Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea.

Ammonia-oxidizing archaea are ubiquitous in marine and terrestrial environments and now thought to be significant contributors to carbon and nitrogen cycling. The isolation of Candidatus "Nitrosopumilus maritimus" strain SCM1 provided the opportunity for linking its chemolithotrophic physiology with a genomic inventory of the globally distributed archaea. Here we report the 1,645,259-bp closed genome of strain SCM1, revealing highly copper-dependent systems for ammonia oxidation and electron transport that are distinctly different from known ammonia-oxidizing bacteria. Consistent with in situ isotopic studies of marine archaea, the genome sequence indicates N. maritimus grows autotrophically using a variant of the 3-hydroxypropionate/4-hydroxybutryrate pathway for carbon assimilation, while maintaining limited capacity for assimilation of organic carbon. This unique instance of archaeal biosynthesis of the osmoprotectant ectoine and an unprecedented enrichment of multicopper oxidases, thioredoxin-like proteins, and transcriptional regulators points to an organism responsive to environmental cues and adapted to handling reactive copper and nitrogen species that likely derive from its distinctive biochemistry. The conservation of N. maritimus gene content and organization within marine metagenomes indicates that the unique physiology of these specialized oligophiles may play a significant role in the biogeochemical cycles of carbon and nitrogen.

Evidence for involvement of copper ions and redox state in regulation of butane monooxygenase in Pseudomonas butanovora.

Pseudomonas butanovora possesses an alcohol-inducible alkane monooxygenase, butane monooxygenase (BMO), that initiates growth on C(2)-C(9) alkanes. A lacZ transcriptional reporter strain, P. butanovora bmoX::lacZ, in which the BMO promoter controls the expression of beta-galactosidase activity, was used to show that 1-butanol induced the BMO promoter in the presence or absence of O(2) when lactate-grown, BMO-repressed cells were washed free of lactate and incubated in NH(4)Cl-KNa phosphate buffer. In contrast, when lactate-grown cells of the reporter strain were incubated in phosphate buffer containing the mineral salts of standard growth medium, 1-butanol-dependent induction was significantly repressed at low O(2) (1 to 2% [vol/vol]) and totally repressed under anoxic conditions. The repressive effect of the mineral salts was traced to its copper content. In cells exposed to 1% (vol/vol) O(2), CuSO(4) (0.5 microM) repressed 1-butanol-dependent induction of beta-galactosidase activity. Under oxic conditions (20% O(2) [vol/vol]), significantly higher concentrations of CuSO(4) (2 microM) were required for almost complete repression of induction in lactate-grown cells. A combination of the Cu(2+) reducing agent Na ascorbate (100 microM) and CuSO(4) (0.5 microM) repressed the induction of beta-galactosidase activity under oxic conditions to the same extent that 0.5 microM CuSO(4) alone repressed it under anoxic conditions. Under oxic conditions, 2 microM CuSO(4) repressed induction of the BMO promoter less effectively in butyrate-grown cells of the bmoX::lacZ strain and of an R8-bmoX::lacZ mutant reporter strain with a putative BMO regulator, BmoR, inactivated. Under anoxic conditions, CuSO(4) repression remained highly effective, regardless of the growth substrate, in both BmoR-positive and -negative reporter strains.

Propionate inactivation of butane monooxygenase activity in 'Pseudomonas butanovora': biochemical and physiological implications.

