Probing the diversity of chloromethane-degrading bacteria by comparative genomics and isotopic fractionation

Chloromethane (CH3Cl) is produced on earth by a variety of abiotic and biological processes. It is the most important halogenated trace gas in the atmosphere, where it contributes to ozone destruction. Current estimates of the global CH3Cl budget are uncertain and suggest that microorganisms might play a more important role in degrading atmospheric CH3Cl than previously thought. Its degradation by bacteria has been demonstrated in marine, terrestrial, and phyllospheric environments. Improving our knowledge of these degradation processes and their magnitude is thus highly relevant for a better understanding of the global budget of CH3Cl. The cmu pathway, for chloromethane utilisation, is the only microbial pathway for CH3Cl degradation elucidated so far, and was characterized in detail in aerobic methylotrophic Alphaproteobacteria. Here, we reveal the potential of using a two-pronged approach involving a combination of comparative genomics and isotopic fractionation during CH3Cl degradation to newly address the question of the diversity of chloromethane-degrading bacteria in the environment. Analysis of available bacterial genome sequences reveals that several bacteria not yet known to degrade CH3Cl contain part or all of the complement of cmu genes required for CH3Cl degradation. These organisms, unlike bacteria shown to grow with CH3Cl using the cmu pathway, are obligate anaerobes. On the other hand, analysis of the complete genome of the chloromethane-degrading bacterium Leisingera methylohalidivorans MB2 showed that this bacterium does not contain cmu genes. Isotope fractionation experiments with L. methylohalidivorans MB2 suggest that the unknown pathway used by this bacterium for growth with CH3Cl can be differentiated from the cmu pathway. This result opens the prospect that contributions from bacteria with the cmu and Leisingera-type pathways to the atmospheric CH3Cl budget may be teased apart in the future.

The only pathway for CH 3 Cl 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 CH 3 Cl methyl group to a corrinoid cofactor, and CmuB, another methyltransferase which catalyzes the transfer of the methyl group from the methylated corrinoid to tetrahydrofolate (H 4 F). Methyl-H 4 F is then oxidized to methylene-H 4 F and further to CO 2 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 CH 3 Cl 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 CH 3 Cl Czapiewski et al., 2002;Keppler et al., 2004Keppler et al., , 2005Saito and Yokouchi, 2008;Greule et al., 2012;Redeker and Kalin, 2012). Degradation of CH 3 Cl 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 . Thus, isotopic approaches combined with genomic approaches may prove decisive in constraining the bacterial contribution to the global CH 3 Cl 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 CH 3 Cl. In parallel and as a proof of concept for the potential of isotope methods to characterize yet unknown pathways for CH 3 Cl degradation, we determined hydrogen and carbon isotopic fractionation patterns of CH 3 Cl 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.
The headspace of cultures was sampled regularly (0.1 mL) for determination of CH 3 Cl concentration by gas chromatography, and 1 mL headspace samples were also taken at each point and conserved in 12 ml Exetainers® (Labco Limited, Lampeter, UK) for subsequent isotopic measurements. Concentration of chloride was measured in supernatants of cultures using the spectrophotometric method of Jörg and Bertau (2004), except for L. methylohalidivorans MB2 because of the high chloride content of MAMS medium.

ANALYSIS OF CONCENTRATIONS AND STABLE ISOTOPE VALUES OF CHLOROMETHANE
Concentration and stable carbon and hydrogen isotope values for CH 3 Cl were performed by gas chromatography coupled with flame ionization detector (GC-FID) and isotope ratio mass spectrometry (IRMS), respectively, as described previously , except that helium flow entering the gas chromatograph in isotopic analysis was increased to 1.8 ml min −1 .
The conventional "delta" notation, which expresses the isotopic composition of a material relative to that of a standard on a per mil (‰) deviation basis, was used. Values of δ 2 H (‰) are relative to that for V-SMOW (Vienna Standard Mean Ocean Water), and values of δ 13 C (‰) are relative to that for V-PDB (Vienna Pee Dee Belemnite). Carbon and hydrogen isotope fractionations associated with CH 3 Cl degradation by L. methylohalidivorans MB2, M. extorquens CM4 and H. sp. MC1 were determined from the slopes (b C and b H ) of the linear regression of isotope variation ( 13 C and δ 2 H) of CH 3 Cl on the logarithm of the remaining CH 3 Cl concentration (ln f): Fractionation factors α C and α H were calculated as α = 1,000/(b+1,000), and also expressed as isotope enrichment factors (ε C and ε H ), calculated as ε = (α−1)10 3 . Errors represent 95% confidence intervals calculated on the least-squares regression.

