Françoise Bringel
email: francoise.bringel@unistra.fr

Ludovic Besaury

Pierre Amato

Eileen Kröber

Steffen Kolb

Frank Keppler

Stéphane Vuilleumier

Thierry Nadalig

Ludovic Besaury

Methylotrophs and methylotroph populations for chloromethane degradation

Keywords:

la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

tributes to the degradation of stratospheric ozone. A better knowledge of chloromethane sources (production) and sinks (degradation) is a prerequisite to estimate its atmospheric budget in the context of global warming. The degradation of chloromethane by methylotrophic communities in terrestrial environments is a major underestimated chloromethane sink. Methylotrophs isolated from soils, marine environments and more recently from the phyllosphere have been grown under laboratory conditions using chloromethane as the sole carbon source. In addition to anaerobes that degrade chloromethane, the majority of cultivated strains were isolated in aerobiosis for their ability to use chloromethane as sole carbon and energy source. Among those, the Proteobacterium Methylobacterium (recently reclassified as Methylorubrum) harbours the only characterized 'chloromethane utilization' (cmu) pathway, so far. This pathway may not be representative of chloromethane-utilizing populations in the environment as cmu genes are rare in metagenomes. Recently, combined 'omics' biological approaches with chloromethane carbon and hydrogen stable isotope fractionation measurements in microcosms, indicated that microorganisms in soils and the phyllosphere (plant aerial parts) represent major sinks of chloromethane in contrast to more recently recognized microbe-inhabited environments, such as clouds. Cultivated chloromethane-degraders lacking cmu genes display a singular isotope fractionation signature of chloromethane. Moreover, 13 CH 3 Cl labelling of active methylotrophic communities by stable isotope probing in soils identify taxa that differ from those known for chloromethane degradation. These observations suggest that new biomarkers for detecting active microbial chloromethane-utilizers in the environment are needed to assess the contribution of microorganisms to the global chloromethane cycle.

Introduction

Chloromethane and stratospheric ozone depletion Chloromethane (methyl chloride, CH 3 Cl) is the most abundant organohalogen in the Earth atmosphere. Its global production is estimated at 4-5 megatons per year, with main sources stemming from terrestrial vegetation [START_REF] Keppler | New insight into the atmospheric chloromethane budget gained using stable carbon isotope ratios[END_REF]. Photolytic degradation of chloromethane releases a halogen radical, which catalyses the destruction of ozone. Thus, chloromethane contributes to depletion of the stratospheric ozone layer (altitude of approximately 20 to 30 km), which constitutes the Earth's natural protective shield that absorbs the solar UVC and partially UVB radiation dangerous for living organisms. Until the 1990s, chloromethane was used as a refrigerant under the name Freon 40. Chloromethane has a stratospheric lifetime of about one year, much shorter than most other chlorofluorocarbons (CFCs), solvents and halons also banned by the international agreement of 1987 known as the Montreal Protocol on substances that deplete the ozone layer (see web resource section). There are still some uncertainties about chloromethane anthropogenic emissions (e.g. coal combustion, feedstock for chemical industries) [START_REF] Li | Emission estimates of methyl chloride from industrial sources in China based on high frequency atmospheric observations[END_REF]. Chloromethane is responsible alone for approximately 16% of stratospheric chlorine-catalysed ozone destruction [START_REF] Carpenter | Update on Ozone-Depleting Substances (ODSs) and other gases of interest to the Montreal protocol[END_REF]. A detailed understanding of its sources and sinks will be essential to predict changes in atmospheric chloromethane fluxes in the context of global climate change.

Chloromethane sources and sinks

Chloromethane formation in plants and soil involves biotic and abiotic processes. Chloride ion can be alkylated during the abiotic oxidation of organic matter by an electron acceptor such as Fe(III) in soils and sediments [START_REF] Keppler | Halocarbons produced by natural oxidation processes during degradation of organic matter[END_REF]. Abiotic chloromethane mainly results from the conversion of plant methoxyl groups (ether-or ester-bonded methyl groups) and their reaction with chloride ion [START_REF] Keppler | Halocarbons produced by natural oxidation processes during degradation of organic matter[END_REF][START_REF] Hamilton | Chloride methylation by plant pectin: an efficient environmentally significant process[END_REF][START_REF] Sailaukhanuly | Mechanistic aspects of the nucleophilic substitution of pectin. On the formation of chloromethane[END_REF]. This process occurs in terrestrial ecosystems at ambient temperatures [START_REF] Derendorp | Methyl chloride emissions from halophyte leaf litter: dependence on temperature and chloride content[END_REF][START_REF] Keppler | Chloromethane release from carbonaceous meteorite affords new insight into Mars lander findings[END_REF], but it is much more efficient at higher temperatures such as those reached during pyrolysis and biomass burning [START_REF] Hamilton | Chloride methylation by plant pectin: an efficient environmentally significant process[END_REF][START_REF] Keppler | New insight into the atmospheric chloromethane budget gained using stable carbon isotope ratios[END_REF][START_REF] Mcroberts | Seasonal changes in chlorine and methoxyl content of leaves of deciduous trees and their impact on release of chloromethane and methanol at elevated temperatures[END_REF]. Chloromethane release was also detected during thermal conversion of Martian soils by the Mars landers Viking [START_REF] Biemann | Search for organic and volatile inorganic compounds in two surface samples from the chryse planitia region of Mars[END_REF] and Curiosity [START_REF] Ming | Volatile and organic compositions of sedimentary rocks in Yellowknife Bay, Gale crater[END_REF], indicating the presence of endogenous organic matter on Mars. Chloromethane emission profiles during thermal treatment of soils sampled from other hyperarid environments hostile microbial life such as the Atacama desert were almost identical to those recorded by the Curiosity rover on Mars [START_REF] Schulze-Makuch | Transitory microbial habitat in the hyperarid Atacama Desert[END_REF]. Furthermore, chloromethane formation was observed from thermal conversion of extraterrestrial material such as carbonaceous meteorites [START_REF] Keppler | Chloromethane release from carbonaceous meteorite affords new insight into Mars lander findings[END_REF] and in protostellar environments [START_REF] Fayolle | Protostellar and cometary detections of organohalogens[END_REF]. These recent observations contributed to the emergence of an 'astronomical' fundamental interest in the understanding of chloromethane's cycle in sun-like stars and on Earth.

On Earth, plants (alive or decaying) are a major biotic source of chloromethane. Chloromethane is produced by enzymatic chloride ion methylation, as shown in higher plants affiliated to the Brassicaceae family [START_REF] Attieh | Purification and characterization of a novel methyltransferase responsible for biosynthesis of halomethanes and methanethiol in Brassica oleracea[END_REF][START_REF] Rhew | Genetic control of methyl halide production in Arabidopsis[END_REF] and wood-degrading fungi [START_REF] Harper | Chloromethane, methyl donor in veratryl alcohol biosynthesis in Phanerochaete chrysosporium and other lignin-degrading fungi[END_REF]. The S-adenosyl-l-methionine-dependent halide ion methyltransferase is encoded by gene HOL1 (Harmless to Ozone Layer) in Arabidopsis thaliana [START_REF] Nagatoshi | Arabidopsis HARMLESS TO OZONE LAYER protein methylates a glucosinolate breakdown product and functions in resistance to Pseudomonas syringae pv. maculicola[END_REF]. HOL1 gene disruption correlates with decreased pathogen defence, possibly due to reduced production of methyl thiocyanate (CH 3 SCN) from glucosinolate-derived thiocyanate by HOL1 [START_REF] Manley | Phytogenesis of halomethanes: A product of selection or a metabolic accident?[END_REF][START_REF] Rhew | Genetic control of methyl halide production in Arabidopsis[END_REF][START_REF] Nagatoshi | Arabidopsis HARMLESS TO OZONE LAYER protein methylates a glucosinolate breakdown product and functions in resistance to Pseudomonas syringae pv. maculicola[END_REF]. Chloromethane production is considered a byproduct of plant thiocyanate metabolism. Such methyltransferases have also been detected in other crop and seaside plants [START_REF] Itoh | Involvement of S-adenosylmethionine-dependent halide/thiol methyltransferase (HTMT) in methyl halide emissions from agricultural plants: isolation and characterization of an HTMT-coding gene from Raphanus sativus (daikon radish)[END_REF] including marine algae [START_REF] Wuosmaa | Methyl chloride transferase: a carbocation route for biosynthesis of halometabolites[END_REF][START_REF] Ohsawa | Purification and characterization of a monohalomethane-producing enzyme S-adenosyl-Lmethionine: halide ion methyltransferase from a marine microalga, Pavlova pinguis[END_REF][START_REF] Toda | Isolation and characterization of a gene encoding a S-adenosyl-l-methioninedependent halide/thiol methyltransferase (HTMT) from the marine diatom Phaeodactylum tricornutum: Biogenic mechanism of CH 3 I emissions in oceans[END_REF].