Butane monooxygenase (BMO) catalyses the oxidation of alkanes to alcohols in the alkane-utilizing bacterium 'Pseudomonas butanovora'. Incubation of alkane-grown 'P. butanovora' with butyrate or propionate led to irreversible time- and O2-dependent loss of BMO activity. In contrast, BMO activity was unaffected by incubation with lactate or acetate. Chloramphenicol inhibited the synthesis of new BMO, but did not change the kinetics of propionate-dependent BMO inactivation, suggesting that the propionate effect was not simply due to it acting as a repressor of BMO transcription. BMO was protected from propionate-dependent inactivation by the presence of its natural substrate, butane. Although both the time and O2 dependency of propionate inactivation of BMO imply that propionate might be a suicide substrate, no evidence was obtained for BMO-dependent propionate consumption, or 14C labelling of BMO polypeptides by [2-(14)C]propionate during inactivation. Propionate-dependent BMO inactivation was also explored in mutant strains of 'P. butanovora' containing single amino acid substitutions in the alpha-subunit of the BMO hydroxylase. Propionate-dependent BMO inactivation in two mutant strains with amino acid substitutions close to the catalytic site differed from wild-type (one was more sensitive and the other less), providing further evidence that propionate-dependent inactivation involves interaction with the BMO catalytic site. A putative model is presented that might explain propionate-dependent inactivation of BMO when framed within the context of the catalytic cycle of the closely related enzyme, soluble methane monooxygenase.

Product repression of alkane monooxygenase expression in Pseudomonas butanovora.

Physiological and regulatory mechanisms that allow the alkane-oxidizing bacterium Pseudomonas butanovora to consume C2 to C8 alkane substrates via butane monooxygenase (BMO) were examined. Striking differences were observed in response to even- versus odd-chain-length alkanes. Propionate, the downstream product of propane oxidation and of the oxidation of other odd-chain-length alkanes following beta-oxidation, was a potent repressor of BMO expression. The transcriptional activity of the BMO promoter was reduced with as little as 10 microM propionate, even in the presence of appropriate inducers. Propionate accumulated stoichiometrically when 1-propanol and propionaldehyde were added to butane- and ethane-grown cells, indicating that propionate catabolism was inactive during growth on even-chain-length alkanes. In contrast, propionate consumption was induced (about 80 nmol propionate consumed.min(-1).mg protein(-1)) following growth on the odd-chain-length alkanes, propane and pentane. The induction of propionate consumption could be brought on by the addition of propionate or pentanoate to the growth medium. In a reporter strain of P. butanovora in which the BMO promoter controls beta-galactosidase expression, only even-chain-length alcohols (C2 to C8) induced beta-galactosidase following growth on acetate or butyrate. In contrast, both even- and odd-chain-length alcohols (C3 to C7) were able to induce beta-galactosidase following the induction of propionate consumption by propionate or pentanoate.

Effects of dichloroethene isomers on the induction and activity of butane monooxygenase in the alkane-oxidizing bacterium "Pseudomonas butanovora".

We examined cooxidation of three different dichloroethenes (1,1-DCE, 1,2-trans DCE, and 1,2-cis DCE) by butane monooxygenase (BMO) in the butane-utilizing bacterium "Pseudomonas butanovora." Different organic acids were tested as exogenous reductant sources for this process. In addition, we determined if DCEs could serve as surrogate inducers of BMO gene expression. Lactic acid supported greater rates of oxidation of the three DCEs than the other organic acids tested. The impacts of lactic acid-supported DCE oxidation on BMO activity differed among the isomers. In intact cells, 50% of BMO activity was irreversibly lost after consumption of approximately 20 nmol mg protein(-1) of 1,1-DCE and 1,2-trans DCE in 0.5 and 5 min, respectively. In contrast, a comparable loss of activity required the oxidation of 120 nmol 1,2-cis DCE mg protein(-1). Oxidation of similar amounts of each DCE isomer ( approximately 20 nmol mg protein(-1)) produced different negative effects on lactic acid-dependent respiration. Despite 1,1-DCE being consumed 10 times faster than 1,2,-trans DCE, respiration declined at similar rates, suggesting that the product(s) of oxidation of 1,2-trans DCE was more toxic to respiration than 1,1-DCE. Lactate-grown "P. butanovora" did not express BMO activity but gained activity after exposure to butane, ethene, 1,2-cis DCE, or 1,2-trans DCE. The products of BMO activity, ethene oxide and 1-butanol, induced lacZ in a reporter strain containing lacZ fused to the BMO promoter, whereas butane, ethene, and 1,2-cis DCE did not. 1,2-trans DCE was unique among the BMO substrates tested in its ability to induce lacZ expression.

