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Soluble butane monooxygenase (sBMO), a three-component di-iron monooxygenase complex expressed by the [C.sub.2]-[C.sub.9] alkane-utilizing bacterium Thauera butanivorans, was kinetically characterized by measuring substrate specificities for [C.sub.1]-[C.sub.5] alkanes and product inhibition profiles, sBMO has high sequence homology with soluble methane monooxygenase (sMMO) and shares a similar substrate range, including gaseous and liquid alkanes, aromatics, alkenes and halogenated xenobiotics. Results indicated that butane was the preferred substrate (defined by [[kappa].sub.cat]: [[kappa].sub.m] ratios). Relative rates of oxidation for [C.sub.1]-[C.sub.5] alkanes differed minimally, implying that substrate specificity is heavily influenced by differences in substrate [[kappa].sub.m] values. The low micromolar [[kappa].sub.m] for linear [C.sub.2]-[C.sub.5] alkanes and the millimolar [[kappa].sub.m] for methane demonstrate that sBMO is two to three orders of magnitude more specific for physiologically relevant substrates of T. butanivorans. Methanol, the product of methane oxidation and also a substrate itself, was found to have similar [[kappa].sub.m] and [[kappa].sub.cat] values to those of methane. This inability to kinetically discriminate between the [C.sub.1] alkane and [C.sub.1] alcohol is observed as a steady-state concentration of methanol during the two-step oxidation of methane to formaldehyde by sBMO. Unlike methanol, alcohols with chain length [C.sub.2]-[C.sub.5] do not compete effectively with their respective alkane substrates. Results from product inhibition experiments suggest that the geometry of the active site is optimized for linear molecules four to five carbons in length and is influenced by the regulatory protein component B (butane monooxygenase regulatory component; BMOB). The data suggest that alkane oxidation by sBMO is highly specialized for the turnover of [C.sub.3]-[C.sub.5] alkanes and the release of their respective alcohol products. Additionally, sBMO is particularly efficient at preventing methane oxidation during growth on linear alkanes [greater than or equal to] [C.sub.2], despite its high sequence homology with sMMO. These results represent, to the best of our knowledge, the first kinetic in vitro characterization of the closest known homologue of sMMO.

Nitrobacter hamburgensis X14 is a facultative lithoautotroph that conserves energy from the oxidation of nitrite (N[O.sup.-.sub.2]) and fixes carbon dioxide (C[O.sub.2]) as its sole source of carbon. The availability of the N. hamburgensis X14 genorne sequence initiated a re-examination of its mixotrophic and organotrophic potential, as genes encoding three flavin-dependent oxidases were identified that may function to oxidize lactate, providing energy and carbon for growth. The response of N. harnburgensis to D- and L-lactate in the presence (mixotrophy) and absence (organotrophy) of N[O.sup.-.sub.2] was examined. L-Lactate did not support organotrophic growth or stimulate mixotrophic growth. In contrast, D-lactate enhanced the growth rate and yield of N. hamburgensis in the presence of N[O.sup.-.sub.2], and served as the sole carbon and energy source for growth in the absence of N[O.sup.-.sub.2] with ammonium as the sole nitrogen source. Lithoautotrophically grown cells immediately consumed D-lactate, suggesting that a lactate metabolic pathway is constitutively expressed. Nevertheless, a physiological adaptation to lactate occurred, as D-lactate-grown cells consumed and assimilated lactate at a faster rate than N[O.sup.-.sub.2]-grown cells, and the D-lactate-dependent [O.sub.2] uptake rate was significantly greater in cells grown either organotrophically or mixotrophically compared with cells grown lithoautotrophically. Although D-lactate was assimilated and metabolized to C[O.sub.2] in the presence or absence of N[O.sup.-.sub.2], exposure to atmospheric C[O.sub.2] or the addition of 0.75 mM sodium carbonate was required for mixotrophic growth and for optimum organotrophic growth on D-lactate.

