A corrinoid-dependent catabolic pathway for growth of a Methylobacterium strain with chloromethane. (1/34)

Methylobacterium sp. strain CM4, an aerobic methylotrophic alpha-proteobacterium, is able to grow with chloromethane as a carbon and energy source. Mutants of this strain that still grew with methanol, methylamine, or formate, but were unable to grow with chloromethane, were previously obtained by miniTn5 mutagenesis. The transposon insertion sites in six of these mutants mapped to two distinct DNA fragments. The sequences of these fragments, which extended over more than 17 kb, were determined. Sequence analysis, mutant properties, and measurements of enzyme activity in cell-free extracts allowed the definition of a multistep pathway for the conversion of chloromethane to formate. The methyl group of chloromethane is first transferred by the protein CmuA (cmu: chloromethane utilization) to a corrinoid protein, from where it is transferred to H4folate by CmuB. Both CmuA and CmuB display sequence similarity to methyltransferases of methanogenic archaea. In its C-terminal part, CmuA is also very similar to corrinoid-binding proteins, indicating that it is a bifunctional protein consisting of two domains that are expressed as separate polypeptides in methyl transfer systems of methanogens. The methyl group derived from chloromethane is then processed by means of pterine-linked intermediates to formate by a pathway that appears to be distinct from those already described in Methylobacterium. Remarkable features of this pathway for the catabolism of chloromethane thus include the involvement of a corrinoid-dependent methyltransferase system for dehalogenation in an aerobe and a set of enzymes specifically involved in funneling the C1 moiety derived from chloromethane into central metabolism.  (+info)

Properties of the methylcobalamin:H4folate methyltransferase involved in chloromethane utilization by Methylobacterium sp. strain CM4. (2/34)

Methylobacterium sp. strain CM4 is a strictly aerobic methylotrophic proteobacterium growing with chloromethane as the sole carbon and energy source. Genetic evidence and measurements of enzyme activity in cell-free extracts have suggested a multistep pathway for the conversion of chloromethane to formate. The postulated pathway is initiated by a corrinoid-dependent methyltransferase system involving methyltransferase I (CmuA) and methyltransferase II (CmuB), which transfer the methyl group of chloromethane onto tetrahydrofolate (H4folate) [Vannelli et al. (1999) Proc. Natl Acad. Sci. USA 96, 4615-4620]. We report the overexpression in Escherichia coli and the purification to apparent homogeneity of methyltransferase II. This homodimeric enzyme, with a subunit molecular mass of 33 kDa, catalyzed the conversion of methylcobalamin and H4folate to cob(I)alamin and methyl-H4folate with a specific activity of 22 nmol x min-1 x (mg protein)-1. The apparent kinetic constants for H4folate were: Km = 240 microM, Vmax = 28.5 nmol x min-1 x (mg protein)-1. The reaction appeared to be first order with respect to methylcobalamin at concentrations up to 2 mM, presumably reflecting the fact that methylcobalamin is an artificial substitute for the methylated methyltransferase I, the natural substrate of the enzyme. Tetrahydromethanopterin, a coenzyme also present in Methylobacterium, did not serve as a methyl group acceptor for methyltransferase II. Purified methyltransferase II restored chloromethane dehalogenation by a cell free extract of a strain CM4 mutant defective in methyltransferase II.  (+info)

Halomethane:bisulfide/halide ion methyltransferase, an unusual corrinoid enzyme of environmental significance isolated from an aerobic methylotroph using chloromethane as the sole carbon source. (3/34)

