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Martin G Klotz - One of the best experts on this subject based on the ideXlab platform.

  • A novel family of functional operons encoding methane/Ammonia Monooxygenase-related proteins in gammaproteobacterial methanotrophs.
    Environmental microbiology reports, 2011
    Co-Authors: Patricia L. Tavormina, Victoria J. Orphan, Marina G. Kalyuzhnaya, Mike S. M. Jetten, Martin G Klotz
    Abstract:

    Genomes of alphaproteobacterial and verrucomicrobial methane-oxidizing bacteria (MOB) encode sequence-divergent copies of particulate methane Monooxygenase [pMMO = (PmoABC); pmoCAB]. In contrast, sequenced gammaproteobacterial MOB (Gamma-MOB) genomes contain single or multiple near-identical copies of pmoCAB operons. In betaproteobacterial Ammonia-oxidizing bacteria (Beta-AOB), near-identical amoCAB operons encode Ammonia Monooxygenase (AMO), a homologue of pMMO. Here, we report that Gamma-MOB in the genera Methylomonas, Methylobacter and Methylomicrobium also encode a sequence-divergent particulate Monooxygenase (pXMO). Whereas all known genes encoding pMMO or AMO cluster in the order ‘CAB’, the genes encoding pXMO are uniquely organized in the non-canonical form ‘pxmABC.’ Steady state pxm mRNA was detected in cultures of Methylomonas sp. as well as in freshwater creek sediment samples, demonstrating that pxm genes are expressed in culture and in situ. Inclusion of PxmA and PxmB proteins in phylogenetic analyses of the Pmo/Amo protein superfamilies created trifurcated trees with three major clades: (i) Pmo of Alpha- and Gamma-MOB and Amo of Gamma-AOB; (ii) Amo of Beta-AOB, Pmo of putative ethane-oxidizing Gamma-MOB and Pxm of Gamma-MOB; and (iii) verrucomicrobial Pmo and Amo of Ammonia-oxidizing Archaea. These data support but do not prove the hypothesis that oxygen-dependent methane and Ammonia Monooxygenases evolved from a substrate-promiscuous ancestor after horizontal transfer while being integrated into the catabolic contexts of their extant hosts.

  • a novel family of functional operons encoding methane Ammonia Monooxygenase related proteins in gammaproteobacterial methanotrophs
    Environmental Microbiology Reports, 2011
    Co-Authors: Patricia L. Tavormina, Victoria J. Orphan, Marina G. Kalyuzhnaya, Mike S. M. Jetten, Martin G Klotz
    Abstract:

    Genomes of alphaproteobacterial and verrucomicrobial methane-oxidizing bacteria (MOB) encode sequence-divergent copies of particulate methane Monooxygenase [pMMO = (PmoABC); pmoCAB]. In contrast, sequenced gammaproteobacterial MOB (Gamma-MOB) genomes contain single or multiple near-identical copies of pmoCAB operons. In betaproteobacterial Ammonia-oxidizing bacteria (Beta-AOB), near-identical amoCAB operons encode Ammonia Monooxygenase (AMO), a homologue of pMMO. Here, we report that Gamma-MOB in the genera Methylomonas, Methylobacter and Methylomicrobium also encode a sequence-divergent particulate Monooxygenase (pXMO). Whereas all known genes encoding pMMO or AMO cluster in the order ‘CAB’, the genes encoding pXMO are uniquely organized in the non-canonical form ‘pxmABC.’ Steady state pxm mRNA was detected in cultures of Methylomonas sp. as well as in freshwater creek sediment samples, demonstrating that pxm genes are expressed in culture and in situ. Inclusion of PxmA and PxmB proteins in phylogenetic analyses of the Pmo/Amo protein superfamilies created trifurcated trees with three major clades: (i) Pmo of Alpha- and Gamma-MOB and Amo of Gamma-AOB; (ii) Amo of Beta-AOB, Pmo of putative ethane-oxidizing Gamma-MOB and Pxm of Gamma-MOB; and (iii) verrucomicrobial Pmo and Amo of Ammonia-oxidizing Archaea. These data support but do not prove the hypothesis that oxygen-dependent methane and Ammonia Monooxygenases evolved from a substrate-promiscuous ancestor after horizontal transfer while being integrated into the catabolic contexts of their extant hosts.