Biodegradation of monohalogenated alkanes by soil NH(3)-oxidizing bacteria.

Although cooxidative biodegradation of monohalogenated hydrocarbons has been well studied in the model NH(3)-oxidizing bacterium, Nitrosomonas europaea, virtually no information exists about cooxidation of these compounds by native populations of NH(3)-oxidizing bacteria. To address this subject, nitrifying activity was stimulated to 125-400 nmol NO(3)(-) produced g(-1) soil h(-1) by first incubating a Ca(OH)(2)-amended, silt loam soil (pH 7.0+/-0.2) at field capacity (270 g H(2)O kg(-1) soil) with 10 micro mol NH(4)(+) g(-1) soil for 14 days, followed by another 10 days of incubation in a shaken slurry (2:1 water:soil, v/w) with periodic pH adjustment and maintenance of 10 mM NH(4)(+). These slurries actively degraded both methyl bromide (MeBr) and ethyl chloride (EtCl) at maximum rates of 20-30 nmol ml(-1) h(-1) that could be sustained for approximately 12 h. Although the MeBr degradation rates were linear for the first 10-12 h of incubation, they could not be sustained regardless of NH(4)(+) level and declined to zero over 20 h of incubation. The transformation capacity of the slurry enrichments (~1 micro mol MeBr ml(-1) soil slurry) was similar to the value measured previously in cell suspensions of N. europaea with similar NH(3)-oxidizing activity. Several MeBr-degrading characteristics of the nitrifying enrichments were found to be similar to those documented in the literature for MeBr-degrading methanotrophs and facultatively methylotrophic bacteria.

Requirement of DNA repair mechanisms for survival of Burkholderia cepacia G4 upon degradation of trichloroethylene.

A Tn5-based mutagenesis strategy was used to generate a collection of trichloroethylene (TCE)-sensitive (TCS) mutants in order to identify repair systems or protective mechanisms that shield Burkholderia cepacia G4 from the toxic effects associated with TCE oxidation. Single Tn5 insertion sites were mapped within open reading frames putatively encoding enzymes involved in DNA repair (UvrB, RuvB, RecA, and RecG) in 7 of the 11 TCS strains obtained (4 of the TCS strains had a single Tn5 insertion within a uvrB homolog). The data revealed that the uvrB-disrupted strains were exceptionally susceptible to killing by TCE oxidation, followed by the recA strain, while the ruvB and recG strains were just slightly more sensitive to TCE than the wild type. The uvrB and recA strains were also extremely sensitive to UV light and, to a lesser extent, to exposure to mitomycin C and H(2)O(2). The data from this study establishes that there is a link between DNA repair and the ability of B. cepacia G4 cells to survive following TCE transformation. A possible role for nucleotide excision repair and recombination repair activities in TCE-damaged cells is discussed.

Molecular and cellular fundamentals of aerobic cometabolism of trichloroethylene.

Cometabolism recognizes that microorganisms can transform non-growth-supporting substrates. The term "cometabolism" was first introduced over 30 years ago and has been redefined, criticized, and used widely ever since. In this review we have examined the aerobic cometabolism of chlorinated solvents, with a particular emphasis on the cometabolism of trichloroethylene. Monooxygenases or dioxygenases with relaxed substrate ranges initiate these transformations. The physiological role of the oxygenases is to initiate the metabolism of growth-supporting substrates (e.g., methane, propane, butane, toluene, ethylene, and ammonia). Diverse enzymes catalyze these oxidative reactions with chlorinated solvents. Synthesis of most of these enzymes is induced by the presence of the growth-supporting substrate and is largely regulated at the level of gene transcription. The genes that code for a given oxygenase are usually clustered together in a single operon and often share homology with counterparts that code for the subunits of related oxygenases in other bacteria. During cometabolism the growth-supporting and non-growth-supporting substrates can both bind to the oxygenase. Transformation of chlorinated solvents by these enzymes presents the cell with a new set of compounds. Some of these compounds are toxic to the cells, others are stable products that are expelled from the cell, and in a few cases the cells utilize the products. The combined effects of cometabolism can have a profound influence on a cell.