Identified chloromethane sinks are dominated by abiotic loss processes in the atmosphere involving reaction with OH radicals, or via chlorine radicals in the marine atmospheric boundary layer (see web resource section). The extent of consumption of chloromethane under the control of biological processes, especially by microorganisms, constitutes one of the largest uncertainties regarding the global budget of chloromethane [START_REF] Harper | The global cycles of the naturally-occurring monohalomethanes[END_REF][START_REF] Keppler | New insight into the atmospheric chloromethane budget gained using stable carbon isotope ratios[END_REF].

Microbial degradation: an underestimated sink in the global chloromethane budget

The study of ecology and diversity of chloromethane and other methyl halide-degrading microorganisms remain a challenging field of environmental biology. Key microbial enzymes of (de)halogenation activity and active microorganisms in chlorine and other halogens (fluorine, bromine, iodine) cycling remain largely unknown [START_REF] Weigold | A metagenomic-based survey of microbial (de) halogenation potential in a German forest soil[END_REF]. The global impact of microorganisms on the chloromethane sink is difficult to quantify due to concomitant processes of production and degradation of chloromethane in the environment. For instance, highly fluctuating chloromethane emissions occur in fern plants as recently discussed ( Jaeger et al., 2018b). Both production and degradation of chloromethane may coexist within a single organism as reported for lignin-degrading fungi. Unlike most other wood-rotting fungi, in Phanerochaete chrysosporium, Phlebia radiata, and Coriolus versicolor, chloromethane serves as an endogenous methyl donor in veratryl alcohol biosynthesis so that no chloromethane is emitted during lignin degradation and growth [START_REF] Harper | Chloromethane, methyl donor in veratryl alcohol biosynthesis in Phanerochaete chrysosporium and other lignin-degrading fungi[END_REF][START_REF] Coulter | Evidence for the existence of independent chloromethane-and S-adenosylmethionine-utilizing systems for methylation in Phanerochaete chrysosporium[END_REF]. However, so far, methyl halide (CH 3 X; chloromethane, bromomethane and iodomethane) cycling studies focused on chloromethane degradation by bacteria rather than by fungi [START_REF] Leisinger | Bacterial growth with chlorinated methanes[END_REF][START_REF] Harper | The global chloromethane cycle: biosynthesis, biodegradation and metabolic role[END_REF][START_REF] Mcdonald | A review of bacterial methyl halide degradation: biochemistry, genetics and molecular ecology[END_REF][START_REF] Schäfer | Bacterial cycling of methyl halides[END_REF][START_REF] Cox | Diversity of methyl halidedegrading microorganisms in oceanic and coastal waters[END_REF]. While the possible implications of fungal populations on the chloromethane cycle remain to be assessed using 'omics' approaches, the aim of this review is primarily to discuss recent investigations of bacterial populations associated with chloromethane sinks in contrasting terrestrial environments, these populations potentially differing from the so far characterized microbes with known pathways for chloromethane degradation. This possibility raises questions about the composition, distribution, functioning and evolution of chloromethane-utilizing populations in response to highly fluctuating emissions of this volatile halogenated compound at the soil-plant-atmosphere interfaces.

Assessing bacterial chloromethane sinks

Recent investigations of plant and soil samples suggest that chloromethane-degraders represent a minor fraction of microbial communities [START_REF] Chaignaud | Methanol consumption drives the bacterial chloromethane sink in a forest soil[END_REF]Jaeger et al., 2018a,b) that are estimated as 10 7 cells per cm 2 of leaf surface to 10 9 cells per g of soil [START_REF] Vorholt | Microbial life in the phyllosphere[END_REF]. In air, microbial concentrations range from a few tens to billions per cubic metre depending on location, altitude above ground, and time of the day and year (Amato et al., 2017a;[START_REF] Dassarma | Survival of microbes in Earth's stratosphere[END_REF], representing a total of 10 19 bacterial cells in the atmosphere [START_REF] Whitman | Prokaryotes: the unseen majority[END_REF]. Microorganisms collected from aerosols, cloud and rain waters retain metabolic activity under cloud-like conditions and are able to utilize atmospheric organic compounds such as formaldehyde and formate present at relatively high concentrations in cloud droplets (≈ 1-100 µM) [START_REF] Amato | A fate for organic acids, formaldehyde and methanol in cloud water: their biotransformation by micro-organisms[END_REF][START_REF] Delort | A short overview of the microbial population in clouds: potential roles in atmospheric chemistry and nucleation processes[END_REF][START_REF] Vaïtilingom | Potential impact of microbial activity on the oxidant capacity and organic carbon budget in clouds[END_REF][START_REF] Šantl-Temkiv | High-flow-rate impinger for the study of concentration, viability, metabolic activity, and ice-nucleation activity of airborne bacteria[END_REF]. While cloud-borne microbial assemblages include active facultative methylotrophs [START_REF] Amato | Active microorganisms thrive among extremely diverse communities in cloud water[END_REF], no isolate grew on chloromethane, no chloromethane consumption was observed (Fig. 8.1), and, consistently, no cmuA gene was detected using a highly sensitive capture hybridization method (see below). In contrast, viable methanotrophs (Methylosinus and Methylocystis) isolated from atmospheric samples (aerosol and rain) [START_REF] Šantl-Temkiv | Viable methanotrophic bacteria enriched from air and rain can oxidize methane at cloudlike conditions[END_REF] were able to use methane under cloud-like physicochemical conditions at concentrations relevant for the atmospheric environment (≈ 1.5 ppm). We concluded that the microbiota thriving in the investigated clouds do not represent a significant sink for chloromethane, unlike soils, aerial parts of plants (phyllosphere), or marine environments [START_REF] Goodwin | Description of toluene inhibition of methyl bromide biodegradation in seawater and isolation of a marine toluene oxidizer that degrades methyl bromide[END_REF][START_REF] Schäfer | Bacterial cycling of methyl halides[END_REF][START_REF] Chaignaud | Methanol consumption drives the bacterial chloromethane sink in a forest soil[END_REF]Jaeger et al., 2018a,b). [START_REF] Amato | Active microorganisms thrive among extremely diverse communities in cloud water[END_REF]. Airtight microcosms used for testing chloromethane (≈ 10 ppm) degradation of the collected cloud water samples (E). Two independent cloud water samples (626 ml and 740 ml, respectively, containing ≈ 10 6 cells pre-concentrated into 20 ml) showed no detectable temporal decrease of chloromethane under the tested conditions at 17°C (F). M. extorquens CM4 added in sterile rainwater spiked with 10 mM of chloromethane was used as positive control (data not shown). No isolate grew on chloromethane supplied as the sole source of carbon and energy (data not shown) in enrichment cultures of cloud and rain samples, performed as previously described [START_REF] Nadalig | Detection and isolation of chloromethane-degrading bacteria from the Arabidopsis thaliana phyllosphere, and characterization of chloromethane utilization genes[END_REF].

UNCORRECTED PROOF

Cultivated chloromethanedegrading methylotrophs

Methylotrophic bacteria capable of utilizing chloromethane as the only source of carbon and/or energy have been isolated [START_REF] Schaefer | Leisingera methylohalidivorans gen. nov., sp. nov., a marine methylotroph that grows on methyl bromide. I[END_REF][START_REF] Nadalig | Probing the diversity of chloromethane-degrading bacteria by comparative genomics and isotopic fractionation[END_REF] from contrasting environments such as soil, plants, freshwater and marine environments (Table 8.1). Chloromethane degraders are found among facultative aerobes, obligate anaerobes, obligate and facultative methylotrophs, and conditional utilizers of chloromethane. This diversity is encompassed by organisms possessing genomes ranging in size from very small (1.3 Mb) to relatively large (6.2 Mb), G + C contents ranging from 38% to 68%, and chloromethane utilization genes can be chromosome-or plasmidborne (Table 8.1).

Under strictly anoxic conditions, the only known pure culture that uses chloromethane as the sole energy source is the Gram-positive homoacetogenic autotrophic bacterium, Acetobacterium dehalogenans MC [START_REF] Traunecker | Isolation and characterization of a methyl chloride utilizing, strictly anaerobic bacterium[END_REF]. In this strain, chloromethane dehalogenation releases methyl-tetrahydrofolate (H 4 F) that is further metabolized to acetate [START_REF] Meßmer | Methyl chloride metabolism of the strictly anaerobic, methyl chloride-utilizing homoacetogen strain MC[END_REF][START_REF] Meßmer | Studies on methyl chloride dehalogenase and O-demethylase in cell extracts of the homoacetogen strain MC based on a newly developed coupled enzyme assay[END_REF]. Pseudomonas aeruginosa NB1 uses chloromethane as the sole source of carbon and energy under either anoxic or aerobic conditions upon switching from nitrate to oxygen as the terminal electron acceptor [START_REF] Freedman | Biodegradation of chloromethane by Pseudomonas aeruginosa strain NB1 under nitratereducing and aerobic conditions[END_REF]. Another case of anoxic chloromethane-utilization was recently reported and provides the first example of co-utilization of two chlorinated methane substrates in a mixed culture. In the absence of dichloromethane, chloromethane was not degraded as the chloromethane-degrader partner is thought to consume H 2 generated by Candidatus Dichloromethanomonas elyunquensis upon dichloromethane degradation (Chen et al., 2017) (Fig. 8.2A).

