteins involved in ammonia oxidation in Nitrosomonas europaea has been investigated. The amino acid sequence of the N-terminal region and four heme-containing peptides produced by proteolysis of the tetraheme cytochrome c554 of N. europaea were determined by Edman degradation. The gene (cycA) encoding this cytochrome is present in three copies per genome (H. McTavish, F. LaQuier, D. Arciero, M. Logan, G. Mundfrom, J.A. Fuchs, and A. B. Hooper, J. Bacteriol. 175:2445-2447, 1993). Three clones, representing at least two copies of cycA, were isolated and sequenced by the dideoxy-chain termination procedure. In both copies, the sequences of 211 amino acids derived from the gene sequence are identical and include all amino acids predicted by the proteolytic peptides. In two copies, the cycA open reading frame (ORF) is followed closely (three bases in one copy) by a second ORF predicted to encode a 28-kDa tetraheme c cytochrome not previously characterized but similar to the nirT gene product of Pseudomonas stutzeri. In one copy of the cycA gene cluster, the second ORF is absent.

Amino Acid Sequence↗
PubMed1994

Characterization of the gene encoding hydroxylamine oxidoreductase in Nitrosomonas europaea.

Hydroxylamine oxidoreductase (HAO) catalyzes the oxidation of hydroxylamine to nitrite in Nitrosomonas europaea. The electrons released in the reaction are partitioned to ammonium monooxygenase and to the respiratory chain. The immediate acceptor of electrons from HAO is believed to be cytochrome c-554 (Cyt c-554). We have isolated a genomic DNA fragment containing the structural gene encoding HAO (hao) and a part of the gene for Cyt c-554. The nucleotide sequence of hao was determined, and its transcription was analyzed. The open reading frame (ORF) encodes amino acid sequences matching the purified peptides of HAO. A 64.28-kDa protein is encoded in this ORF, in close agreement with the empirically determined molecular mass of 63 kDa. The N terminus was located 24 amino acids from the start codon, suggesting the presence of a leader sequence. The putative eight heme-binding peptides were localized in this ORF. The gene for Cyt c-554 was located 1,200 bp downstream from the 3' end of hao. An ORF was identified in the upstream region from hao and may encode a protein of unknown function. Data bank searches did not reveal proteins with substantial similarities to HAO, but they did reveal similarities between Cyt c-554 and other c-type cytochromes.

Amino Acid Sequence↗
PubMed1995

Effects of ammonia on the de novo synthesis of polypeptides in cells of Nitrosomonas europaea denied ammonia as an energy source.

The effects of ammonium on the de novo synthesis of polypeptides in the soil-nitrifying bacterium Nitrosomonas europaea have been investigated. Cells were incubated in the presence of both acetylene and NH4+. Under these conditions, the cells were unable to utilize NH4+ as an energy source. Energy to support protein synthesis was supplied by the oxidation of hydroxylamine or other alternative substrates for hydroxylamine oxidoreductase. De novo protein synthesis was detected by 14C incorporation from 14CO2 into polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. In the presence of NH4+, acetylene-treated cells synthesized the 27-kDa polypeptide of ammonia monoxygenase (AMO) and two other major polypeptides (with sizes of 55 and 65 kDa). The synthesis of these polypeptides was completely inhibited by chloramphenicol and attenuated by rifampin. The optimal concentration of hydroxylamine for the in vivo 14C-labeling reaction was found to be 2 mM. The effect of NH4+ concentration was also examined. It was shown to cause a saturable response with a Ks of approximately 2.0 mM NH4+. Labeling studies conducted at different pH values suggest cells respond to NH3 rather than NH4+. No other compounds tested were able to influence the synthesis of the 27-kDa component of AMO, although we have also demonstrated that this polypeptide can be synthesized under anaerobic conditions in cells utilizing pyruvate- or hydrazine-dependent nitrite reduction as an energy source. We conclude that ammonia has a regulatory effect on the synthesis of a subunit of AMO in addition to providing nitrogen for protein synthesis.

Acetylene↗
PubMed1996

Mutagenesis of hydroxylamine oxidoreductase in Nitrosomonas europaea by transformation and recombination.