RESULTS
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 . Two types of organization of cmu genes were identified . The usual gene organization involves a putative cmuBCA operon and was found in all Frontiers in Microbiology | Terrestrial Microbiology Kaksonen et al.
Kaksonen et al. experimentally characterized chloromethane-degrading bacteria with the cmu pathway except the reference chloromethanedegrading 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.

COMPARATIVE GENOMICS
An exhaustive survey of the presence of cmu genes in available sequenced bacterial genomes was carried out, yielding several novel insights (Table 2). First, all strains with cmu homologs contained all three genes cmuA, cmuB, and cmuC essential for growth with CH 3 Cl using the cmu pathway. Second, these three genes were detected as a cmuBCA gene cluster (Figure 1) in the genomes of five bacterial strains that had not been reported to possess cmu genes or CH 3 Cl degradation activity. Strikingly and in contrast to all strains growing with CH 3 Cl using the cmu pathway described so far, all these strains are anaerobes. Three of them are Gram-negative bacteria from the class Deltaproteobacteria, i.e., Desulfotomaculum alcoholivorax (Kaksonen et al., 2008), Desulfurispora thermophila (Kaksonen et al., 2007) and Desulfomonile tiedjei (DeWeerd et al., 1990), and two belong to the class Clostridia, i.e., the Gram-positive Thermincola potens (Byrne-Bailey et al., 2010) and the Gram-negative Thermosediminibacter oceani (Pitluck et al., 2010). Notably, Desulfomonile tiedjei has a second cmu cluster containing only cmuB and cmuA (Figure 1) 6 kb away from a cmuBCA gene cluster. Levels of identity with homologs of the CM4 strain at the protein level are high, and range between 64 and 84%, 60 and 64%, and 34 and 39% for cmuA, cmuB and cmuC gene products, respectively ( Table 2). Pairwise identity comparisons of the proteins encoded by cmu genes show that homologs of strains Desulfotomaculum alcoholivorax, Desulfurispora thermophila, Thermincola potens and Thermosediminibacter oceani are most related to each other, with identities at the protein level between 84-93%, 82-92%, and 66-87% for cmuA, cmuB, and cmuC gene products, respectively, and that Frontiers in Microbiology | Terrestrial Microbiology

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CM4 homologs represent outliers for all three genes. It is interesting to note that the gene products of two copies of cmuA and cmuB of Desulfomonile tiedjei (78 and 77% protein identity, respectively) are not each others' closest homologs. In addition, no evidence for substantial identity at the DNA level was detected between the two cmu gene clusters of this strain (data not shown). Further, CmuA encoded by the cmuBCA cluster of Desulfomonile tiedjei is closer to homologs from other strains (>80% identity at the protein level) than that encoded by the partial cmu cluster cmuBA (<75% identity). Analysis of the L. methylohalidivorans MB2 genome (Buddruhs et al., 2013) confirmed the original report of Schäfer et al. (2007) that this CH 3 Cl strain did not contain bona fide cmu genes. As mentioned in the genome report, the closest homolog to cmuA is a gene coding a short (232 residues) corrinoid methyltransferase protein (MtbC) with only 32% identity to the C-terminal domain of CmuA (Table 2). However, no full-length homologs to cmuB and cmuC were detected in the genome sequence ( Table 2). Taken together, these data confirm that the metabolic pathway used by L. methylohalidivorans MB2 to grow with CH 3 Cl is different to that of other known chloromethane-degrading strains with the cmu pathway.
The presence of downstream genes in the cmu pathway in strains containing cmuABC genes was also evaluated ( Table 2). Genes potentially involved in the tetrahydrofolate (H 4 F) dependent pathway for oxidation of methyl-H 4 F to formate via methylene-H 4 F are present in all genomes ( Table 2), but close homologs of metF encoding methylene-H 4 F reductase were not detected except in strain MC1. Notably, only Alphaproteobacterial strains CM4, MC1, and L. methylohalidivorans MB2 possess the genes involved in the serine and ethylmalonyl-CoA pathways involved in growth of strains CM4 and MC1 with C1 compounds. Moreover, the tetrahydromethanopterin (H 4 MPT) pathway crucial for growth of Methylobacterium with methanol (Marx et al., 2005), but thought to be dispensable for growth with CH 3 Cl (Studer et al., 2002), is only present in M. extorquens CM4 and Hyphomicrobium sp. MC1 which also grow with methanol, but absent in L. methylohalidivorans MB2, which is unable to grow with methanol, as well as in all other strains containing cmu genes investigated here. Finally, a search for genes common to chloromethane-degrading strains (including or excluding L. methylohalidivorans MB2) failed to reveal genes other than essential housekeeping genes (data not shown). This suggests that identification of the genes involved in CH 3 Cl degradation or in adaptation to CH 3 Cl metabolism is not possible by comparative genomics analysis alone.

