Halocarbons such as chloromethane (CH₃Cl) and bromomethane are known for their ozone depletion potential (Harper, 2000). CH₃Cl, the most abundant volatile halocarbon in the atmosphere (∼600 ppt), is responsible for approximately 15% of halogen-dependent ozone destruction in the stratosphere (Harper, 2000; Montzka and Reimann, 2011). The largest sources of CH₃Cl emissions to the atmosphere include terrestrial vegetation (Hamilton et al., 2003; Yoshida et al., 2004; Keppler et al., 2005) and in particular the phyllosphere (i.e., aboveground parts of vegetation, Saito and Yokouchi, 2008), biomass burning, and the oceans (Montzka and Reimann, 2011). Conversely, the dominant sink for CH₃Cl is via reaction with hydroxyl radicals in the troposphere and represents 84% of the total, estimated at 4.1 Tg Cl yr^-1 (Yoshida et al., 2004). However, certain methylotrophic bacteria capable of using CH₃Cl as their sole source of carbon and energy for growth may also participate in this process, but the magnitude of their contribution remains to be characterized. Chloromethane-degrading bacteria are quite widespread, with representatives affiliated to the genera Aminobacter, Hyphomicrobium, Leisingera, Methylobacterium, Roseovarius (Alpha-Proteobacteria), Pseudomonas (Gamma-Proteobacteria) and Acetobacterium (Actinobacteria), isolated from diverse environments such as soils (Doronina et al., 1996; Miller et al., 1997; Coulter et al., 1999; McAnulla et al., 2001), activated sludge (Hartmans et al., 1986; Traunecker et al., 1991; Freedman et al., 2004), freshwaters (McAnulla et al., 2001), and seawater (Schäfer et al., 2005).

The only pathway for CH₃Cl degradation known so far is corrinoid- and tetrahydrofolate-dependent, and was characterized in detail for the aerobic facultative methylotrophic strain Methylobacterium extorquens CM4 (Vannelli et al., 1999). This pathway, termed cmu (abbreviation for chloromethane utilization), involves a set of genes that were subsequently detected in several other chloromethane-degrading strains (reviewed in Schäfer et al., 2007; also see Nadalig et al., 2011). The first step of the cmu pathway involves the methyltransferase/corrinoid-binding CmuA protein, which transfers the CH₃Cl methyl group to a corrinoid cofactor, and CmuB, another methyltransferase which catalyzes the transfer of the methyl group from the methylated corrinoid to tetrahydrofolate (H₄F). Methyl-H₄F is then oxidized to methylene-H₄F and further to CO₂ via formate to conserve energy, or exploited for biomass production. However, other yet to be characterized metabolic pathways may be involved in the degradation of CH₃Cl in the environment. For example, Leisingera methylohalidivorans MB2 grows with methyl halides but was reported not to contain close homologs of cmu genes (Schäfer et al., 2007).

Evidence for a given metabolic pathway may be obtained through the use of stable isotope techniques, and this has been used to distinguish different sources and sinks for CH₃Cl (Harper et al., 2001, 2003; Czapiewski et al., 2002; Keppler et al., 2004, 2005; Saito and Yokouchi, 2008; Greule et al., 2012; Redeker and Kalin, 2012). Degradation of CH₃Cl by cell suspensions of strains with the cmu pathway is also associated with specific carbon fractionation (Miller et al., 2001) but also with hydrogen isotope fractionation (Nadalig et al., 2013). Thus, isotopic approaches combined with genomic approaches may prove decisive in constraining the bacterial contribution to the global CH₃Cl budget.

In the present study, we review available bacterial genome sequences for the presence of cmu genes, thereby uncovering several bacteria that have not been described to degrade CH₃Cl. In parallel and as a proof of concept for the potential of isotope methods to characterize yet unknown pathways for CH₃Cl degradation, we determined hydrogen and carbon isotopic fractionation patterns of CH₃Cl during growth of the chloromethane-degrading strain L. methylohalidivorans MB2 lacking cmu genes, as compared to that observed for cmu pathway strains M. extorquens CM4 and Hyphomicrobium sp. MC1.