Mutagenesis of Nitrosomonas europaea was achieved by electroporation and recombination. To demonstrate this, an aminoglycoside 3'-phosphotransferase (kan) gene was specifically inserted into each of the three gene copies of hao individually. Southern hybridizations and PCR analysis showed the incorporation of the kan gene at the chosen genetic loci. The isolation of mutant strains was achieved in 7 to 14 days when the strains were grown on solid medium. The induced mutations were stable even in the absence of kanamycin-selective pressure for periods of up to 45 days in culture. The mutant strains did not show an observable phenotype different from that of the wild type when grown under the same conditions.

Base Sequence↗
PubMed1998

Mutagenesis and expression of amo, which codes for ammonia monooxygenase in Nitrosomonas europaea.

Nitrosomonas europaea has two copies of the operon encoding ammonia monooxygenase (AMO). The nucleotide sequences of the two copies of amoA were obtained, and they were found to differ by one nucleotide. To determine if both copies of amoA were functional, insertional mutagenesis was performed to inactivate either copy of amoA alone. A DNA cassette containing the lacZ and kan genes inserted into amoA was constructed. Mutagenesis was done by using transformation and homologous recombination to mobilize the cassette into the chromosomal copies of amoA. Mutations were obtained in both copies of amoA. Either copy of amoA was sufficient to support growth when the other copy was disrupted. However, inactivation of one copy of amoA, but not the other, resulted in slower growth. Measurements of ammonia-dependent O2 consumption, which depends on AMO, confirmed that the slower-growing mutant had lower activity while the faster-growing mutant had near wild-type levels of activity. Similarly, as measured by [14C]acetylene label incorporation, there was less active AMO present in the slower-growing mutant than in the faster-growing mutant or in the wild type. Northern blot analysis of transcription likewise showed that the slower-growing mutant had less full-sized AMO mRNA.

Acetylene↗
PubMed2000

Physical map location of the multicopy genes coding for ammonia monooxygenase and hydroxylamine oxidoreductase in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11.

Pulsed-field gel electrophoresis of PmeI digests of the Nitrosomonas sp. strain ENI-11 chromosome produced four bands ranging from 1,200 to 480 kb in size. Southern hybridizations suggested that a 487-kb PmeI fragment contained two copies of the amoCAB genes, coding for ammonia monooxygenase (designated amoCAB(1) and amoCAB(2)), and three copies of the hao gene, coding for hydroxylamine oxidoreductase (hao(1), hao(2), and hao(3)). In this DNA fragment, amoCAB(1) and amoCAB(2) were about 390 kb apart, while hao(1), hao(2), and hao(3) were separated by at least about 100 kb from each other. Interestingly, hao(1) and hao(2) were located relatively close to amoCAB(1) and amoCAB(2), respectively. DNA sequence analysis revealed that hao(1) and hao(2) shared 160 identical nucleotides immediately upstream of each translation initiation codon. However, hao(3) showed only 30% nucleotide identity in the 160-bp corresponding region.

Chromosome Mapping↗
PubMed2001

Transcript analysis of multiple copies of amo (encoding ammonia monooxygenase) and hao (encoding hydroxylamine oxidoreductase) in Nitrosomonas europaea.

The genes encoding ammonia monooxygenase (amoCAB), hydroxylamine oxidoreductase (hao), and the c-type cytochrome c-554 (hcy) are present in multiple copies in the genome of Nitrosomonas europaea. The upstream regions of the two copies of amoC, the three copies of hao, and one copy of hcy were cloned and sequenced. Primer extension reactions were done to identify transcription start sites for these genes, as well as for amoA. Putative sigma(70) promoter sequences were found associated with all but one of the mapped transcription start sites. Primer extensions were done with amoC primers using RNA harvested from cells incubated with and without ammonium. The experiments suggested that N. europaea cells may be able to use different promoters in the presence and absence of ammonium.

Ammonia↗
PubMed2002

Nitrite reductase of Nitrosomonas europaea is not essential for production of gaseous nitrogen oxides and confers tolerance to nitrite.

A gene that encodes a periplasmic copper-type nitrite reductase (NirK) was identified in Nitrosomonas europaea. Disruption of this gene resulted in the disappearance of Nir activity in cell extracts. The nitrite tolerance of NirK-deficient cells was lower than that of wild-type cells. Unexpectedly, NirK-deficient cells still produced nitric oxide (NO) and nitrous oxide (N(2)O), the latter in greater amounts than that of wild-type cells. This demonstrates that NirK is not essential for the production of NO and N(2)O by N. europaea. Inactivation of the putative fnr gene showed that Fnr is not essential for the expression of nirK.

Bacterial Proteins↗