Under aerobic conditions, cometabolic degradation of chloromethane was observed by nitrifying bacteria [START_REF] Rasche | Factors limiting aliphatic chlorocarbon degradation by Nitrosomonas europaea: cometabolic inactivation of ammonia monooxygenase and substrate specificity[END_REF] and methanotrophs [START_REF] Han | Chloromethane stimulates growth of Methylomicrobium album BG8 on methanol[END_REF]. This is attributed to the activity of ammonium-or methane monooxygenase, respectively. When chloromethane is provided at low concentration in presence of another C 1 substrate, the growth of the facultative methanotroph Methylomicrobium album BG8 is enhanced (Fig. 8.2C). Another case of conditional degradation of chloromethane was found for the growth of the abundant coastal Betaproteobacterium strain HTCC2181. This obligate methylotroph utilizes chloromethane for energy only when methanol is supplied as the carbon source [START_REF] Halsey | Synergistic metabolism of a broad range of C1 compounds in the marine methylotrophic bacterium HTCC2181[END_REF] by a yet uncharacterized methyltransferase, with possible methyl transfer from chloromethane to H 4 F (Fig. 8.2B; discussed in substrate co-utilization section).

Microbial utilization of chloromethane as the sole carbon and energy source (Fig. 8.2A) was first described in Hyphomicrobium sp. strain MC1, isolated from a sewage treatment plant in Switzerland in 1986 [START_REF] Hartmans | Methyl chloride: naturally occurring toxicant and C-1 growth substrate[END_REF]. Strains affiliated with other taxonomic groups were subsequently recovered from soils (Aminobacter, Hyphomicrobium, Methylorubrum), marine environments (Roseovarius, Leisingera) and plants (Hyphomicrobium) (Table 8.1). Measured doubling time during aerobic growth at 30°C with chloromethane (10-15 mM) ranges from ≈ 5 hours (Hyphomicrobium strains CM2, MC1 and AT2) to ≈ 19 hours (Hyphomicrobium strains AT3 and AT4) [START_REF] Nadalig | Detection and isolation of chloromethane-degrading bacteria from the Arabidopsis thaliana phyllosphere, and characterization of chloromethane utilization genes[END_REF]. M. extorquens strain CM4 became the reference organism of chloromethane utilization (see below).

The cmu pathway for chloromethane degradation Biochemistry and genetics of aerobic chloromethane utilization have been elucidated in detail in strain CM4 affiliated to Methylobacterium extorquens, recently reclassified as Methylorubrum extorquens [START_REF] Green | Review of the genus Methylobacterium and closely related organisms: a proposal that some Methylobacterium species be reclassified into a new genus, Methylorubrum gen[END_REF]. Using random mutagenesis of strain CM4 with a miniTn5 transposon, mutants unable to grow with chloromethane were used to identify chloromethane utilization (cmu) genes [START_REF] Vannelli | Chloromethane metabolism by Methylobacterium sp. strain CM4[END_REF], 1999) (Fig. 8.2A). The first step involves chloromethane dehalogenation with the transfer of its methyl group to the C 1 carrier tetrahydrofolate (H 4 F) to produce methyl-H 4 F, and one chloride and one proton. The fact that the primary product of chloromethane demethylation is methyl-H 4 F rather than formaldehyde differs this pathway from C 1 oxidation pathways, including the methanol oxidation pathway [START_REF] Chistoserdova | Modularity of methylotrophy, revisited[END_REF][START_REF] Studer | Chloromethane-induced genes define a third C1 utilization pathway in Methylobacterium chloromethanicum CM4[END_REF]. Chloromethane dehalogenase consists of the corrinoid methyltransferase CmuA and the H 4 F-dependent methyltransferase CmuB [START_REF] Studer | Properties of the methylcobalamin:H 4 folate methyltransferase involved in chloromethane utilization by Methylobacterium sp. strain CM4[END_REF][START_REF] Studer | Chloromethane: tetrahydrofolate methyl transfer by two proteins from Methylobacterium chloromethanicum strain CM4[END_REF] chloromethane [START_REF] Vannelli | Chloromethane metabolism by Methylobacterium sp. strain CM4[END_REF]. Both proteins CmuB [START_REF] Studer | Properties of the methylcobalamin:H 4 folate methyltransferase involved in chloromethane utilization by Methylobacterium sp. strain CM4[END_REF] and CmuA [START_REF] Studer | Chloromethane: tetrahydrofolate methyl transfer by two proteins from Methylobacterium chloromethanicum strain CM4[END_REF] are required for growth-supporting dehalogenation of chloromethane. CmuA acts as a bifunctional enzyme (possessing methyltransferase and the corrinoid-binding domains), and it also serves as an intermediate methyl carrier. In a second reaction, CmuB methyltransferase transfers the methyl group from CmuA to H 4 F [START_REF] Studer | Chloromethane: tetrahydrofolate methyl transfer by two proteins from Methylobacterium chloromethanicum strain CM4[END_REF]. Thus, besides H 4 F, chloromethane dehalogenation requires a corrinoid cofactor, which explains an absolute need for cobalt for growth with methyl halides [START_REF] Studer | Chloromethane: tetrahydrofolate methyl transfer by two proteins from Methylobacterium chloromethanicum strain CM4[END_REF]. This uncharacterized corrinoid cofactor possibly differs from the chromosome-encoded cobalamin compound [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes chloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: a proteomic and bioinformatics study[END_REF]. In M. extorquens strain CM4, all genes of the cmu pathway and cob genes are present on a 380 kb plasmid called pCMU01. Comparison of complete genome sequences of strain CM4 with those of other M. extorquens strains unable to grow with chloromethane showed that plasmid pCMU01 harbours unique genes without homologues in the compared genomes (bluB2, btuB, cobA, cbiD), as well as 13 duplicated genes with homologues of chromosome-borne genes involved in cobalamin-associated biosynthesis and transport, or in H 4 F-dependent metabolism (folC2) [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes chloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: a proteomic and bioinformatics study[END_REF]. The clustering of genes coding for dehalogenase enzymes and for biosynthesis of associated cofactors suggests a history of coupled enzyme/coenzyme gene acquisition related to chloromethane utilization in strain CM4.