Two distinct monooxygenases for alkane oxidation in Nocardioides sp. strain CF8.

Alkane monooxygenases in Nocardioides sp. strain CF8 were examined at the physiological and genetic levels. Strain CF8 can utilize alkanes ranging in chain length from C(2) to C(16). Butane degradation by butane-grown cells was strongly inhibited by allylthiourea, a copper-selective chelator, while hexane-, octane-, and decane-grown cells showed detectable butane degradation activity in the presence of allylthiourea. Growth on butane and hexane was strongly inhibited by 1-hexyne, while 1-hexyne did not affect growth on octane or decane. A specific 30-kDa acetylene-binding polypeptide was observed for butane-, hexane-, octane-, and decane-grown cells but was absent from cells grown with octane or decane in the presence of 1-hexyne. These results suggest the presence of two monooxygenases in strain CF8. Degenerate primers designed for PCR amplification of genes related to the binuclear-iron-containing alkane hydroxylase from Pseudomonas oleovorans were used to clone a related gene from strain CF8. Reverse transcription-PCR and Northern blot analysis showed that this gene encoding a binuclear-iron-containing alkane hydroxylase was expressed in cells grown on alkanes above C(6). These results indicate the presence of two distinct monooxygenases for alkane oxidation in Nocardioides sp. strain CF8.

Induction of butane consumption in Pseudomonas butanovora.

The induction of the enzyme activities involved in butane metabolism in Pseudomonas butanovora was characterized. P. butanovora was grown on butane or its metabolites, both singly and in mixtures with other growth substrates. Cells grown in each of the butane metabolites readily consumed the growth substrate and downstream metabolites, but consumed the upstream butane metabolites more slowly. Upstream activities in the butane metabolism could be induced by downstream metabolites, but to much lower levels than with the primary substrate. The induction of butane oxidation was not repressed when P. butanovora was grown or incubated in a mixture of butane and 1-butanol, butyraldehyde or butyrate. However, no induction of butane consumption was observed in a mixture of butane and lactate, which is indicative of catabolite repression. In lactate-grown cells that were rid of the growth substrate and incubated with butane and acetylene (to inactivate newly formed butane monooxygenase), the consumption of butane, 1-butanol and butyraldehyde consumption was not induced. The overall results suggest an independent regulatory mechanism for each of the enzyme activities in butane metabolism. In addition, a low, constitutive butane oxidation was observed in cells grown on substrates other than butane metabolites.

Cytotoxicity associated with trichloroethylene oxidation in Burkholderia cepacia G4.

The effects of trichloroethylene (TCE) oxidation on toluene 2-monooxygenase activity, general respiratory activity, and cell culturability were examined in the toluene-oxidizing bacterium Burkholderia cepacia G4. Nonspecific damage outpaced inactivation of toluene 2-monooxygenase in B. cepacia G4 cells. Cells that had degraded approximately 0.5 micromol of TCE (mg of cells(-1)) lost 95% of their acetate-dependent O(2) uptake activity (a measure of general respiratory activity), yet toluene-dependent O(2) uptake activity decreased only 35%. Cell culturability also decreased upon TCE oxidation; however, the extent of loss varied greatly (up to 3 orders of magnitude) with the method of assessment. Addition of catalase or sodium pyruvate to the surfaces of agar plates increased enumeration of TCE-injured cells by as much as 100-fold, indicating that the TCE-injured cells were ultrasensitive to oxidative stress. Cell suspensions that had oxidized TCE recovered the ability to grow in liquid minimal medium containing lactate or phenol, but recovery was delayed substantially when TCE degradation approached 0.5 micromol (mg of cells(-1)) or 66% of the cells' transformation capacity for TCE at the cell density utilized. Furthermore, among B. cepacia G4 cells isolated on Luria-Bertani agar plates from cultures that had degraded approximately 0.5 micromol of TCE (mg of cells(-1)), up to 90% were Tol(-) variants, no longer capable of TCE degradation. These results indicate that a toxicity threshold for TCE oxidation exists in B. cepacia G4 and that once a cell suspension has exceeded this toxicity threshold, the likelihood of reestablishing an active, TCE-degrading biomass from the cells will decrease significantly.