'Pseudomonas butanovora' uses an alcohol-inducible alkane monooxygenase (BMO) to grow on [C.sub.2]--[C.sub.9] n-alkanes. Five ORFs were identified flanking the BMO structural genes. Two of the ORFs, bmoR, encoding a putative [[sigma].sup.54]-transcriptional regulator BmoR, and bmoG, encoding a putative GroEL chaperonin BmoG, were analysed by gene-inactivation experiments. The BmoR-deficient mutant grew at slower growth rates than the wild-type on [C.sub.2]-[C.sub.5] n-alkanes and showed little to no growth on [C.sub.6]-[C.sub.8] n-alkanes within 7 days. A BmoR-deficient mutant was constructed in the 'P. butanovora' bmoX::lacZ reporter strain and used to test whether bmoR was involved in bmoX induction after growth on [C.sub.2]-[C.sub.8] carbon sources. In acetate- or lactate-grown cells, [C.sub.2]-[C.sub.8] n-alcohols failed to induce [beta]-galactosidase activity. In contrast, in propionate-, butyrate- or pentanoate-grown cells, n-butanol induced ~45 % of the [beta]-galactosidase activity observed in the control bmoX::lacZ strain. In propionate-grown cells, [C.sub.2]-[C.sub.5] n-alcohols induced [beta]-galactosidase activity, whereas [C.sub.7] and [C.sub.8] n-alcohols did not. BmoR may act as a [[sigma].sup.54]-transcriptional regulator of bmo that is controlled by the n-alcohol produced in the alkane oxidation. During growth on short-chain-length fatty acids, however, another BMO regulatory system seems to be activated to promote transcription of bmo by short-chain-length alcohols (i.e. [less than or equal to][C.sub.6]). The bmoG-deficient mutant did not grow on [C.sub.2]-[C.sub.8] n-alkanes; however, it was capable of transcribing bmoX and bmoC of the BMO operon. BmoG may act as a chaperonin to assemble competent BMO.

The chemolithoautotroph Nitrosomonas europaea has two genes predicted to encode outer-membrane (OM) ferrioxamine transporters. Expression of the ferrioxamine uptake system required induction, as shown by the shorter lag phase in ferrioxamine-containing cultures when ferrioxamine-exposed cells were used as an inoculum. The two OM ferrioxamine siderophore transporters encoded by fox[A.sub.1] (NE1097) and fox[A.sub.2] (NE1088) were produced only in cells grown in Fe-limited ferrioxamine-containing medium. The inactivation of fox[A.sub.1], singly or in combination with fox[A.sub.2], prevented growth in Fe-limited medium containing excess desferrioxamine (DFX). The fox[A.sub.2]-disrupted single mutant grew poorly in the regular Fe-limited (0.2 [micro]M) medium with 10 [micro]M DFX, but grew well when the Fe level was raised to 1.0 [micro]M with 10 [micro]M DFX. For efficient acquisition of Fe-Ioaded ferrioxamine, N. europaea needs both femoxamine transporters Fox[A.sub.1] and Fox[A.sub.2]. Fox[A.sub.1] probably regulates its own production, and it controls the production of Fox[A.sub.2] as well.

The properties of oxidation of dichloroethene (DCE) and trichloroethylene (TCE) by three mutant strains of Pseudomonas butanovora containing single amino acid substitutions in the [alpha]-subunit of butane monooxygenase hydroxylase (BMOH-[alpha]) were compared to the properties of the wild-type strain (Rev WT). The rates of oxidation of three chloroethenes ICEs) were reduced in mutant strain Gll3N and corresponded with a lower maximum rate of butane oxidation. The rate of TCE degradation was reduced by one-half in mutant strain L279F, whereas the rates of DCE oxidation were the same as those in Rev WT. Evidence was obtained that the composition of products of CE oxidation differed between Rev WT and some of the mutant strains. For example, while Rev WT released nearly all available chlorine stoichiometrically during CE oxidation, strain F321Y released about 40% of the chlorine during 1,2-cis-DCE and TCE oxidation, and strain Gll3N released between 14 and 25% of the available chlorine during oxidation of DCE and 56% of the available chlorine during oxidation of TCE. Whereas Rev WT, strain L279F, and strain F321Y formed stoichiometric amounts of 1,2-cis-DCE epoxide during oxidation of 1,2-cis-DCE, only about 50% of the 1,2-cis-DCE oxidized by strain G 113N was detected as the epoxide. Evidence was obtained that 1,2-cis-DCE epoxide was a substrate for butane monooxygenase (BMO) that was oxidized after the parent compound was consumed. Yet all of the mutant strains released less than 40% of the available 1,2-cis-DCE chlorine, suggesting that they have altered activity towards the epoxide. In addition, strain GI13N was unable to degrade the epoxide. TCE epoxide was detected during exposure of Rev WT and strain F321Y to TCE but was not detected with strains L279F and G113N. Lactate-dependent [O.sub.2] uptake rates were differentially affected by DCE degradation in the mutant strains, providing evidence that some products released by the altered BMOs reduced the impact of CE on cellular toxicity. The use of CEs as substrates in combination with P. butanovora BMOH-[alpha] mutants might allow insights into the catalytic mechanism of BMO to be obtained.