Coping with chloromethane upon methylotrophic growth Bacteria that grow on chloromethane need to accommodate multiple stresses such as intracellular production of protons and chloride, adjustment of metabolism towards higher demand for dehalogenase-requiring cofactors (H 4 F and cobalamin-related compounds), and the necessity to regulate their metabolism for effective chloromethane utilization [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes chloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: a proteomic and bioinformatics study[END_REF][START_REF] Michener | Transfer of a catabolic pathway for chloromethane in Methylobacterium strains highlights different limitations for growth with chloromethane or with dichloromethane[END_REF][START_REF] Chaignaud | Genomic and transcriptomic analysis of growthsupporting dehalogenation of chlorinated methanes in Methylobacterium[END_REF]. The limiting factors were inferred from experimental observations: (i) growth with chloromethane is slower than with methanol [START_REF] Chaignaud | Genomic and transcriptomic analysis of growthsupporting dehalogenation of chlorinated methanes in Methylobacterium[END_REF]; (ii) difference in growth rates under chloromethane utilization are not due to differences in chloromethane dehalogenase activity in cellfree extracts of taxonomically-related strains [START_REF] Nadalig | Detection and isolation of chloromethane-degrading bacteria from the Arabidopsis thaliana phyllosphere, and characterization of chloromethane utilization genes[END_REF]; and (iii) transfer of a cmu catabolic pathway gene cassette (plasmid pJM105) in 'naïve' Methylorubrum strains conferred poor growth ability on chloromethane supplied as the sole energy and carbon source [START_REF] Michener | Transfer of a catabolic pathway for chloromethane in Methylobacterium strains highlights different limitations for growth with chloromethane or with dichloromethane[END_REF]. Among potential limiting factors, intracellular release of protons and chloride ions is likely, [START_REF] Vannelli | A corrinoid-dependent catabolic pathway for growth of a Methylobacterium strain with chloromethane[END_REF]McAnulla et al., 2001b); (B) Bacterioplankton HTCC2181, adapted from [START_REF] Giovannoni | The small genome of an abundant coastal ocean methylotroph[END_REF]; (C) Methylomicrobium album BG8, adapted from [START_REF] Han | Chloromethane stimulates growth of Methylomicrobium album BG8 on methanol[END_REF]; and (D) Culture RM, adapted from [START_REF] Chen | Mutualistic interaction between dichloromethane-and chloromethanedegrading bacteria in an anaerobic mixed culture[END_REF]. Chloromethane and dichloromethane are each degraded by a different organism in a mutualism interaction. Chloromethane could not substitute for dichloromethane as the sole source of energy. It remains unknown how chloromethane enters the C 1 cycle of the chloromethane-degrading homoacetogen strain performing H 2 /CO 2 reductive acetogenesis (Kleindienst et al., 2017). For Candidatus Dichloromethanomonas elyunquensis, dichloromethane is a source of energy. No dichloromethane degradation occurs in medium without CO 2 (bicarbonate) supplementation [START_REF] Kleindienst | Candidatus Dichloromethanomonas elyunquensis' gen. nov., sp. nov., a dichloromethane-degrading anaerobe of the Peptococcaceae family[END_REF]. Enzymes involved in dichloromethane degradation and CO 2 assimilation have not been characterized. since dehalogenation of chloromethane, as of dichloromethane, initiates growth-supporting degradation. Nonetheless, each of these chlorinated methanes has specific adaptive responses for chloride export. For example, in Methylorubrum, the chromosome-encoded proton/chloride antiporter ClcA had a significant fitness impact for growth with dichloromethane but not with chloromethane (Michener et al., 2014a[START_REF] Michener | Transfer of a catabolic pathway for chloromethane in Methylobacterium strains highlights different limitations for growth with chloromethane or with dichloromethane[END_REF]. Adaptive responses to growth with chloromethane compared with growth with methanol were detected using complementary approaches of random mutagenesis [START_REF] Vannelli | A corrinoid-dependent catabolic pathway for growth of a Methylobacterium strain with chloromethane[END_REF], genome sequencing [START_REF] Marx | Complete genome sequences of six strains of the genus Methylobacterium[END_REF], comparative genomics, proteomics and transcriptomics [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes chloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: a proteomic and bioinformatics study[END_REF][START_REF] Chaignaud | Genomic and transcriptomic analysis of growthsupporting dehalogenation of chlorinated methanes in Methylobacterium[END_REF], and experimental evolution [START_REF] Michener | Transfer of a catabolic pathway for chloromethane in Methylobacterium strains highlights different limitations for growth with chloromethane or with dichloromethane[END_REF]. A summary of the global response to growth with chloromethane in M. extorquens strain CM4 is proposed in Fig. 8.3.

According to this model, the adaptive response to chloromethane is complex and includes general oxidative stress response functions (Fig. 8.3) such as molecular chaperones, DNA repair and reactive oxygen species (ROS) removal functions, as well as a dehalogenation-specific response to intracellular hydrochloric acid production (membrane-bound proton translocating pyrophosphatase HppA and a putative chloride/proton antiporter ClcA2 encoded by plasmid pCMU01). The pnt gene cluster encoding a NADH/NADPH transhydrogenase with cross-membrane proton translocation activity, most likely plays a role in maintaining both the intracellular pools of NAD + and NADP +, and the internal cellular pH with the efflux of protons during chloromethane dehalogenation. Higher expression of genes (enzymes and transporters) for biosynthesis of methyltransferase cofactors were detected, especially bluB2 (sharing 38% amino acid identity with the chromosomal-encoded bluB gene). BluB catalyses both the production of a lower ligand of cobalamin (5,6-dimethylbenzimidazole) and of a H 4 F precursor (erythrose 4P), thus linking together the biosynthesis of the two cofactors of chloromethane dehalogenation [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes chloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: a proteomic and bioinformatics study[END_REF]. Coping with chloromethane upon methylotrophic growth. In M. extorquens CM4, the cmu pathwayencoding genes and associated genes are plasmid pCMU01-borne (gene names in green). In cultures grown on chloromethane compared with methanol, more abundant RNAs (green bullets) [START_REF] Chaignaud | Genomic and transcriptomic analysis of growthsupporting dehalogenation of chlorinated methanes in Methylobacterium[END_REF] and proteins (black-circled bullets) [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes chloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: a proteomic and bioinformatics study[END_REF] were found. Essential genes in chloromethane assimilation or dechlorination are marked with a star [START_REF] Vannelli | A corrinoid-dependent catabolic pathway for growth of a Methylobacterium strain with chloromethane[END_REF][START_REF] Michener | Transfer of a catabolic pathway for chloromethane in Methylobacterium strains highlights different limitations for growth with chloromethane or with dichloromethane[END_REF]. For the putative role of the acetone carboxylase components encoded by the plasmid-borne acxABC gene cluster chloromethane assimilation, refer to [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes chloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: a proteomic and bioinformatics study[END_REF]. In conclusion, the ability to utilize chloromethane likely involves adaptation to oxidative stress, production of reducing equivalents, conversion of H 4 F-bound C 1 units, and central metabolism. The adaptive response to growth with chloromethane has little overlap with other toxic halogenated compounds [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes chloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: a proteomic and bioinformatics study[END_REF]Michener et al., 2014b[START_REF] Michener | Transfer of a catabolic pathway for chloromethane in Methylobacterium strains highlights different limitations for growth with chloromethane or with dichloromethane[END_REF][START_REF] Chaignaud | Genomic and transcriptomic analysis of growthsupporting dehalogenation of chlorinated methanes in Methylobacterium[END_REF], and possibly with chloromethane degradation by cmu-independent pathways.

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

The strategy of combining stable isotope approaches (Compound-Specific Isotope Analysis; CSIA) with comparative genomics helps to explore the diversity of microbial degradation of chloromethane [START_REF] Nadalig | Probing the diversity of chloromethane-degrading bacteria by comparative genomics and isotopic fractionation[END_REF]. CSIA is a chemical method to determine the ratio of isotopes of different elements (e.g. C, N, H, O, Cl) in environmental samples [START_REF] Nijenhuis | Stable isotope fractionation concepts for characterizing biotransformation of organohalides[END_REF]. This ratio can change during the course of microbial degradation. For example, carbon large fractionation shifts (δ 13 C from an initial value of -60‰ to -30‰) were observed when 90% of the chloromethane was degraded by high-cell-density suspensions of methylotrophic bacteria [START_REF] Miller | Large carbon isotope fractionation associated with oxidation of methyl halides by methylotrophic bacteria[END_REF]. Combined measurements of carbon and hydrogen isotope fractionation of the remaining untransformed chloromethane provide further evidence of biotic degradation and clues about the metabolic pathway and enzymes involved (Nadalig et al., 2013) (Table 8.2). When genomes of experimentally validated chloromethane-degrading methylotrophs were searched for the presence of cmuA, two groups of bacteria were detected: (i) M. extorquens CM4 and Hyphomicrobium sp. MC1 isolated from soils share the cmu pathway genes; (ii) Leisingera methylohalidovorans MB2 and Roseovarius sp. 217 isolated from marine environments harbour no cmu genes and degrade chloromethane by a yet uncharacterized mechanism (Table 8.1). Cultures of L. methylohalidovorans MB2 displayed clear differences of both carbon and hydrogen isotope signatures upon chloromethane dehalogenation compared with cmu-containing strain cultures of Aminobacter, Hyphomicrobium and Methylorubrum [START_REF] Nadalig | Probing the diversity of chloromethane-degrading bacteria by comparative genomics and isotopic fractionation[END_REF].