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.

Differential regulation of amoA and amoB gene copies in Nitrosomonas europaea.

Nitrosomonas europaea contains two nearly identical copies of the operon, amoCAB, which encodes the ammonia monooxygenase (AMO) enzyme. Cells of N. europaea containing single mutations in either amoA or amoB gene copies were incubated in ammonium both prior to and after exposure to acetylene or light. For each strain, the O(2) consumption rates and amounts of AmoA polypeptide, the active site-containing subunit of AMO, produced in each strain were determined. Strains carrying a mutation in either the amoA(2) or amoB(2) genes responded similarly to wild-type cells, but the strains carrying mutations in the amoA(1) or amoB(1) genes responded differently from the wild-type, or from each other. These results suggest that the copies of amoA and amoB are differentially regulated upon exposure to different external stimuli.

New insights into methyl bromide cooxidation by Nitrosomonas europaea obtained by experimenting with moderately low density cell suspensions.

We examined the rates and sustainability of methyl bromide (MeBr) oxidation in moderately low density cell suspensions ( approximately 6 x 10(7) cells ml(-1)) of the NH(3)-oxidizing bacterium Nitrosomonas europaea. In the presence of 10 mM NH(4)(+) and 0.44, 0. 22, and 0.11 mM MeBr, the initial rates of MeBr oxidation were sustained for 12, 12, and 24 h, respectively, despite the fact that only 10% of the NH(4)(+), 18% of the NH(4)(+), and 35% of the NH(4)(+), respectively, were consumed. Although the duration of active MeBr oxidation generally decreased as the MeBr concentration increased, similar amounts of MeBr were oxidized with a large number of the NH(4)(+)-MeBr combinations examined (10 to 20 micromol mg [dry weight] of cells(-1)). Approximately 90% of the NH(3)-dependent O(2) uptake activity and the NO(2)(-)-producing activity were lost after N. europaea was exposed to 0.44 mM MeBr for 24 h. After MeBr was removed and the cells were resuspended in fresh growth medium, NO(2)(-) production increased exponentially, and 48 to 60 h was required to reach the level of activity observed initially in control cells that were not exposed to MeBr. It is not clear what percentage of the cells were capable of cell division after MeBr oxidation because NO(2)(-) accumulated more slowly in the exposed cells than in the unexposed cells despite the fact that the latter were diluted 10-fold to create inocula which exhibited equal initial activities. The decreases in NO(2)(-)-producing and MeBr-oxidizing activities could not be attributed directly to NH(4)(+) or NH(3) limitation, to a decrease in the pH, to the composition of the incubation medium, or to toxic effects caused by accumulation of the end products of oxidation (NO(2)(-) and formaldehyde) in the medium. Additional cooxidation-related studies of N. europaea are needed to identify the mechanism(s) responsible for the MeBr-induced loss of cell activity and/or viability, to determine what percentages of cells damaged by cooxidative activities are culturable, and to determine if cooxidative activity interferes with the regulation of NH(3)-oxidizing activity.

Effects of soil and water content on methyl bromide oxidation by the ammonia-oxidizing bacterium Nitrosomonas europaea.