Several chloromethane-degrading bacteria with the cmu pathway have been characterized (Schäfer et al., 2007), and a complete and assembled genome sequence is available for two of them, i.e., M. extorquens CM4 (Marx et al., 2012) and Hyphomicrobium sp. MC1 (Vuilleumier et al., 2011). Two types of organization of cmu genes were identified (Nadalig et al., 2011). The usual gene organization involves a putative cmuBCA operon and was found in all experimentally characterized chloromethane-degrading bacteria with the cmu pathway except the reference chloromethane-degrading strain M. extorquens CM4, which harbors cmu genes in two clusters (Figure 1). The chloromethane-degrading strain L. methylohalidivorans MB2, in contrast, was known to lack cmu genes (Schäfer et al., 2007), so the recent report of its assembled genome sequence (Buddruhs et al., 2013) was of particular interest.

The presence of cmu genes in recently completed genome sequences was somewhat expected, but their detection in exclusively anaerobic bacteria came as a surprise considering that they had so far only been found in aerobic chloromethane-degrading bacteria. Anaerobic chloromethane-degrading bacteria reported so far use a different, although in one case at least also corrinoid-dependent, pathway (Traunecker et al., 1991; Messmer et al., 1993; Freedman et al., 2004). Worthy of note, CH₃Cl dehalogenation by the cmu pathway does not require aerobic conditions and is actually sensitive to oxygen (Studer et al., 2001). It is thus possible that anaerobic bacteria with cmu genes identified here (Table 2) are actually able to use CH₃Cl as a carbon and energy source, although this remains to be tested experimentally.

The conserved cmuBCA gene organization (Figure 1) in CH₃Cl-degrading bacteria (Nadalig et al., 2011), and the high level of identity between the protein sequences encoded by cmu genes of Thermincola potens, Desulfurispora thermophila, Thermosediminibacter oceani, Desulfotomaculum alcoholivorax, and Desulfomonile tiejdei (>81, >74, and >58% for cmuA, cmuB, and cmuC, respectively), suggests a common evolutionary origin for these genes and their dissemination in the environment by horizontal gene transfer. The presence of an excisionase in the immediate environment of cmu genes in these five strains (Figure 1) further supports acquisition of cmu genes by horizontal transfer in these strains, as does the presence of two cmu gene clusters in Desulfomonile tiedjei whose sequences are not closer related to each other than to those of other chloromethane-degrading strains. To our knowledge, however, potential sources of CH₃Cl that would support dissemination of cmu genes in anaerobic environments have not yet been reported.

Incidentally, our analysis also confirms the particular status in the cmu pathway of cmuC, a gene shown to be essential for growth of strain CM4 with CH₃Cl (Studer et al., 2002; Roselli et al., 2013) but whose function remains elusive. Indeed, sequence conservation among the proteins encoded by genes cmuA, cmuB, and cmuC are lowest for the CmuC gene product (Table 2). Moreover, the probable loss of a cmuC homolog in one of the two cmu gene clusters of Desulfomonile tiedjei strain CM4 (Figure 1) also hints at its possibly lesser role in CH₃Cl metabolism.

As to the chloromethane-degrading strain L. methylohalidivorans MB2, analysis of its genome (Buddruhs et al., 2013) confirms the initial report (Schäfer et al., 2007) that it lacks the cmu pathway. The best partial hit to CmuA (32% amino acid identity) is a 211-residue corrinoid protein, and cmuB or cmuC homologs were not detected in the L. methylohalidivorans MB2 genome (Table 2). However, downstream genes of the H₄F-dependent cmu pathway (metF, folD, purU) were all found, so an H₄F-dependent metabolic pathway for growth of L. methylohalidivorans MB2 with CH₃Cl remains a possibility.