The cmu gene organization is unique in strain CM4 [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes chloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: a proteomic and bioinformatics study[END_REF], where the genes are plasmid-borne and located in two distant regions [START_REF] Vannelli | Chloromethane metabolism by Methylobacterium sp. strain CM4[END_REF]. In other strains, when detected by comparative genomics and targeted PCR amplifications, cmu genes (Fig. 8.4) were conserved on a single chromosomal cluster (McAnulla et al., 2001b;[START_REF] Nadalig | Probing the diversity of chloromethane-degrading bacteria by comparative genomics and isotopic fractionation[END_REF]. In particular, a trio of adjacent genes (cmuBCA) is conserved in genomes of aerobic chloromethane-degraders isolated from soils and plants, genomes of Alphaproteobacteria isolated from deep-sea (Celeribacter indicus P73 and Roseovarius pacificus 81-2), as well as of obligate anaerobes (Desulfobacula sp. TS; Desulfomonile tiedjei DSM 6799, Desulfurispora thermophila DSM 16022, Desulfotomaculum alcoholivorax DSM 16058, Thermincola potens JR and Thermosediminibacter oceani DSM 16646). These anaerobic strains harbour cmuA homologues closely related to each other (identities at the protein level between 84-93%) [START_REF] Nadalig | Probing the diversity of chloromethane-degrading bacteria by comparative genomics and isotopic fractionation[END_REF] but none of these strains has yet been reported to degrade chloromethane. The overall conserved cmuBCA trio suggests a functional link between the encoded CmuA, CmuB and CmuC. While CmuA and CmuB are two methyltransferases essential for chloromethane dehalogenation [START_REF] Studer | Chloromethane: tetrahydrofolate methyl transfer by two proteins from Methylobacterium chloromethanicum strain CM4[END_REF], the function of CmuC remains uncharacterized. CmuC, a putative third methyltransferase-like protein most similar to MtaA (corrinoid:co enzyme M methyltransferase), is dispensable for dehalogenation but needed for chloromethane assimilation [START_REF] Vannelli | A corrinoid-dependent catabolic pathway for growth of a Methylobacterium strain with chloromethane[END_REF]. Mobile genetic elements (black arrows in Fig. 8.4) in the vicinity of conserved cmu clusters potentially suggest their acquisition by horizontal gene transfer. Duplicated cmu genes found in strain CM4 (two homologous cmuC copies adjacent to either cmuB or cmuA) and in D. tiedjei DSM 6799 (a cmuAB copy in addition to the conserved cmuBCA) suggest specific strain adaptation. An 'orphan' cmuA-like gene (encoding for a protein sharing 43% amino acid identity with M. extorquens CM4 CmuA) with no other detected genes of the cmu pathway in its genome, raises the question of whether the marine Gammaproteobacterium Vibrio orientalis CIP 102891 in able to degrade chloromethane. No chloromethane degradation was observed for V. orientalis CIP 102891 resting cells (Fig. 8.5) or by actively growing cell cultures supplied with chloromethane in addition to another carbon source (data not shown). This suggests that the V. orientalis CmuA-like homolog might play a yet unknown function, possibly different from chloromethane dehalogenation.

Methylotrophic populations of the chloromethane sink

Chloromethane-degrading populations reduce chloromethane emissions to the atmosphere In contrast to live soils, sterilized soils or plants do not degrade chloromethane [START_REF] Chaignaud | Genomic and transcriptomic analysis of growthsupporting dehalogenation of chlorinated methanes in Methylobacterium[END_REF]Jaeger et al., 2018a,b) suggesting a microbial sink of chloromethane. Compared to chloromethane degrading methylotrophs (Table 8.2), the carbon isotope enrichment factor (ε C ) of remaining chloromethane was similar for fern leaves (mean of -43 ± 12‰) and lower for soils (ranging from -38 to 11 ±3‰ in agricultural, grassland and forest soils, respectively). Stable isotope hydrogen fractionation (ε H ) observed during degradation experiments with fern leaves (-8 ± 19‰), and soils (-50 ± 19‰) was similar to reported ε H for chloromethane-degrading bacterial strains (Table 8.2). No change in stable isotope fractionation signature was observed when the investigated environmental samples were sterilized, suggesting a biotic microbially driven process [START_REF] Chaignaud | Genomic and transcriptomic analysis of growthsupporting dehalogenation of chlorinated methanes in Methylobacterium[END_REF]Jaeger et al., 2018a,b). This hypothesis is in agreement with the recent finding that the occurrence and the diversity of bacterial chloromethane degradation (cmu gene expression) is correlated with differential expression of the plant gene HOL1 involved in the production of chloromethane [START_REF] Farhan Ul Haque | Correlated production and consumption of chloromethane in the Arabidopsis thaliana phyllosphere[END_REF]. Thus, a relation was established between chloromethane production by plants and relative abundance of leave-associated chloromethane-degrading bacteria. This suggests that chloromethane-degrading bacteria co-determine the extent of chloromethane emissions by plants to the atmosphere, and that methylotrophic microorganisms are involved in the terrestrial chloromethane sink. Rare occurrence of cmuA in the environment Of the genes of the cmu pathway, the cmuA gene is the most conserved [START_REF] Farhan Ul Haque | Correlated production and consumption of chloromethane in the Arabidopsis thaliana phyllosphere[END_REF][START_REF] Nadalig | Detection and isolation of chloromethane-degrading bacteria from the Arabidopsis thaliana phyllosphere, and characterization of chloromethane utilization genes[END_REF]. Thus, cmuA has been used to study the diversity of methyl halide-degrading organisms in the environment [START_REF] Miller | Degradation of methyl bromide and methyl chloride in soil microcosms: Use of stable C isotope fractionation and stable isotope probing to identify reactions and the responsible microorganisms[END_REF][START_REF] Schäfer | Evidence for the presence of a CmuA methyltransferase pathway in novel marine methyl halide-oxidizing bacteria[END_REF][START_REF] Nadalig | Detection and isolation of chloromethane-degrading bacteria from the Arabidopsis thaliana phyllosphere, and characterization of chloromethane utilization genes[END_REF][START_REF] Cox | Diversity of methyl halidedegrading microorganisms in oceanic and coastal waters[END_REF][START_REF] Chaignaud | Methanol consumption drives the bacterial chloromethane sink in a forest soil[END_REF]. With increasing numbers of cmuA sequences in databases, oligonucleotide primers have been modified for more efficient PCR amplification and improved detection of cmuA (see details in Chaignaud et al., 2018, supplemental material). No PCR products were retrieved from environmental DNA prior to enrichment steps of forest soil [START_REF] Chaignaud | Methanol consumption drives the bacterial chloromethane sink in a forest soil[END_REF] and plant leaves ( Jaeger et al., 2018b). When amplified, a low diversity of cmuA sequences was found in samples of soils, plant leaves and large volumes of oceanic and coastal waters [START_REF] Cox | Diversity of methyl halidedegrading microorganisms in oceanic and coastal waters[END_REF][START_REF] Farhan Ul Haque | Correlated production and consumption of chloromethane in the Arabidopsis thaliana phyllosphere[END_REF][START_REF] Chaignaud | Methanol consumption drives the bacterial chloromethane sink in a forest soil[END_REF]. This suggested that cmuA-harbouring bacteria might represent a small component of the investigated environmental microbiota.

Recently, an extremely sensitive molecular tool for cmuA detection by 'targeted gene capture' was developed [START_REF] Gasc | Sequence capture by hybridization to explore modern and ancient genomic diversity in model and nonmodel organisms[END_REF][START_REF] Ribière | Targeted gene capture by hybridization to illuminate ecosystem functioning[END_REF]. Its detection limit of 0.00006% makes it particularly adapted to investigate rare genes and biological functions in the environment [START_REF] Gasc | Hybridization capture reveals microbial diversity missed using current profiling methods[END_REF]. It consists of utilizing metal beads for recovering the sequences of interest within a community (or within nucleic acid extracts) by hybridization. The beads are coated with biotinylated RNA sequences of ≈ 80 bp, complementary to the targeted gene sequence and potential orthologs. Using KASpOD software [START_REF] Parisot | Probe design strategies for oligonucleotide microarrays[END_REF], such 80-mer sequences have been successfully used to design specific probes based on large sequence datasets to reveal microbial diversity missed using current profiling methods. Based on known cmuA gene sequences, gene capture method was attempted in different environments [e.g. soil ( Jaeger et al., 2018a), plant leaves ( Jaeger et al., 2018b) and cloud microbial communities (L. Besaury, P. Amato, P. Peyret, C. Gasc, unpublished data)] with no success. When homologous sequences were searched in metagenomic databases in a wider range of The remaining chloromethane was quantified by gas chromatography (GC-FID) at different time points. Cell suspensions of V. orientalis CIP 102891 (empty square) were harvested from exponential-grown phase as described for L. methylohalidovorans MB2 saline cultures [START_REF] Nadalig | Probing the diversity of chloromethane-degrading bacteria by comparative genomics and isotopic fractionation[END_REF]. For V. orientalis CIP 102891, the amount of chloromethane remaining was similar to that of the negative controls without cells (empty circle, saline phosphate buffer; empty diamond, phosphate buffer) but significantly different from the positive degraders M. extorquens CM4 in (black diamond) and L. methylohalidovorans MB2 (black square). environments (Table 8.3), only 11 cmuA sequences were retrieved, of which three originated from the rhizosphere of the plant A. thaliana, and the others from marine environments (Table 8.3). Basing on these results, cmuA is estimated to represent around one DNA sequence out of 3 billion, and can be considered as a rare gene [START_REF] Kislyuk | Genomic fluidity: an integrative view of gene diversity within microbial populations[END_REF]. It may be necessary for future sampling campaigns to assess if environmental niche partitioning of microbial minorities consuming chloromethane is found as for methane [START_REF] Bodelier | Microbial minorities modulate methane consumption through niche partitioning[END_REF]. Using Blast searches, cmuA DNA sequences were retrieved from environmental metagenomes, translated to proteins and aligned to reference 'sensu stricto' proteins of functionally characterized CmuA proteins of Alphaproteobacteria cultivated under aerobic conditions, to uncharacterized proteins of the clade called 'CmuA-anaerobe' found in strict anaerobes and of 'CmuA-like' proteins found in bacteria lacking cmuBC (as defined in Fig. 8.6). Of the 11 metagenomic cmuA sequences, one was closely related to the 'sensu stricto CmuA' clade, one to the 'CmuA-anaerobe', and the remaining belonged to the most-distant CmuA-like clade. Remarkably, chloromethane dehalogenation by 'sensu stricto CmuA' does not require aerobic conditions and is even sensitive to oxygen [START_REF] Studer | Chloromethane: tetrahydrofolate methyl transfer by two proteins from Methylobacterium chloromethanicum strain CM4[END_REF]. Thus, anaerobic bacteria with cmu genes may be able to use chloromethane as a source of carbon and energy, as previously discussed [START_REF] Nadalig | Probing the diversity of chloromethane-degrading bacteria by comparative genomics and isotopic fractionation[END_REF]. Nonetheless, the ability to degrade methyl halides under environmental conditions remains to be confirmed for bacterial populations coding for 'CmuA-anaerobe' and CmuA-like proteins.