  • Ammonia dependent differential regulation of the gene cluster that encodes Ammonia Monooxygenase in nitrosococcus oceani atcc 19707
    Environmental Microbiology, 2008
    Co-Authors: Amal El F Sheikh, Martin G Klotz
    Abstract:

    Summary Molecular analysis of the Ammonia Monooxygenase-encoding gene cluster in Nitrosococcus oceani revealed that the amo genes are differentially expressed from three promoters dependent on the external Ammonia concentration. Whereas a distal amoRCABD operon promoter, pC1, is active only in the presence of Ammonia, identified proximal amoC (pC2) and amoA (pA) sigma-70-type promoters are constitutive. Promoter region pC2 also contains a consensus sequence for binding of RpoN, usually involved in nitrogen starvation response. Additional regulation of transcript stability by anti-termination and mRNA degradation are proposed and discussed.

  • Ammonia‐dependent differential regulation of the gene cluster that encodes Ammonia Monooxygenase in Nitrosococcus oceani ATCC 19707
    Environmental Microbiology, 2008
    Co-Authors: Amal F. El Sheikh, Martin G Klotz
    Abstract:

    Summary Molecular analysis of the Ammonia Monooxygenase-encoding gene cluster in Nitrosococcus oceani revealed that the amo genes are differentially expressed from three promoters dependent on the external Ammonia concentration. Whereas a distal amoRCABD operon promoter, pC1, is active only in the presence of Ammonia, identified proximal amoC (pC2) and amoA (pA) sigma-70-type promoters are constitutive. Promoter region pC2 also contains a consensus sequence for binding of RpoN, usually involved in nitrogen starvation response. Additional regulation of transcript stability by anti-termination and mRNA degradation are proposed and discussed.

  • Diversity of Ammonia Monooxygenase operon in autotrophic Ammonia-oxidizing bacteria.
    Archives of microbiology, 2001
    Co-Authors: Jeanette M. Norton, J.javier Alzerreca, Yuichi Suwa, Martin G Klotz
    Abstract:

    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 β- and the γ-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 β-subdivision representatives but in single copies in the γ-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 β-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.

Daniel J. Arp - One of the best experts on this subject based on the ideXlab platform.

  • Disruption of sucA, Which Encodes the E1 Subunit of α-Ketoglutarate Dehydrogenase, Affects the Survival of Nitrosomonas europaea in Stationary Phase
    Journal of bacteriology, 2006
    Co-Authors: Norman G Hommes, Luis A. Sayavedra-soto, Elizabeth G. Kurth, Daniel J. Arp
    Abstract:

    Although Nitrosomonas europaea lacks measurable α-ketoglutarate dehydrogenase activity, the recent completion of the genome sequence revealed the presence of the genes encoding the enzyme. A knockout mutation was created in the sucA gene encoding the E1 subunit. Compared to wild-type cells, the mutant strain showed an accelerated loss of Ammonia Monooxygenase and hydroxylamine oxidoreductase activities upon entering stationary phase. In addition, unlike wild-type cells, the mutant strain showed a marked lag in the ability to resume growth in response to pH adjustments in late stationary phase.

  • Inhibition of Membrane-Bound Methane Monooxygenase and Ammonia Monooxygenase by Diphenyliodonium: Implications for Electron Transfer
    Journal of bacteriology, 2004
    Co-Authors: Andrew K. Shiemke, Daniel J. Arp, Luis A. Sayavedra-soto
    Abstract:

    Diphenyliodonium (DPI) is known to irreversibly inactivate flavoproteins. We have found that DPI inhibits both membrane-bound methane Monooxygenase (pMMO) from Methylococcus capsulatus and Ammonia Monooxygenase (AMO) of Nitrosomonas europaea. The effect of DPI on NADH-dependent pMMO activity in vitro is ascribed to inactivation of NDH-2, a flavoprotein which we proposed catalyzes reduction of the quinone pool by NADH. DPI is a potent inhibitor of type 2 NADH:quinone oxidoreductase (NDH-2), with 50% inhibition occurring at ≈5 μM. Inhibition of NDH-2 is irreversible and requires NADH. Inhibition of NADH-dependent pMMO activity by DPI in vitro is concomitant with inhibition of NDH-2, consistent with our proposal that NDH-2 mediates reduction of pMMO. Unexpectedly, DPI also inhibits pMMO activity driven by exogenous hydroquinols, but with ≈100 μM DPI required to achieve 50% inhibition. Similar concentrations of DPI are required to inhibit formate-, formaldehyde-, and hydroquinol-driven pMMO activities in whole cells. The pMMO activity in DPI-treated cells greatly exceeds the activity of NDH-2 or pMMO in membranes isolated from those cells, suggesting that electron transfer from formate to pMMO in vivo can occur independent of NADH and NDH-2. AMO activity, which is known to be independent of NADH, is affected by DPI in a manner analogous to pMMO in vivo: ≈100 μM is required for 50% inhibition regardless of the nature of the reducing agent. DPI does not affect hydroxylamine oxidoreductase activity and does not require AMO turnover to exert its inhibitory effect. Implications of these data for the electron transfer pathway from the quinone pool to pMMO and AMO are discussed.