A novel dehalogenating/transhalogenating enzyme, halomethane:bisulfide/halide ion methyltransferase, has been isolated from the facultatively methylotrophic bacterium strain CC495, which uses chloromethane (CH(3)Cl) as the sole carbon source. Purification of the enzyme to homogeneity was achieved in high yield by anion-exchange chromatography and gel filtration. The methyltransferase was composed of a 67-kDa protein with a corrinoid-bound cobalt atom. The purified enzyme was inactive but was activated by preincubation with 5 mM dithiothreitol and 0.5 mM CH(3)Cl; then it catalyzed methyl transfer from CH(3)Cl, CH(3)Br, or CH(3)I to the following acceptor ions (in order of decreasing efficacy): I(-), HS(-), Cl(-), Br(-), NO(2)(-), CN(-), and SCN(-). Spectral analysis indicated that cobalt in the native enzyme existed as cob(II)alamin, which upon activation was reduced to the cob(I)alamin state and then was oxidized to methyl cob(III)alamin. During catalysis, the enzyme shuttles between the methyl cob(III)alamin and cob(I)alamin states, being alternately demethylated by the acceptor ion and remethylated by halomethane. Mechanistically the methyltransferase shows features in common with cobalamin-dependent methionine synthase from Escherichia coli. However, the failure of specific inhibitors of methionine synthase such as propyl iodide, N(2)O, and Hg(2+) to affect the methyltransferase suggests significant differences. During CH(3)Cl degradation by strain CC495, the physiological acceptor ion for the enzyme is probably HS(-), a hypothesis supported by the detection in cell extracts of methanethiol oxidase and formaldehyde dehydrogenase activities which provide a metabolic route to formate. 16S rRNA sequence analysis indicated that strain CC495 clusters with Rhizobium spp. in the alpha subdivision of the Proteobacteria and is closely related to strain IMB-1, a recently isolated CH(3)Br-degrading bacterium (T. L. Connell Hancock, A. M. Costello, M. E. Lidstrom, and R. S. Oremland, Appl. Environ. Microbiol. 64:2899-2905, 1998). The presence of this methyltransferase in bacterial populations in soil and sediments, if widespread, has important environmental implications.  (+info)

Oxidation of methyl halides by the facultative methylotroph strain IMB-1. (4/34)

Washed cell suspensions of the facultative methylotroph strain IMB-1 grown on methyl bromide (MeBr) were able to consume methyl chloride (MeCl) and methyl iodide (MeI) as well as MeBr. Consumption of >100 microM MeBr by cells grown on glucose, acetate, or monomethylamine required induction. Induction was inhibited by chloramphenicol. However, cells had a constitutive ability to consume low concentrations (<20 nM) of MeBr. Glucose-grown cells were able to readily oxidize [(14)C]formaldehyde to (14)CO(2) but had only a small capacity for oxidation of [(14)C]methanol. Preincubation of cells with MeBr did not affect either activity, but MeBr-induced cells had a greater capacity for [(14)C]MeBr oxidation than did cells without preincubation. Consumption of MeBr was inhibited by MeI, and MeCl consumption was inhibited by MeBr. No inhibition of MeBr consumption occurred with methyl fluoride, propyl iodide, dibromomethane, dichloromethane, or difluoromethane, and in addition cells did not oxidize any of these compounds. Cells displayed Michaelis-Menten kinetics for the various methyl halides, with apparent K(s) values of 190, 280, and 6,100 nM for MeBr, MeI, and MeCl, respectively. These results suggest the presence of a single oxidation enzyme system specific for methyl halides (other than methyl fluoride) which runs through formaldehyde to CO(2). The ease of induction of methyl halide oxidation in strain IMB-1 should facilitate its mass culture for the purpose of reducing MeBr emissions to the atmosphere from fumigated soils.  (+info)

Emissions of methyl halides and methane from rice paddies. (5/34)

Methyl halide gases are important sources of atmospheric inorganic halogen compounds, which in turn are central reactants in many stratospheric and tropospheric chemical processes. By observing emissions of methyl chloride, methyl bromide, and methyl iodide from flooded California rice fields, we estimate the impact of rice agriculture on the atmospheric budgets of these gases. Factors influencing methyl halide emissions are stage of rice growth, soil organic content, halide concentrations, and field-water management. Extrapolating our data implies that about 1 percent of atmospheric methyl bromide and 5 percent of methyl iodide arise from rice fields worldwide. Unplanted flooded fields emit as much methyl chloride as planted, flooded rice fields.  (+info)