UNCORRECTED PROOF

Substrate co-utilization as a strategy to sustain growth and resilience of chloromethane-utilizing methylotrophic populations

In the troposphere, chloromethane is a trace gas with a global mean concentration of ≈ 500 to 600 parts per trillion by volume (pptv). In environments with low nutrient levels, methylotroph populations growing on different C 1 substrates [START_REF] Neufeld | Substrate-specific clades of active marine methylotrophs associated with a phytoplankton bloom in a temperate coastal environment[END_REF][START_REF] Gifford | Quantitative transcriptomics reveals the growth-and nutrient-dependent response of a streamlined marine methylotroph to methanol and naturally occurring dissolved organic matter[END_REF] would benefit from simultaneous utilization of several C 1 compounds as a strategy to enhance their growth. In coastal ecosystems, methanol is released during phytoplankton blooms [START_REF] Heikes | Atmospheric methanol budget and ocean implication[END_REF]. When methanol is present in seawater, the methanol-assimilating methylotroph HTCC 2181 uses chloromethane as source of energy [START_REF] Halsey | Synergistic metabolism of a broad range of C1 compounds in the marine methylotrophic bacterium HTCC2181[END_REF]. Its genome, one of the smallest known for bacteria, lacks the tetrahydromethanopterin (H 4 MPT) C 1 transfer module, so C 1 transfer can only rely on H 4 F [START_REF] Giovannoni | The small genome of an abundant coastal ocean methylotroph[END_REF]. Strain HTCC 2181 may have evolved by reducing its C 1 abilities (loss of the genes for the C 1 carrier, H 4 MPT synthesis) and thus reducing its metabolic versatility in favour of specialization on carbon compounds at low amounts [START_REF] Giovannoni | The small genome of an abundant coastal ocean methylotroph[END_REF].

To our knowledge, no data are available on chloromethane concentration in upper soil of terrestrial ecosystems, which are highly active sinks [START_REF] Borodina | Use of DNA-stable isotope probing and functional gene probes to investigate the diversity of methyl chloride-utilizing bacteria in soil[END_REF][START_REF] Chaignaud | Methanol consumption drives the bacterial chloromethane sink in a forest soil[END_REF]. In the laboratory, high chloromethane ratios (in the percent range) have been used to isolate and grow pure chloromethane-degrading cultures that originated from soil [START_REF] Schäfer | Bacterial cycling of methyl halides[END_REF] in order to obtain enough biomass for growth and chloromethane degradation studies. The identification of natural microbial populations actively assimilating carbon from chloromethane was first achieved using stable isotope probing (DNA SIP) [START_REF] Miller | Degradation of methyl bromide and methyl chloride in soil microcosms: Use of stable C isotope fractionation and stable isotope probing to identify reactions and the responsible microorganisms[END_REF].

Studies in soil revealed that bacterial population members of soil microbial communities take up carbon from amended chloromethane [START_REF] Miller | Degradation of methyl bromide and methyl chloride in soil microcosms: Use of stable C isotope fractionation and stable isotope probing to identify reactions and the responsible microorganisms[END_REF][START_REF] Borodina | Use of DNA-stable isotope probing and functional gene probes to investigate the diversity of methyl chloride-utilizing bacteria in soil[END_REF][START_REF] Chaignaud | Genomic and transcriptomic analysis of growthsupporting dehalogenation of chlorinated methanes in Methylobacterium[END_REF]. Moreover, it has been demonstrated that methanotrophs, as investigated with a pure culture of Methylomicrobium album BG8, can assimilate carbon from chloromethane when a surplus of methanol occurs, although chloromethane alone was not assimilated [START_REF] Han | Chloromethane stimulates growth of Methylomicrobium album BG8 on methanol[END_REF]. These findings suggest a possible co-metabolic lifestyle of some methanotrophs regarding chloromethane degradation. In a forest soil, the rate of chloromethane degradation was recently shown to be enhanced when methanol was present [START_REF] Chaignaud | Methanol consumption drives the bacterial chloromethane sink in a forest soil[END_REF]. In this study, Beijerinckiceae (i.e. phylotypes affiliating closely with Methylovirgula ligni), assimilated carbon from methanol or from chloromethane as suggested by SIP experiments of soil microcosms amended with labelled or unlabelled methanol, chloromethane or both simultaneously [START_REF] Chaignaud | Genomic and transcriptomic analysis of growthsupporting dehalogenation of chlorinated methanes in Methylobacterium[END_REF][START_REF] Morawe | Acidotolerant bacteria and fungi as a sink of methanol-derived carbon in a deciduous forest soil[END_REF].

Methylotrophic co-utilization of chloromethane with other C 1 compounds such as methanol, which is found in larger amounts in the environment and utilized by most methylotrophs, may thus represent an important component of chloromethane sink activity. The utilization of methanol as an alternate substrate to chloromethane may favour the persistence of chloromethane-degrading populations in the context of shortage of chloromethane in natural habitats. The presence of pollutants such as toluene in ocean water was found to inhibit chloromethane and bromomethane uptake, thereby challenging the persistence of methyl halide-degrading populations and subsequent methyl halide oceanic uptake, which would in turn disturb an important component of the biogeochemical cycle of these ozone-depleting compounds [START_REF] Goodwin | Description of toluene inhibition of methyl bromide biodegradation in seawater and isolation of a marine toluene oxidizer that degrades methyl bromide[END_REF].

Future trends

Low-and high-affinity degraders One of the key aspects of future explorations of the methylotrophic chloromethane sink is the need to unravel the enzymes and pathways that define the global sink of halocarbons not only in bacteria but also in fungi. Future research needs to grasp the diversity of chloromethane-degrading-associated metabolisms and taxa associated with the microbial chloromethane sink. Much effort has focused on molecular processes for methylotrophic growth under laboratory conditions. Owing to technical The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model [START_REF] Jones | The rapid generation of mutation data matrices from protein sequences[END_REF]. Bootstrap values were calculated from 1000 replicates,% values ≥ 50 are presented. Analyses were conducted using the software MEGA7 [START_REF] Kumar | MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets[END_REF] [START_REF] Borodina | Use of DNA-stable isotope probing and functional gene probes to investigate the diversity of methyl chloride-utilizing bacteria in soil[END_REF]. limitations, so far biological chloromethane sinks have been assessed using higher than natural concentrations. Future investigations should focus on environmentally relevant chloromethane levels (picomolar). High variations in chloromethane flux emissions have been reported ( Jaeger et al., 2018) so it can be postulated that low and high affinity chloromethane-degrading populations may be found in the environment, as for methane [START_REF] Maxfield | Substantial high-affinity methanotroph populations in Andisols effect high rates of atmospheric methane oxidation[END_REF].