  • Metabolism of Inorganic N Compounds by Ammonia-Oxidizing Bacteria
    Critical reviews in biochemistry and molecular biology, 2003
    Co-Authors: Daniel J. Arp, Lisa Y Stein
    Abstract:

    Ammonia oxidizing bacteria extract energy for growth from the oxidation of Ammonia to nitrite. Ammonia Monooxygenase, which initiates Ammonia oxidation, remains enigmatic given the lack of purified preparations. Genetic and biochemical studies support a model for the enzyme consisting of three subunits and metal centers of copper and iron. Knowledge of hydroxylamine oxidoreductase, which oxidizes hydroxylamine formed by Ammonia Monooxygenase to nitrite, is informed by a crystal structure and detailed spectroscopic and catalytic studies. Other inorganic nitrogen compounds, including NO, N2O, NO2, and N2 can be consumed and/or produced by Ammonia-oxidizing bacteria. NO and N2O can be produced as byproducts of hydroxylamine oxidation or through nitrite reduction. NO2 can serve as an alternative oxidant in place of O2 in some Ammonia-oxidizing strains. Our knowledge of the diversity of inorganic N metabolism by Ammonia-oxidizing bacteria continues to grow. Nonetheless, many questions remain regarding the enzymes and genes involved in these processes and the role of these pathways in Ammonia oxidizers.

  • Transcript analysis of multiple copies of amo (encoding Ammonia Monooxygenase) and hao (encoding hydroxylamine oxidoreductase) in Nitrosomonas europaea.
    Journal of bacteriology, 2001
    Co-Authors: Norman G Hommes, Luis A. Sayavedra-soto, Daniel J. Arp
    Abstract:

    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.

  • Loss of Ammonia Monooxygenase Activity in Nitrosomonas europaea upon Exposure to Nitrite
    Applied and environmental microbiology, 1998
    Co-Authors: Lisa Y Stein, Daniel J. Arp
    Abstract:

    Nitrosomonas europaea, an obligate Ammonia-oxidizing bacterium, lost an increasing amount of Ammonia oxidation activity upon exposure to increasing concentrations of nitrite, the primary product of Ammonia-oxidizing metabolism. The loss of activity was specific to the Ammonia Monooxygenase (AMO) enzyme, as confirmed by a decreased rate of NH4+-dependent O2 consumption, some loss of active AMO molecules observed by polypeptide labeling with 14C2H2, the protection of activity by substrates of AMO, and the requirement for copper. The loss of AMO activity via nitrite occurred under both aerobic and anaerobic conditions, and more activity was lost under alkaline than under acidic conditions except in the presence of large concentrations (20 mM) of nitrite. These results indicate that nitrite toxicity in N. europaea is mediated by a unique mechanism that is specific for AMO.

Alan B Hooper - One of the best experts on this subject based on the ideXlab platform.

  • differential inhibition in vivo of Ammonia Monooxygenase soluble methane Monooxygenase and membrane associated methane Monooxygenase by phenylacetylene
    Environmental Microbiology, 2000
    Co-Authors: Sonny Lontoh, Alan B Hooper, Alan A. Dispirito, Cinder L. Krema, Mark Whittaker, Jeremy D. Semrau
    Abstract:

    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.

  • Differential inhibition in vivo of Ammonia Monooxygenase, soluble methane Monooxygenase and membrane‐associated methane Monooxygenase by phenylacetylene
    Environmental microbiology, 2000
    Co-Authors: Sonny Lontoh, Alan B Hooper, Alan A. Dispirito, Cinder L. Krema, Mark Whittaker, Jeremy D. Semrau
    Abstract:

    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.