Chloromethane utilization gene cluster from Hyphomicrobium chloromethanicum strain CM2(T) and development of functional gene probes to detect halomethane-degrading bacteria. (6/34)

Hyphomicrobium chloromethanicum CM2(T), an aerobic methylotrophic member of the alpha subclass of the class proteobacteria, can grow with chloromethane as the sole carbon and energy source. H. chloromethanicum possesses an inducible enzyme system for utilization of chloromethane, in which two polypeptides (67-kDa CmuA and 35-kDa CmuB) are expressed. Previously, four genes, cmuA, cmuB, cmuC, and purU, were shown to be essential for growth of Methylobacterium chloromethanicum on chloromethane. The cmuA and cmuB genes were used as probes to identify homologs in H. chloromethanicum. A cmu gene cluster (9.5 kb) in H. chloromethanicum contained 10 open reading frames: folD (partial), pduX, orf153, orf207, orf225, cmuB, cmuC, cmuA, fmdB, and paaE (partial). CmuA from H. chloromethanicum (67 kDa) showed high identity to CmuA from M. chloromethanicum and contains an N-terminal methyltransferase domain and a C-terminal corrinoid-binding domain. CmuB from H. chloromethanicum is related to a family of methyl transfer proteins and to the CmuB methyltransferase from M. chloromethanicum. CmuC from H. chloromethanicum shows identity to CmuC from M. chloromethanicum and is a putative methyltransferase. folD codes for a methylene-tetrahydrofolate cyclohydrolase, which may be involved in the C(1) transfer pathway for carbon assimilation and CO(2) production, and paaE codes for a putative redox active protein. Molecular analyses and some preliminary biochemical data indicated that the chloromethane utilization pathway in H. chloromethanicum is similar to the corrinoid-dependent methyl transfer system in M. chloromethanicum. PCR primers were developed for successful amplification of cmuA genes from newly isolated chloromethane utilizers and enrichment cultures.  (+info)

Hyphomicrobium chloromethanicum sp. nov. and Methylobacterium chloromethanicum sp. nov., chloromethane-utilizing bacteria isolated from a polluted environment. (7/34)

Two chloromethane-utilizing facultatively methylotrophic bacteria, strains CM2T and CM4T, were isolated from soil at a petrochemical factory. On the basis of their morphological, physiological and genotypical properties, strain CM2T (= VKM B-2176T = NCIMB 13687T) is proposed as a new species of the genus Hyphomicrobium, Hyphomicrobium chloromethanicum, and strain CM4T (= VKM B-2223T = NCIMB 13688T) as a new species of the genus Methylobacterium, Methylobacterium chloromethanicum.  (+info)

Large carbon isotope fractionation associated with oxidation of methyl halides by methylotrophic bacteria. (8/34)

The largest biological fractionations of stable carbon isotopes observed in nature occur during production of methane by methanogenic archaea. These fractionations result in substantial (as much as approximately 70 per thousand) shifts in delta(13)C relative to the initial substrate. We now report that a stable carbon isotopic fractionation of comparable magnitude (up to 70 per thousand) occurs during oxidation of methyl halides by methylotrophic bacteria. We have demonstrated biological fractionation with whole cells of three methylotrophs (strain IMB-1, strain CC495, and strain MB2) and, to a lesser extent, with the purified cobalamin-dependent methyltransferase enzyme obtained from strain CC495. Thus, the genetic similarities recently reported between methylotrophs, and methanogens with respect to their pathways for C(1)-unit metabolism are also reflected in the carbon isotopic fractionations achieved by these organisms. We found that only part of the observed fractionation of carbon isotopes could be accounted for by the activity of the corrinoid methyltransferase enzyme, suggesting fractionation by enzymes further along the degradation pathway. These observations are of potential biogeochemical significance in the application of stable carbon isotope ratios to constrain the tropospheric budgets for the ozone-depleting halocarbons, methyl bromide and methyl chloride.  (+info)