Unknowns of the chloromethane cycle

How plant chloromethane emissions vary during their developmental stage or their physiological states (chloride stress, pathogen invasion, exposure to chlorinated pesticides), and the role of chloromethane production by plants, is unknown [START_REF] Bringel | Plant-pesticide interactions and the global chloromethane budget[END_REF]. Host-associated microbiota may elicit a response that modulates the interactive interplay between host/microbial cells/insects as found for other plant-produced VOCs (review by [START_REF] Bringel | Pivotal roles of phyllosphere microorganisms at the interface between plant functioning and atmospheric trace gas dynamics[END_REF]. For example, chloromethane regenerates veratryl alcohol during degradation of lignin by some white-rot fungi [START_REF] Harper | Chloromethane, methyl donor in veratryl alcohol biosynthesis in Phanerochaete chrysosporium and other lignin-degrading fungi[END_REF], and one may speculate that associated bacterial communities may interfere with this process. Similarly, methylotroph populations may interfere with chlorine output through volatilization via chloromethane emissions, as white-rot fungi and higher plants methylate chloride [START_REF] Oberg | The natural chlorine cycle -fitting the scattered pieces[END_REF]. On the bacterial side, even with M. extorquens strain CM4, in the in-labo best-characterized chloromethane degrader, our current understanding is not sufficient to predict its behaviour under environmental conditions. Key actors remain uncharacterized: the regulator of the cmu pathway, the chemical nature of the native corrinoid cofactor of the CmuA methyltransferase from chloromethane to H 4 F, many genes involved in corrinoid cofactor metabolism [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes chloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: a proteomic and bioinformatics study[END_REF], and conserved genes adjacent to cmuA such as cmuC (Fig. 8.4) [START_REF] Michener | Transfer of a catabolic pathway for chloromethane in Methylobacterium strains highlights different limitations for growth with chloromethane or with dichloromethane[END_REF]. In fact, all the details of the dehalogenation reactions and carbon funnelling to central metabolism remain to be investigated physiologically, biochemically on wild-type and mutants. Unfortunately, reverse genetics for site-directed mutagenesis has not been successful so far in M. extorquens strain CM4 [START_REF] Michener | Transfer of a catabolic pathway for chloromethane in Methylobacterium strains highlights different limitations for growth with chloromethane or with dichloromethane[END_REF] and Hyphomicrobium sp. Nonetheless, complementary 'omics' technologies such metabolomics, fluxomics and experimental evolution are available for Methylorubrum studies [START_REF] Ochsner | Methylobacterium extorquens: methylotrophy and biotechnological applications[END_REF].

Biomarkers of chloromethane sinks

Gene cmuA is the only known biomarker for bacterial chloromethane utilization. It is rarely found in metagenomes (Table 8.2) ( Jaeger et al., 2018a,b), and is absent in the genome of some chloromethane utilizers [START_REF] Buddruhs | Complete genome sequence of the marine methyl-halide oxidizing Leisingera methylohalidivorans type strain (DSM 14336(T)), a representative of the Roseobacter clade[END_REF] (Table 8.1). Together, these observations reveal the need to define cmuA-independent biomarkers of the chloromethane microbial sinks. In open oceans [START_REF] Goodwin | Description of toluene inhibition of methyl bromide biodegradation in seawater and isolation of a marine toluene oxidizer that degrades methyl bromide[END_REF] and soils [START_REF] Rasche | Factors limiting aliphatic chlorocarbon degradation by Nitrosomonas europaea: cometabolic inactivation of ammonia monooxygenase and substrate specificity[END_REF][START_REF] Chaignaud | Methanol consumption drives the bacterial chloromethane sink in a forest soil[END_REF], co-substrate consumption may be a major driver of chloromethane microbial sinks. Thus, new strategies should be explored for investigating microbial cmu-independent chloromethane-degrading pathways, such as experiments on co-consumption of chloromethane with mixtures of other environmentally relevant carbon sources, co-cultures of mixed populations [START_REF] Nai | From axenic to mixed cultures: technological advances accelerating a paradigm shift in microbiology[END_REF], and cultivation-independent approaches using single-cell labelling and identification, such as microfluidic cell sorting combined with 'omics' methods and Raman-FISH [START_REF] Musat | Detecting metabolic activities in single cells, with emphasis on nanoSIMS[END_REF][START_REF] Hol | Zooming in to see the bigger picture: microfluidic and nanofabrication tools to study bacteria[END_REF]. This quest for new biomarkers will help explore the dynamics and diversity of chloromethane-degrading microorganisms active in situ in natural and anthropogenic environments.

Web resources

Data reporting on production and consumption of ozone depleting substances

The Montreal Protocol on Substances that Deplete the Ozone Layer: http://ozone.unep.org/en/treaties-and-decisions/ montreal-protocol-substances-deplete-ozone-layer Scientific Assessments of Ozone Depletion conducted under the auspices of the World Meteorological Organization (WMO) and the United Nations Environment Programme (UNEP): https://www.esrl.noaa.gov/csd/assessments/ ozone/and http://www.wmo.int/pages/prog/ arep/gaw/ozone/

Figure 8 . 1

 81 Figure 8.1 Assessing cloud water microorganisms for chloromethane degradation. Cloud water samples were collected under sterile conditions from the atmospheric station at Puy de Dôme mountain summit (1465 m above see level, France) (A) at the roof platform of a historical meteorological station (B) using cloud droplet impactors (C and D) as previously described[START_REF] Amato | Active microorganisms thrive among extremely diverse communities in cloud water[END_REF]. Airtight microcosms used for testing chloromethane (≈ 10 ppm) degradation of the collected cloud water samples (E). Two independent cloud water samples (626 ml and 740 ml, respectively, containing ≈ 10 6 cells pre-concentrated into 20 ml) showed no detectable temporal decrease of chloromethane under the tested conditions at 17°C (F). M. extorquens CM4 added in sterile rainwater spiked with 10 mM of chloromethane was used as positive control (data not shown). No isolate grew on chloromethane supplied as the sole source of carbon and energy (data not shown) in enrichment cultures of cloud and rain samples, performed as previously described[START_REF] Nadalig | Detection and isolation of chloromethane-degrading bacteria from the Arabidopsis thaliana phyllosphere, and characterization of chloromethane utilization genes[END_REF].

Figure 8 . 4

 84 Figure 8.4 Organisation of cmu genes in bacteria. Names of experimentally validated chloromethane-degrading bacteria are written in bold. Arrows represent protein-coding genes, and annotations above arrows indicate gene names. Homologous genes are given with identical colour except for arrows in black, pink and grey, which indicate mobile elements, transcriptional regulators, and genes not involved in chloromethane degradation, respectively. The percentage of amino acid sequence identity with the reference Hyphomicrobium sp. MC1 CmuA protein is indicated. Gene clusters are drawn to scale.

Figure 8 . 5

 85 Figure 8.5 Assessing the ability of Vibrio orientalis CIP 102891 to degrade chloromethane. Triplicates of resting cells were exposed to chloromethane in gastight flasks at room temperature. The remaining chloromethane was quantified by gas chromatography (GC-FID) at different time points. Cell suspensions of V. orientalis CIP 102891 (empty square) were harvested from exponential-grown phase as described for L. methylohalidovorans MB2 saline cultures[START_REF] Nadalig | Probing the diversity of chloromethane-degrading bacteria by comparative genomics and isotopic fractionation[END_REF]. For V. orientalis CIP 102891, the amount of chloromethane remaining was similar to that of the negative controls without cells (empty circle, saline phosphate buffer; empty diamond, phosphate buffer) but significantly different from the positive degraders M. extorquens CM4 in (black diamond) and L. methylohalidovorans MB2 (black square).

  Uncultured bacterium clone Tocil Wood soil 36 (AY934440) Uncultured bacterium clone Gibbet Hill soil SIP 44 (AY934460) Uncultured bacterium clone Tocil Wood soil 2 (AY934446]) Uncultured marine bacterium clone 28 (AY439204) Uncultured marine bacterium clone 20 (AY439202) Roseovarius pacificus 81-2 (SAMN05444398 12431) Uncultured bacterium beech soil 7 (LT674497.1) Celeribacter indicus P73 (P73 3965_ID:30976086)

Figure 8 . 6

 86 Figure8.6 (see p.164) Phylogenetic trees of partial 16S rRNA and partial protein CmuA sequences associated with chloromethane-degradation of cultivated strains and environmental samples. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model[START_REF] Jones | The rapid generation of mutation data matrices from protein sequences[END_REF]. Bootstrap values were calculated from 1000 replicates,% values ≥ 50 are presented. Analyses were conducted using the software MEGA7[START_REF] Kumar | MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets[END_REF]. Sequences of experimentally validated chloromethanedegraders are written in bold. Sequences marked with a 13 C icon result from DNA-SIP ( 13 CH 3 Cl) studies in soil microcosms from a deciduous German forest dominated by beech (16S RNA: LT674486.1 to LT674490.1; CmuA: LT674491.1 to LT674498.1 in[START_REF] Chaignaud | Methanol consumption drives the bacterial chloromethane sink in a forest soil[END_REF]), clone libraries from Californian agricultural soils (16S RNA: AY439185.1 to AY439194.1; CmuA: AY439202 to AY439204 in[START_REF] Miller | Degradation of methyl bromide and methyl chloride in soil microcosms: Use of stable C isotope fractionation and stable isotope probing to identify reactions and the responsible microorganisms[END_REF]), and CmuA from clone libraries from English woodland soil (AY934428 to AY934484)[START_REF] Borodina | Use of DNA-stable isotope probing and functional gene probes to investigate the diversity of methyl chloride-utilizing bacteria in soil[END_REF]. (A) 16S rRNA tree based on 197 nucleotide sequences. Thermotoga maritima sequence served as outgroup. (B) CmuA tree based on 60 amino acid sequences. Oceanicola batsensis HTCC2597 and Pelagibaca bermudensis HTCC2601 sequences served as outgroup. Pink triangles indicate sequences retrieved from metagenome JGI database. Condensed branch of 11 sequences in cluster 1 (uncultured bacterium clone Warwickshire enrichment 3 (AY934432), 25 (AY934433), 36 (AY934430) and 42 (AY934428); Uncultured bacterium clone Warwickshire garden soil 37 (AY934481); Uncultured bacterium clone Gibbet Hill soil 9 (AY934448), 16 (AY934452), and 37 (AY93448); Uncultured bacterium clone Tocil Wood soil 17 (AY934484), 25 (AY934444), and 38 (AY934439)). Condensed branch of 4 marine sediment metagenome sequences in cluster 2 (GAF77597.1, GAF84365.1, GAG35382.1, GAG79179.1 and GAI71391.1).