  • Evidence for an iron center in the Ammonia Monooxygenase from Nitrosomonas europaea
    FEBS letters, 1996
    Co-Authors: James A. Zahn, Alan B Hooper, David M. Arciero, Alan A. Dispirito
    Abstract:

    Binding of the ligand, nitric oxide, in the presence of reductant was used to identify a ferrous S=3/2 signal, characteristic of a ferrous nitrosyl complex, and a g=2.03 copper of iron signal in membranes of the Ammonia-oxidizing bacterium, Nitrosomonas europaea. The same ferrous S=3/2 signal is thought to be a component of the membrane-associated methane Monooxygenase (pMMO) of Methylococcus capsulatus Bath, since it is seen in the membrane fraction of cells expressing pMMO and in the purified enzyme, but not in the membrane fraction of cells expressing the soluble MMO [Zahn, J.A. and DiSpirito, A.A. (1996) J. Bacteriol. 178, 1018–1029]. Treatment of resting membranes or cells of N. europaea with nitrapyrin, 2-chloro,6-trichloromethylpyridine, resulted in the increase in magnitude of a g = 6, high-spin ferric iron signal. In the presence of NO and reductant, nitrapyrin prevented the formation of the S=3/2 nitrosyl-iron complex while increasing the intensity of the g=6 signal. Nitrapyrin is a specific inhibitor of, and is reduced by, the Ammonia monoxygenase (AMO) [Bedard, C. and Knowles, R. (1989) Microbiol. Rev. 53, 38–83]. Taken together the data suggest that iron capable of forming the S=3/2 complex is a catalytic component of AMO of N. europaea, possibly a part of the oxygen-activating center. Inactivation of the membrane-associated AMO with acetylene did not diminish the S=3/2 nitrosyl-iron signal, the g=6 signal, or the g=6 signal.

  • Sequence of the gene, amoB, for the 43-kDa polypeptide of Ammonia monoxygenase of Nitrosomonas europaea.
    Biochemical and biophysical research communications, 1994
    Co-Authors: D.j. Bergmann, Alan B Hooper
    Abstract:

    Abstract The sequence for the 3′ portion of amo B, a gene encoding a 43-kDa polypeptide component Ammonia Monooxygenase of Nitrosomonas europaea , is presented. The derived polypeptide has no homology with other known proteins. Amo A and amo B are the only open reading frames in the putative amo operon.

  • Sequence of the gene coding for Ammonia Monooxygenase in Nitrosomonas europaea.
    Journal of Bacteriology, 1993
    Co-Authors: Hugh Mctavish, James A. Fuchs, Alan B Hooper
    Abstract:

    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. Images

Jeanette M. Norton - One of the best experts on this subject based on the ideXlab platform.

  • Diversity of Ammonia Monooxygenase operon in autotrophic Ammonia-oxidizing bacteria.
    Archives of microbiology, 2001
    Co-Authors: Jeanette M. Norton, J.javier Alzerreca, Yuichi Suwa, Martin G Klotz
    Abstract:

    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 β- and the γ-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 β-subdivision representatives but in single copies in the γ-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 β-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.

  • Multiple copies of Ammonia Monooxygenase (amo) operons have evolved under biased AT/GC mutational pressure in Ammonia‐oxidizing autotrophic bacteria
    FEMS microbiology letters, 1998
    Co-Authors: Martin G Klotz, Jeanette M. Norton
    Abstract:

    The recent availability of complete sequences of Ammonia Monooxygenase (16 amoA, 5 amoB and 5 amoC gene sequences) and particulate methane Monooxygenase (2 pmoA, pmoB and pmoC gene sequences each) genes allowed for a detailed analysis of their relatedness. Nucleotide sequence analysis was performed in order to identify the origins of the nearly identical operon copies within a given nitrosofier/methanotroph strain. Our data suggest that amo-homologous gene evolution has occurred in individual strains (orthology) under biased AT/GC pressure rather than by horizontal transfer. The multiple operon copies within individual strains are the result of operon duplication (paralogy). While the near identity of the multiple operon copies makes it impossible to determine whether paralogous gene expansion occurred in the last common ancestor of Ammonia oxidizers or after speciation took place, we conclude that the duplication events were not recent events. We propose that the elimination of third basepair degeneracy between copies within one organism is implemented by a rectification mechanism resulting in concerted evolution.