Little information exists on the potential of NH(3)-oxidizing bacteria to cooxidize halogenated hydrocarbons in soil. A study was conducted to examine the cooxidation of methyl bromide (MeBr) by an NH(3)-oxidizing bacterium, Nitrosomonas europaea, under soil conditions. Soil and its water content modified the availability of NH(4)(+) and MeBr and influenced the relative rates of substrate (NH(3)) and cosubstrate (MeBr) oxidations. These observations highlight the complexity associated with characterizing soil cooxidative activities when soil and water interact to differentially affect substrate and cosubstrate availabilities.

The hydrogenase cytochrome b heme ligands of Azotobacter vinelandii are required for full H(2) oxidation capability.

The hydrogenase in Azotobacter vinelandii, like other membrane-bound [NiFe] hydrogenases, consists of a catalytic heterodimer and an integral membrane cytochrome b. The histidines ligating the hemes in this cytochrome b were identified by H(2) oxidation properties of altered proteins produced by site-directed mutagenesis. Four fully conserved and four partially conserved histidines in HoxZ were substituted with alanine or tyrosine. The roles of these histidines in HoxZ heme binding and hydrogenase were characterized by O(2)-dependent H(2) oxidation and H(2)-dependent methylene blue reduction in vivo. Mutants H33A/Y (H33 replaced by A or Y), H74A/Y, H194A, H208A/Y, and H194,208A lost O(2)-dependent H(2) oxidation activity, H194Y and H136A had partial activity, and H97Y,H98A and H191A had full activity. These results suggest that the fully conserved histidines 33, 74, 194, and 208 are ligands to the hemes, tyrosine can serve as an alternate ligand in position 194, and H136 plays a role in H(2) oxidation. In mutant H194A/Y, imidazole (Imd) rescued H(2) oxidation activity in intact cells, which suggests that Imd acts as an exogenous ligand. The heterodimer activity, quantitatively determined as H(2)-dependent methylene blue reduction, indicated that the heterodimers of all mutants were catalytically active. H33A/Y had wild-type levels of methylene blue reduction, but the other HoxZ ligand mutants had significantly less than wild-type levels. Imd reconstituted full methylene blue reduction activity in mutants H194A/Y and H208A/Y and partial activity in H194,208A. These results indicate that structural and functional integrity of HoxZ is required for physiologically relevant H(2) oxidation, and structural integrity of HoxZ is necessary for full heterodimer-catalyzed H(2) oxidation.

Isolation and characterization of alkane-utilizing Nocardioides sp. strain CF8.

A butane-utilizing bacterial strain CF8 was isolated and identified as a member of the genus Nocardioides from chemotaxonomic and 16S rDNA sequence analysis. Strain CF8 grew on alkanes ranging from C(2) to C(16) in addition to butane and various other substrates including primary alcohols, carboxylic acids, and phenol. Butane degradation by strain CF8 was inactivated by light, a specific inactivator of copper-containing monooxygenases. The unique thermal aggregation phenomenon of acetylene-binding polypeptides was also observed for strain CF8. These results suggest that butane monooxygenase in strain CF8 is a third example of the copper-containing monooxygenases previously described in ammonia oxidizers and methanotrophs.

Diversity in butane monooxygenases among butane-grown bacteria.

