Halomethane:Bisulfide/Halide Ion Methyltransferase, an Unusual Corrinoid Enzyme of Environmental Significance Isolated from an Aerobic Methylotroph Using Chloromethane as the Sole Carbon Source

Abstract

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.

Methyl chloride utilising bacteria are ubiquitous in the natural environment

“…It is thus feasible that Hyphomicrobium strains arising perhaps from run-o¡ of land or freshwater environments can grow in marine environments, even though the salt concentrations may not be optimal for growth. The growth yield of these organisms on CH 3 Cl was generally high at 8.0^11.5 g dry weight mol 31 C. These values are similar to those reported for other chloromethane utilisers [5,8,14].…”

“…All samples used for enrichment yielded strains capable of growth on CH 3 Cl, consistent with the hypothesis that CH 3 Cl utilising bacteria are widespread in the environment. This is expected since natural processes produce most CH 3 Cl, however it has obvious implications for the cycling of this compound in the environment in that CH 3 Cl utilisers may be a signi¢cant environmental [5] found that long-term culturing of H. chloromethanicum CM2 and M. chloromethanicum CM4 on alternative substrates led to a loss of the ability to grow on CH 3 Cl, so presumably the ability to grow on this compound is advantageous to these isolates in their natural habitat [9,14].…”

“…Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 -1 0 9 7 ( 0 1 ) 0 0 2 5 6 -7 soil [14] (these two strains were shown by 16S rRNA sequence analysis to be of the Aminobacter genus), and strain MB2 isolated from the marine environment on CH 3 Br [15].…”

Hydrologic regulation of gross methyl chloride and methyl bromide uptake from Alaskan Arctic tundra

“…For the Toolik Lake data: (a) r 2 5 0.23, Po0.1, net CH 3 Cl flux 5 À948.51 1 7.13 Â VWC; (b) r 2 5 0.27, Po0.05, net CH 3 Br flux 5 À26.47 1 0.21 Â VWC; (c) r 2 5 0.56, Po0.001, CH 3 Cl uptake 5 À1763.28 1 16.98 Â VWC; (d) r 2 5 0.57, Po0.001, CH 3 Br uptake 5 À42.29 1 0.40 Â VWC. cultural fields, and woodland soils (Coulter et al, 1999;McAnulla et al, 2001;McDonald et al, 2002;Miller et al, 2004). It is possible that the dominant methyl halidedegrading taxa in cold, waterlogged, anaerobic soils differ significantly from those in temperate, welldrained, aerobic environments.…”

Detection and isolation of chloromethane-degrading bacteria from the Arabidopsis thaliana phyllosphere, and characterization of chloromethane utilization genes

“…Certain methylotrophic bacteria are able to use chloromethane as the sole carbon and energy source for growth (McDonald et al, 2002;Trotsenko & Doronina, 2003;reviewed in Schäfer et al, 2007; Table 1). Chloromethanedegrading bacteria have been isolated from diverse environments such as soils (Doronina et al, 1996;Miller et al, 1997;Coulter et al, 1999;McAnulla et al, 2001a), activated sludge (Hartmans et al, 1986;Traunecker et al, 1991;Freedman et al, 2004), freshwater (McAnulla et al, 2001a), and seawater (Schäfer et al, 2005), and include representatives affiliated to a wide range of genera including Aminobacter, Hyphomicrobium, Leisingera, Methylobacterium, Roseovarius (Alphaproteobacteria), Pseudomonas (Gammaproteobacteria) and Acetobacterium (Actinobacteria). However, the association of such bacteria with plants has not yet been investigated.…”