  • multiple copies of Ammonia Monooxygenase amo operons have evolved under biased at gc mutational pressure in Ammonia oxidizing autotrophic bacteria
    Fems Microbiology Letters, 1998
    Co-Authors: Martin G Klotz, Jeanette M. Norton
    Abstract:

    The recent availability of complete sequences of Ammonia Monooxygenase (16 amoA, 5 amoB and 5 amoC gene sequences) and particulate methane Monooxygenase (2 pmoA, pmoB and pmoC gene sequences each) genes allowed for a detailed analysis of their relatedness. Nucleotide sequence analysis was performed in order to identify the origins of the nearly identical operon copies within a given nitrosofier/methanotroph strain. Our data suggest that amo-homologous gene evolution has occurred in individual strains (orthology) under biased AT/GC pressure rather than by horizontal transfer. The multiple operon copies within individual strains are the result of operon duplication (paralogy). While the near identity of the multiple operon copies makes it impossible to determine whether paralogous gene expansion occurred in the last common ancestor of Ammonia oxidizers or after speciation took place, we conclude that the duplication events were not recent events. We propose that the elimination of third basepair degeneracy between copies within one organism is implemented by a rectification mechanism resulting in concerted evolution.

  • A gene encoding a membrane protein exists upstream of the amoA/amoB genes in Ammonia oxidizing bacteria: a third member of the amo operon?
    FEMS microbiology letters, 1997
    Co-Authors: Martin G Klotz, J.javier Alzerreca, Jeanette M. Norton
    Abstract:

    The gene cluster encoding Ammonia Monooxygenase (AMO) in the chemolithotrophic soil bacterium Nitrosospira sp. NpAV was found to contain a third open reading frame, termed amoC, upstream of the genes amoA and amoB that encode the subunits of AMO. The amoC gene and its flanking regions were isolated and sequenced from a 4.4 kb EcoRI fragment that contains one of three copies of the Ammonia Monooxygenase gene cluster. The presence of this gene upstream of the other two amoA gene copies in Nitrosospira NpAV as well as upstream of amoA genes in the genomes of other Ammonia oxidizing nitrifiers (strains in the genera Nitrosomonas, Nitrosospira, Nitrosolobus and Nitrosovibrio) was confirmed using genomic DNA, oligodeoxyribonucleotide primers and the PCR. The amoC gene in Nitrosospira sp. NpAV encodes a 270 amino acid polypeptide of approximately 36 kDa. Topological analysis of the predicted primary structure revealed 6 membrane spanning domains. The amoC gene was expressed in recombinant Escherichia coli from its indigenous promoter.

  • The gene encoding Ammonia Monooxygenase subunit A exists in three nearly identical copies in Nitrosospira sp. NpAV
    FEMS microbiology letters, 1996
    Co-Authors: Jeanette M. Norton, Jackie M. Low, Martin G Klotz
    Abstract:

    The gene encoding Ammonia Monooxygenase subunit A (AmoA) was found in three copies in the genome of the chemolithotrophic soil bacterium, Nitrosospira sp. NpAV. The open reading frame and flanking regions of the three copies were isolated from digested and size fractionated genomic DNA using oligodeoxyribonucleotide primers and polymerase chain reaction. The three gene copies of amoA were sequenced and the sequences compared to each other. The open reading frames and the upstream and downstream flanking regions were nearly identical in the three copies. All three copies were expressed in recombinant Escherichia coli strains from the indigenous promoter producing a product of approximately 30 kDa. All amoA copies encode 274 amino acid polypeptides which have similarity to the Ammonia Monooxygenase acetylene-binding protein from Nitrosomonas europaea.

Michael R. Hyman - One of the best experts on this subject based on the ideXlab platform.

  • Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea
    Applied and Environmental Microbiology, 2016
    Co-Authors: Kristen Bennett, Natalie C. Sadler, Aaron T. Wright, Chris M Yeager, Michael R. Hyman
    Abstract:

    ABSTRACT Nitrosomonas europaea is an aerobic nitrifying bacterium that oxidizes Ammonia (NH3) to nitrite (NO2−) through the sequential activities of Ammonia Monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4+-dependent O2 uptake by N. europaea by 17OD was time- and concentration-dependent. The effects of 17OD were specific for Ammonia-oxidizing activity, and de novo protein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization–tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA.