Butane monooxygenase (sBMO) has been purified to homogeneity from the Gram-negative [beta]-proteobacterium 'Pseudomonas butanovora' and confirmed to be a three-component diiron monooxygenase system. The reconstituted enzyme complex oxidized [C.sub.3]-[C.sub.6] linear and branched aliphatic alkanes, which are growth substrates for 'P. butanovora'. The sBMO complex was composed of an iron-containing hydroxylase (BMOH), a flavo-iron sulfur-containing NADH-oxidoreductase (BMOR) and a small regulatory component protein (BMOB). The physical characteristics of sBMO were remarkably similar to the sMMO family of soluble multicomponent diiron monooxgenases. However, the catalytic properties of sBMO were quantitatively different in regard to inactivation in the presence of substrate and product distribution. BMOH was capable of ethene oxidation when supplied with [H.sub.2][O.sub.2] and ethene (known as the peroxide shunt), but this activity was at least three orders of magnitude less than that observed for the hydroxylase of sMMO of Methylosinus trichosporium OB3b. BMOH and BMOR were efficient in the oxidation of ethene in the absence of BMOB with regard to rate of reaction and product yield. Regiospecificity of sBMO was strongly biased towards primary hydroxylation, with [greater than or equal to] 80% of the hydroxylations occurring at the terminal carbon atom.

The article describes genome-scale analysis for understanding the ammonia-oxidizing bacteria (AOB). The genome sequences helps in knowing the ecology, evolution and environmental roles of these bacteria.

The genome sequencing and analysis of the alphaproteobacterium Nitrobacter winogradskyi revealed a single circular chromosome of 3,402,093 bp encoding 3,143 predicted proteins. Approximately 10 percent of the Nitrobacter winogradskyi genome codes for genes involved in transport and secretion, including the presence of transporters for various organic-nitrogen molecules.

Although Nitrosomonas europaea lacks measurable [alpha]-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.

Nitrosomonas europaea is an aerobic ammonia-oxidizing bacterium that participates in the C and N cycles. N. europaea utilizes C[O.sub.2] as its predominant carbon source, and is an obligate chemolithotroph, deriving all the reductant required for energy and biosynthesis from the oxidation of ammonia (N[H.sub.3]) to nitrite (N[O.sup.-.sub.2]). This bacterium fixes carbon via the Calvin-Benson-Bassham (CBB) cycle via a type I ribulose bisphosphate carboxylase/oxygenase (RubisCO). The RubisCO operon is composed of five genes, cbbLSQON. This gene organization is similar to that of the operon for 'green-like' type I RubisCOs in other organisms. The cbbR gene encoding the putative regulatory protein for RubisCO transcription was identified upstream of cbbL. This study showed that transcription of cbb genes was upregulated when the carbon source was limited, while amo, hao and other energy-harvesting-related genes were downregulated. N. europaea responds to carbon limitation by prioritizing resources towards key components for carbon assimilation. Unlike the situation for amo genes, [NH.sub.3] was not required for the transcription of the cbb genes. All five cbb genes were only transcribed when an external energy source was provided. In actively growing cells, mRNAs from the five genes in the RubisCO operon were present at different levels, probably due to premature termination of transcription, rapid mRNA processing and mRNA degradation.

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 [approximately equal to] 5 [micro]M. 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 [approximately equal to] 100 [micro]M 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: [approximately equal to] 100 [micro] M 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.

The nitrifying bacterium Nitrosomonas europaea can obtain all its carbon for growth from C[O.sub.2] and all its energy and reductant for growth from the oxidation of N[H.sub.3] and is considered an obligate chemolithoautotroph. Previous studies have shown that N. europaea can utilize limited amounts of certain organic compounds, including amino acids, pyruvate, and acetate, although no organic compound has been reported to support the growth of N. europaea. The recently completed genomic sequence of N. europaea revealed a potential permease for fructose. With this in mind, we tested if N. europaea could utilize fructose and other compounds as carbon sources to support growth. Cultures were incubated in the presence of fructose or other organic compounds in sealed bottles purged of C[O.sub.2]. In these cultures, addition of either fructose or pyruvate as the sole carbon source resulted in a two-to threefold increase in optical density and protein content in 3 to 4 days. Studies with [[sub.14]C] fructose showed that >90% of the carbon incorporated by the cells during growth was derived from fructose. Cultures containing mannose, glucose, glycerol, mannitoi, citrate, or acetate showed little or no growth. N. europaea was not able to grow with fructose as an energy source, although the presence of fructose did provide an energy benefit to the cells. These results show that N. europaea can be grown in C[O.sub.2]-free medium by using fructose and pyruvate as carbon sources and may now be considered a facultative chemolithoorganotroph.

Two 1-butanol dehydrogenases, a quinoprotein and a quinohemoprotein mediate two distinct electron transport pathways in Pseudomonas butanovora during 1-butanol-dependent growth. The electrons from 1-butanol oxidation either follow quinoprotein coupled ubiquinone and cyanide-sensitive terminal oxidase pathway or directly transferred to quinohemoprotein-coupled terminal oxidase that is less cyanide sensitive.