Butane monooxygenases of butane-grown Pseudomonas butanovora, Mycobacterium vaccae JOB5, and an environmental isolate, CF8, were compared at the physiological level. The presence of butane monooxygenases in these bacteria was indicated by the following results. (i) O(2) was required for butane degradation. (ii) 1-Butanol was produced during butane degradation. (iii) Acetylene inhibited both butane oxidation and 1-butanol production. The responses to the known monooxygenase inactivator, ethylene, and inhibitor, allyl thiourea (ATU), discriminated butane degradation among the three bacteria. Ethylene irreversibly inactivated butane oxidation by P. butanovora but not by M. vaccae or CF8. In contrast, butane oxidation by only CF8 was strongly inhibited by ATU. In all three strains of butane-grown bacteria, specific polypeptides were labeled in the presence of [(14)C]acetylene. The [(14)C]acetylene labeling patterns were different among the three bacteria. Exposure of lactate-grown CF8 and P. butanovora and glucose-grown M. vaccae to butane induced butane oxidation activity as well as the specific acetylene-binding polypeptides. Ammonia was oxidized by all three bacteria. P. butanovora oxidized ammonia to hydroxylamine, while CF8 and M. vaccae produced nitrite. All three bacteria oxidized ethylene to ethylene oxide. Methane oxidation was not detected by any of the bacteria. The results indicate the presence of three distinct butane monooxygenases in butane-grown P. butanovora, M. vaccae, and CF8.

Inactivation of toluene 2-monooxygenase in Burkholderia cepacia G4 by alkynes.

High concentrations of acetylene (10 to 50% [vol/vol] gas phase) were required to inhibit the growth of Burkholderia cepacia G4 on toluene, while 1% (vol/vol) (gas phase) propyne or 1-butyne completely inhibited growth. Low concentrations of longer-chain alkynes (C5 to C10) were also effective inhibitors of toluene-dependent growth, and 2- and 3-alkynes were more potent inhibitors than their 1-alkyne counterparts. Exposure of toluene-grown B. cepacia G4 to alkynes resulted in the irreversible loss of toluene- and o-cresol-dependent O2 uptake activities, while acetate- and 3-methylcatechol-dependent O2 uptake activities were unaffected. Toluene-dependent O2 uptake decreased upon the addition of 1-butyne in a concentration- and time-dependent manner. The loss of activity followed first-order kinetics, with apparent rate constants ranging from 0.25 min-1 to 2.45 min-1. Increasing concentrations of toluene afforded protection from the inhibitory effects of 1-butyne. Furthermore, oxygen, supplied as H2O2, was required for inhibition by 1-butyne. These results suggest that alkynes are specific, mechanism-based inactivators of toluene 2-monooxygenase in B. cepacia G4, although the simplest alkyne, acetylene, was relatively ineffective compared to longer alkynes. Alkene analogs of acetylene and propyne-ethylene and propylene-were not inactivators of toluene 2-monooxygenase activity in B. cepacia G4 but were oxidized to their respective epoxides, with apparent Ks and Vmax values of 39.7 microM and 112.3 nmol min-1 mg of protein-1 for ethylene and 32.3 microM and 89.2 nmol min-1 mg of protein-1 for propylene.

Kinetic characterization of the inactivation of ammonia monooxygenase in Nitrosomonas europaea by alkyne, aniline and cyclopropane derivatives.

The kinetic mechanisms of seven inactivators of ammonia oxidation activity in cells of the nitrifying bacterium, Nitrosomonas europaea were investigated. The effects of the inactivators were specific for ammonia monooxygenase (AMO) which oxidizes ammonia to hydroxylamine. The aniline derivatives, 1,3-phenylenediamine and p-anisidine, were potent inactivators of AMO while other derivatives were ineffective as inactivators. Two cyclopropane derivatives, 1, 2-dimethylcyclopropane and cyclopropyl bromide, were inactivators while cyclopropane was not an inactivator. The mechanisms of three alkynes, 1-hexyne, 3-hexyne, and acetylene, were also examined. For all seven compounds, the inactivation of AMO was irreversible, time-dependent, first-order, and dependent on catalytic turnover. Saturation of the rate of inactivation was indicated for p-anisidine (kinact=2.85 min-1; KI=1.0 mM) and cyclopropyl bromide (kinact=4.4 min-1; KI=97 microM), but not for any of the remaining five inactivators, including acetylene. Ammonia slowed the rate of inactivation for acetylene and cyclopropyl bromide, but enhanced the rate of inactivation for the remaining inactivators. All seven compounds appear to be mechanism-based inactivators of AMO.