  • Ammonia Monooxygenase from Nitrosomonas europaea
    Microbial Growth on C1 Compounds, 1996
    Co-Authors: Daniel J. Arp, William K. Keener, Sterling A. Russell, Norman G Hommes, Michael R. Hyman, Lisa Y. Juliette, Luis A. Sayavedra-soto
    Abstract:

    Nitrosomonas europaea is an obligate chemolithoautotroph which obtains energy for growth from the oxidation of Ammonia (NH3) to nitrite (NO2 -). This bacterium, along with other bacteria which obtain their energy from the oxidation of NH3, contribute to the biogeochemical cycling of inorganic nitrogen. The product of NH3 oxidation, NO2 -, becomes the growth substrate for bacteria which oxidize NO2 - to nitrate (NO3 -). Together, these two groups of bacteria carry out the process known as nitrification. In croplands where NH3 is applied as a nitrogen fertilizer, nitrification can lead to loss of nitrogen because the product, NO3 -, is readily leached into ground and surface waters and serves as a substrate for denitrification. Gaseous products of nitrification include the greenhouse gas nitrous oxide. In the treatment of sewage, nitrification plays a beneficial role in the conversion of NH3 to NO3 - which is readily denitrified to N2. More recently, a potential role for NH3 oxidizers in the degradation of some environmental pollutants (e.g. chlorinated aliphatics) has been recognized.

  • Oxidation of methyl fluoride and dimethyl ether by Ammonia Monooxygenase in Nitrosomonas europaea.
    Applied and environmental microbiology, 1994
    Co-Authors: Michael R. Hyman, C L Page, D J Arp
    Abstract:

    Methyl fluoride and dimethyl ether were previously identified as inhibitors of Ammonia oxidation and N2O production in autotrophic nitrifying bacteria. We demonstrate that methyl fluoride and dimethyl ether are substrates for Ammonia Monooxygenase and are converted to formaldehyde and a mixture of methanol and formaldehyde, respectively.

  • inhibition of Ammonia oxidation in nitrosomonas europaea by sulfur compounds thioethers are oxidized to sulfoxides by Ammonia Monooxygenase
    Applied and Environmental Microbiology, 1993
    Co-Authors: Lisa Y. Juliette, Michael R. Hyman, Daniel J. Arp
    Abstract:

    Organic sulfur compounds are well-known nitrification inhibitors. The inhibitory effects of dimethylsulfide, dimethyldisulfide, and ethanethiol on Ammonia oxidation by Nitrosomonas europaea were examined. Both dimethylsulfide and dimethyldisulfide were weak inhibitors of Ammonia oxidation and exhibited inhibitory characteristics typical of substrates for Ammonia Monooxygenase (AMO). Depletion of dimethylsulfide required O2 and was prevented with either acetylene or allylthiourea, two inhibitors of AMO. The inhibition of Ammonia oxidation by dimethylsulfide was examined in detail. Cell suspensions incubated in the presence of Ammonia oxidized dimethylsulfide to dimethyl sulfoxide. Depletion of six other thioethers was also prevented by treating cell suspensions with either allylthiourea or acetylene. The oxidative products of three thioethers were identified as the corresponding sulfoxides. The amount of sulfoxide formed accounted for a majority of the amount of sulfide depleted. By using gas chromatography coupled with mass spectrometry, allylmethylsulfide was shown to be oxidized to allylmethylsulfoxide by N. europaea with the incorporation of a single atom of 18O derived from 18O2 into the sulfide. This result supported our conclusion that a Monooxygenase was involved in the oxidation of allylmethylsulfide. The thioethers are concluded to be a new class of substrates for AMO. This is the first report of the oxidation of the sulfur atom by AMO in whole cells of N. europaea. The ability of N. europaea to oxidize dimethylsulfide is not unique among the Ammonia-oxidizing bacteria. Nitrosococcus oceanus, a marine nitrifier, was also demonstrated to oxidize dimethylsulfide to dimethyl sulfoxide.

  • In vitro activation of Ammonia Monooxygenase from Nitrosomonas europaea by copper.
    Journal of Bacteriology, 1993
    Co-Authors: Scott A. Ensign, Michael R. Hyman
    Abstract:

    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. Images