Research has been conducted on the copper-type nitrite reductase in Nitrosomonas europaea. Results indicate that this reductase is not essential for nitrogen oxide production by N. europaea.

Two different enzymes, the NAD(sup)+-independent PQQ alcohol dehydrogenase BOH quinoprotein and BDH quinohemoprotein are involved in the butane oxidation pathway of Pseudomonas butanovora. Data indicate that inactivation of either of the genes, encoding the enzymes, affects the bacterial butane-dependent growth.

Burkholderia cepacia G4 can degrade trichloroethylene (TCE) as long as its DNA repair mechanism is intact. This bacterium is more resistant to the toxic effects of TCE than other bacteria and for this reason is considered a potential agent for TCE biodegradation.

Researchers present evidence that Nocardioides sp. strain CF8 has two different alkane monooxygenases. One is expressed in cells grown on alkanes above C(sub 6).

Trichloroethylene (TCE) oxidation by Burkholderia cepacia G4 is toxic to the bacterium. This occurs because reactive intermediates generated during TCE oxidation can inactivate not only the oxygenase, but also other cellular constituents as well.

Results show that oxidation of butane by 'Pseudomonas butanovora' is mediated by an inducible NAD+ -independent 1-butanol dehydrogenase when grown on butane. Studies on the purified enzyme indicate that it is a quinohaemoprotein containing pyrroloquinoline and haem c as prosthetic groups.

Identification of transcript start sites and putative promoters of ammonia monooxygenase and hydroxylamine oxidoreductase in Nitrosomonas europea is presented. Data reveal transcript start sites contain promoter sequences and cells use different promoters independent of ammonium ion in the incubation medium.

A survey of aerobic cometabolism of chlorinated aliphatic hydrocarbons by a butane-grown mixed culture was performed. The transformation of 1,1-dichloroethylene (1,1-DCE) and cis-l,2-dichloroethylene (cDCE) required O(sub 2) and was inhibited by butane and inactivated by acetylene, indicating that a monooxygenase enzyme was likely involved in the transformations. The initial transformation rates and the quantities of chlorinated aliphatic hydrocarbons transformed were inversely proportional to the chlorine contents within each group of chlorinated methanes, ethanes, and ethenes. Lower quantities of chloroform were transformed than chloromethane and dichloromethane, but chloroform transformation resulted in much higher cell inactivation. For the ethane group, chloroethane was most effectively transformed but caused significant cell inactivation. Dior trichloroethanes that have all chlorines on one carbon were more effectively transformed and caused less cell inactivation than the isomers that have chlorine on both carbons. For chlorinated ethenes, 1,1-DCE was most rapidly transformed, whereas trans-l,2-dichloroethylene was not transformed. Vinyl chloride was transformed to the greatest extent, and very limited transformation of trichloroethylene was observed. The 1,1-DCE transformation caused greater cell inactivation than the transformation of the other chlorinated ethenes. Chloride release studies showed nearly complete oxidative dechlorination of chlorinated methanes and chloroethane, vinyl chloride and c-DCE (86% approximately 100%), and incomplete dechlorination of 1,1-dichloroethane, 1,2-dichloroethane, 1,1,1-trichloroethane, 1,1,2-trichloroethane, and 1,1-DCE (37% approximately 75%) was observed.

Research has been conducted on the methyl bromide oxidation. The rates of this oxidation in bacterium Nitrosomonas europaea low density cell suspensions have been investigated.

Research has been conucted on the hydrogenase in Azotobacter vinelandii. The imidazole rescue of activity in a membrane protein has been reported.

Research has been conducted on the NH (sub)3 -oxidizing bacteria. The cooxidation of methyl bromide by NH (sub)3 -oxidizing bacterium Nitrosomonas europaea under soil conditions has been examined.

Research was conducted to determine the biochemical characteristics of butane monooxygenase in three strains of butane-grown bacteria. Results indicate each of the strains studied contained a distinct form of the enzyme.

It was shown that the route of butane metabolism by butane-grown Pseudomonas butanovora was butane -> 1-butanol -> butyraldehyde -> butyrate. Butane was oxidized at the terminal carbon to generate 1-butanol. Secondary alcohols were then oxidized to the corresponding ketones. Butane consumption was determined in a 1 ml gas-tight syringe with a removed needle. In addition, the O2 consumption by cells during the presence of different compounds was obtained using a Clark-style O2 electrode inserted into a sealed chamber.

Research was conducted to examine the inactivation of toluene 2-monooxygenase in Burkholderia cepacia G4 by alkynes. Results demonstrate that different alkynes serve as potent inhibitors of the toluene-dependent growth of B. cepacia. Findings also reveal that toluene 2-monooxygenase activity is specifically inactivated by the compounds. Therefore, alkynes represent a general class of mechanism-based inactivators for toluene 2-monooxygenase activity in B. cepacia G4.