In our experiments, we showed that L. methylohalidivorans MB2, previously grown with CH₃Cl (0.37 mM; Schaefer et al., 2002), is capable of using this one-carbon compound as sole carbon and energy source at an initial concentration of 10 mM. A direct comparison of its growth behavior with that of strains CM4 and MC1 is prevented by the fact that the latter two strains do not grow in high-salt mineral medium, whereas L. methylohalidivorans MB2 does not grow in the standard low-salt mineral medium used for strains CM4 and MC1. Incidentally, this suggests that salt adaptation may be unrelated to adaptation to intracellular chloride production during dehalogenation, as observed recently for bacteria growing with dichloromethane (Michener et al., 2014).

The differences in CH₃Cl metabolism of L. methylohalidivorans MB2 suggested by comparative genomics were experimentally supported by isotope analysis (Figure 3; Table 3). For L. methylohalidivorans MB2, isotopic enrichment factor for carbon during growth was substantially larger than for CM4 and MC1, indicating a larger primary isotope effect and providing further evidence for operation of another pathway for utilization of CH₃Cl in this strain. In contrast, a previous study on carbon isotopic fractionation of CH₃Cl by cell suspensions of three bacterial strains, including L. methylohalidivorans MB2, gave similar isotopic enrichment values (ranging between 42 and 47‰; Miller et al., 2001). In particular, the value obtained for Aminobacter ciceronei strain IMB1, the only strain so far shown to possess cmuA but not cmuB (Woodall et al., 2001), was similar to those of strains CM4 and MC1 (Miller et al., 2001). This suggests that the corrinoid dehalogenase protein CmuA drives carbon isotopic fractionation in chloromethane-degrading strains with the cmu pathway. Moreover and unlike for carbon, a larger isotope effect than in previous resting cell experiments (Nadalig et al., 2013) was observed for hydrogen during growth in strains CM4 and MC1. However, the most striking finding of the present study was the lack of substantial hydrogen isotope enrichment upon CH₃Cl degradation by L. methylohalidivorans MB2. This suggests that unlike CmuAB chloromethane dehalogenase, the unknown dehalogenase of this strain does not cause hydrogen fractionation during degradation of the chloromethane methyl group. Nevertheless and as a common denominator to all three chloromethane-degrading strains investigated here (Table 3), carbon isotope fractionation (the primary isotope effect in cleavage of the carbon-halogen bond) was more pronounced than hydrogen isotope fractionation (a secondary isotope effect in CH₃Cl dehalogenation), as expected (Elsner et al., 2005).

The observed differences in isotopic fractionation of CH₃Cl carbon and hydrogen between the three strains CM4, MC1, and MB2 are best visualized in Figure 4, which shows the trends in enrichment of the heavier isotope of carbon and hydrogen for the different strains at different time points during growth. As proposed by Elsner et al. (2005), the slopes in these graphs constitute a clear indication that L. methylohalidivorans MB2 uses a different pathway for growth with CH₃Cl than strains CM4 and MC1, which utilize the same pathway.

Measurements of isotopic fractionation for a given environmental compartment will include the overall contribution of the metabolic diversity of chloromethane-degrading bacteria and their relative occurrence in that environment. It is tempting to speculate that chloromethane degradation in the soil environment, for which an isotopic fractionation of 49‰ similar to that found here for strains CM4 and MC1 was obtained in a previous study (Miller et al., 2004), is predominantly performed by bacteria with the cmu pathway. Our results on microbially driven hydrogen and carbon isotope fractionation suggest that using in a two-dimensional isotope scheme might help to confirm this hypothesis. Thus, a combination of genomic studies with physiological and isotopic characterisation of chloromethane-degrading bacterial strains, as performed here, will remain a major objective for the near future in order to constrain the bacterial sink strength of the atmospheric budget of CH₃Cl.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.