GROWTH OF STRAINS WITH CHLOROMETHANE AS SOLE CARBON AND ENERGY SOURCE
Methylobacterium extorquens CM4, Hyphomicrobium sp. MC1, and L. methylohalidivorans MB2 were cultivated with 10 mM CH 3 Cl as sole carbon and energy source in the recommended medium allowing fastest growth, i.e., minimal mineral medium for strains CM4 and MC1, and high-salt mineral medium for strain MB2 (Figure 2A). Chloromethane consumption during growth was measured in the gaseous phase by gas chromatography ( Figure 2B). In cultures of M. extorquens CM4 and H. sp. MC1, CH 3 Cl was completely degraded after 30 h under the chosen growth conditions. In contrast, consumption of CH 3 Cl by the L. methylohalidivorans MB2 culture required a longer time (∼45 h) to proceed to completion, although its growth behavior was similar to that of the other two strains.

CARBON AND HYDROGEN ISOTOPE FRACTIONATION OF CHLOROMETHANE DURING GROWTH
During degradation of CH 3 Cl, δ 13 C values of residual chloromethane increased from approximately −32‰ (initial value) to 55, 9, and 33‰ for strains CM4, MC1, and MB2 respectively ( Figure 3A). No carbon or hydrogen fractionation was observed in abiotic controls with media M3 and MAMS (data not shown). Derived values of isotope fractionation factor (α C ) and of the corresponding enrichment factor were very similar for cmu pathway strains CM4 and MC1, and substantially larger for L. methylohalidivorans MB2 (Table 3).
However, trends were markedly different for the three strains when considering the enrichment of 2 H in residual CH 3 Cl during cultivation. For strains CM4 and MC1, δ 2 H values increased www.frontiersin.org from approximately −124‰ at the start of the experiment to 16‰ and −12‰ for strains CM4 and MC1, respectively ( Figure 3B). In marked contrast, however, no substantial change of δ 2 H was observed during degradation of CH 3 Cl by L. methylohalidivorans MB2 ( Figure 3B). This resulted in large differences of hydrogen stable isotope fractionation factor (α H ) and of the corresponding enrichment factor between strains CM4 and MC1 containing the cmu pathway for CH 3 Cl degradation on the one hand, and strain MB2 lacking the corresponding genes on the other hand (Table 3).

DISCUSSION
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 chloromethanedegrading 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, CH 3 Cl 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 CH 3 Cl as a carbon and energy source, although this remains to be tested experimentally.
The conserved cmuBCA gene organization (Figure 1) in CH 3 Cl-degrading bacteria , 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 CH 3 Cl 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 CH 3 Cl (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 CH 3 Cl 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 H 4 F-dependent cmu pathway (metF, folD, purU ) were all found, so an H 4 F-dependent metabolic Frontiers in Microbiology | Terrestrial Microbiology pathway for growth of L. methylohalidivorans MB2 with CH 3 Cl remains a possibility.
In our experiments, we showed that L. methylohalidivorans MB2, previously grown with CH 3 Cl (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 CH 3 Cl 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 CH 3 Cl in this strain. In contrast, a previous study on carbon isotopic fractionation of CH 3 Cl 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 chloromethanedegrading strains with the cmu pathway. Moreover and unlike for carbon, a larger isotope effect than in previous resting cell experiments  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 CH 3 Cl 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 CH 3 Cl dehalogenation), as expected (Elsner et al., 2005).
The observed differences in isotopic fractionation of CH 3 Cl 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 CH 3 Cl 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 CH 3 Cl.