A novel dehalogenating/transhalogenating enzyme, halomethane:bisulfide/halide ion methyltransferase, has been isolated from the facultatively methylotrophic bacterium strain CC495, which uses chloromethane (CH3Cl) 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 CH3Cl; then it catalyzed methyl transfer from CH3Cl, CH3Br, or CH3I to the following acceptor ions (in order of decreasing efficacy): I−, HS−, Cl−, Br−, NO2 −, 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, N2O, and Hg2+to affect the methyltransferase suggests significant differences. During CH3Cl 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 CH3Br-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.

An enzyme catalyzing the O-methylation of acetovanillone (3-methoxy-4-hydroxyacetophenone) by S-adenosylmethionine was isolated from Phanerochaete chrysosporium and purified 270-fold by ultrafiltration, anion-exchange chromatography, and gel filtration. The enzyme exhibited a pH optimum between 7 and 9 and was rapidly denatured at temperatures above 55'C. The Km values for acetovanillone and S-adenosylmethionine were 34 and 99 ,uM, respectively. S-Adenosylhomocysteine acted as a powerful competitive inhibitor of S-adenosylmethionine, with a Ki of 41 ,uM. The enzyme was also susceptible to inhibition by thiol reagents and low concentrations of heavy metal ions. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was monomeric and had a molecular weight of approximately 53,000. Substrate specificity studies showed that 3-methoxy-and 3,5-dimethoxy-substituted 4-hydroxy-benzaldehydes,-benzoic acids, and-acetophenones were the preferred substrates for the enzyme. The corresponding 3,4-dihydroxy compounds were methylated relatively slowly, while the 3-hydroxy-4-methoxy compounds were almost inactive as substrates. Substituents in both the 2 and 4 positions relative to the hydroxyl group appeared to be essential for significant enzyme attack of a substrate. Provided that certain steric criteria were satisfied, the nature of the substituent was not critical. Hence, xenobiotic compounds such as 2,4-dichlorophenol and 2,4-dibromophenol were methylated almost as readily as acetovanillone. However, an extended side chain in the 4 position was not compatible with activity as a substrate, and neither homovanillic, caffeic, nor ferulic acid was methylated. The substrate range of the O-methyltransferase tends to imply a role in the catabolism or detoxification of lignin degradation products such as vanillic and syringic acids.

The bacterial strains IMB-1 T and CC495 T , which are capable of growth on methyl chloride (CH 3 Cl, chloromethane) and methyl bromide (CH 3 Br, bromomethane), were isolated from agricultural soil in California fumigated with CH 3 Br, and woodland soil in Northern Ireland, respectively. Two pesticide-/herbicide-degrading bacteria, strains ER2 and C147, were isolated from agricultural soil in Canada. Strain ER2 degrades N-methyl carbamate insecticides, and strain C147 degrades triazine herbicides widely used in agriculture. On the basis of their morphological, physiological and genotypic characteristics, these four strains are considered to represent two novel species of the genus Aminobacter, for which the names Aminobacter ciceronei sp. nov. (type strain IMB-1 T =ATCC 202197 T =CIP 108660 T =CCUG 50580 T ; strains ER2 and C147) and Aminobacter lissarensis sp. nov. (type strain CC495 T =NCIMB 13798 T =CIP 108661 T = CCUG 50579 T ) are proposed.

The sources and sinks of the ozone-depleting halocarbons, chloromethane (CH 3 Cl) and bromomethane (CH 3 Br), have been the subject of recent controversy. Considerable uncertainty surrounds the relative contributions of oceanic and terrestrial sources of CH 3 Cl and natural versus anthropogenic fluxes of CH 3 Br. Halogen stable isotope ratios in atmospheric halomethanes could provide a valuable tool in estimating relative magnitudes of sources, particularly those of CH 3 Cl. However, the reliability of such techniques is critically dependent on the conservative nature of the halogens within these atmospheric halomethanes. Here we demonstrate that intact cells of the soil bacterium strain CC495 under anaerobic or microaerophilic conditions rapidly exchange 37 Clwith organically bound chlorine in CH 3 Cl. Since Cloccurs ubiquitously and such bacteria appear to be widespread, any chlorine isotope fractionation during biological or abiotic CH 3 Cl production may therefore not be apparent in atmospheric CH 3 Cl. Cells of strain CC495 also catalyzed transhalogenation of CH 3 Br to CH 3 Cl, suggesting that this transformation may represent a significant sink for atmospheric CH 3 Br in soil.