  Figure8.6 (see p.164) Phylogenetic trees of partial 16S rRNA and partial protein CmuA sequences associated with chloromethane-degradation of cultivated strains and environmental samples. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model[START_REF] Jones | The rapid generation of mutation data matrices from protein sequences[END_REF]. Bootstrap values were calculated from 1000 replicates,% values ≥ 50 are presented. Analyses were conducted using the software MEGA7[START_REF] Kumar | MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets[END_REF]. Sequences of experimentally validated chloromethanedegraders are written in bold. Sequences marked with a 13 C icon result from DNA-SIP ( 13 CH 3 Cl) studies in soil microcosms from a deciduous German forest dominated by beech (16S RNA: LT674486.1 to LT674490.1; CmuA: LT674491.1 to LT674498.1 in[START_REF] Chaignaud | Methanol consumption drives the bacterial chloromethane sink in a forest soil[END_REF]), clone libraries from Californian agricultural soils (16S RNA: AY439185.1 to AY439194.1; CmuA: AY439202 to AY439204 in[START_REF] Miller | Degradation of methyl bromide and methyl chloride in soil microcosms: Use of stable C isotope fractionation and stable isotope probing to identify reactions and the responsible microorganisms[END_REF]), and CmuA from clone libraries from English woodland soil (AY934428 to AY934484)[START_REF] Borodina | Use of DNA-stable isotope probing and functional gene probes to investigate the diversity of methyl chloride-utilizing bacteria in soil[END_REF]. (A) 16S rRNA tree based on 197 nucleotide sequences. Thermotoga maritima sequence served as outgroup. (B) CmuA tree based on 60 amino acid sequences. Oceanicola batsensis HTCC2597 and Pelagibaca bermudensis HTCC2601 sequences served as outgroup. Pink triangles indicate sequences retrieved from metagenome JGI database. Condensed branch of 11 sequences in cluster 1 (uncultured bacterium clone Warwickshire enrichment 3 (AY934432), 25 (AY934433), 36 (AY934430) and 42 (AY934428); Uncultured bacterium clone Warwickshire garden soil 37 (AY934481); Uncultured bacterium clone Gibbet Hill soil 9 (AY934448), 16 (AY934452), and 37 (AY93448); Uncultured bacterium clone Tocil Wood soil 17 (AY934484), 25 (AY934444), and 38 (AY934439)). Condensed branch of 4 marine sediment metagenome sequences in cluster 2 (GAF77597.1, GAF84365.1, GAG35382.1, GAG79179.1 and GAI71391.1).

Table 8 .1

 8 Chloromethane-degrading bacteria isolated from contrasting environments

	Name (taxonomical class)	Origin	Metabolism/trophic type	Genome	Comments
	Acetobacterium dehalogenans MC (Clostridia)	Activated sludge (Traunecker et al., 1991)	Anaerobic homoacetogenic	ns a	Chloromethane dehalogenation forms methyl-H 4 F (Meßmer et al., 1996)
	Acetobacterium sp. in mixed culture RM (with Candidatus Dichloromethanomonas elyunquensis)	Pristine river sediment (Justicia-Leon et al., 2012)	Anaerobic hydrogenotrophic chloromethane-degradation	ns	H 2 -dependent chloromethane degradation (Chen et al., 2017)
	Aminobacter ciceronei IMB1 (Alphaproteobacteria)	Fumigated strawberries 1998) (Hancock et al.,	Aerobic facultative methylotroph	ns	cmuA b
	Aminobacter lissarensis CC495 (Alphaproteobacteria)	Beech woodland soil (Coulter et al., 1999)	Aerobic facultative methylotroph	ns	cmuA b
	Celeribacter indicus P73 (Alphaproteobacteria)	Deep-sea sediment (Lai et al., 2014)	Aerobic facultative methylotroph (genome-based) c	4.5 Mb, five plasmids; G + C content 66% (Cao et al., 2015)	Genome-based detection of cmu genes; 3 folD-purU copies (This work)
	Hyphomicrobium sp. strains AT2, AT3, AT4 (Alphaproteobacteria)	Phyllosphere of Arabidopsis thaliana (Nadalig et al., 2011)	Aerobic facultative methylotroph	ns	cmuA b
	Hyphomicrobium sp. MC1 (Alphaproteobacteria)	Industrial sewage plan (Hartmans et al., 1986)	Aerobic facultative methylotroph	4.7 Mb; G + C content 59% 2011) (Vuilleumier et al.,	Complete cmu pathway
	Hyphomicrobium sp. MC2 (Alphaproteobacteria)	Soil from a petrochemical al., 1996) factory (Doronina et	Aerobic facultative methylotroph	ns	cmuA b
	Leisingera methylohalidovorans MB2 (Alphaproteobacteria)	Marine tide pool (Schaefer et al., 2002)	Aerobic facultative methylotroph	4.6 Mb, two plasmids; G + C content 62% (Buddruhs et al., 2013)	Uncharacterized cmu-independent chloromethane degradation
	Methylomicrobium album BG8 (Gammaproteobacteria)	Freshwater (Whittenbury et al., 1970)	Aerobic facultative methanotroph, obligatory methylotroph	4.5 Mb, one plasmid; G + C content 56% (Kits et al., 2013)	Low chloromethane concentrations enhance growth on methanol (Han and Semrau, 2000). No cmu gene
	Methylorubrum d extorquens CM4 (Alphaproteobacteria)	Soil from a petrochemical factory (Doronina et al., 1996)	Aerobic facultative methylotroph	6.2 Mb, two plasmids; G + C content 68% (Marx et al., 2012)	Plasmid pCMU01-encoded cmu (Roselli et al., 2013)
	Nocardioides sp. SAC-4 (Actinobacteria)	Topsoil (McAnulla et al., 2001a)	Aerobic facultative methylotroph e	ns
	OM43 bacterioplankton clade, strain HTCC2181 (Betaproteobacteria)	Seawater (Giovannoni et al., 2008)	Obligate methylotroph	1.3 Mb (three contigs); G + C content 38% (Giovannoni et al., 2008)	cmu-independent chloromethane use as energy source if methanol is the C source (Halsey et al., 2012)

Date: 12:26 Friday 15 March 2019 File: Methylotrophs 3P Bacterial Chloromethane Degradation | 155

Table 8 .1 Continued

 8

	A	CH 3 Cl	CmuAB HCl H 4 F	MetF2 H 4 F CH 3
							H 4 F CH 2	H 4 F CH FolD	FolD	H 4 F CHO	PurU	HCOO -	CO 2 + Energy
				CO 2 Serine	H 4 F
								cycle
									Biomass
	B	CH 3 Cl	H 4 F	HCl		?
						H 4 F CH 2	H 4 F CH	H 4 F CHO	HCOO -	CO 2 + Energy
					H 4 F	Spont.	H 4 F
		CH 3 OH				CH 2 O
				CO 2 RuMP
						pathway
									Biomass
	C	CH 3 Cl	pMMO O 2	HCl Cl CH 2 OH
								Spont.
		CH 3 OH	CO 2 MDH			RuMP CH 2 O	FADH	HCOO -	CO 2 + Energy
						pathway
									Biomass
	D					H 4 F CH 2	H 4 F CH	H 4 F CHO	HCOO -
		CH 3 Cl	HCl ? ?	H 4 F CH 3	Biomass	H 4 F	CO 2
						CH 3 [Co]-E	CH 3 CO-SCoA	CH 3 COO -
		Acetobacterium sp.			CO	H
		CH 2 Cl 2	? ?	H 4 F CH 2	H 4 F CH	H 4 F CHO	HCOO -	H 2	CO 2
			HCl		H 4 F CH 3	Biomass	H 4 F	e-, H +	CO 2
						CH 3 [Co]-E	CH 3 CO-SCoA	CH 3 COO -
		Candidatus Dichloromethanomonas elyunquensis CO

UNCORRECTED PROOF Date: 12:26 Friday 15 March 2019 File: Methylotrophs 3P 2 Figure 8.2 Examples of methylotrophic pathways for chloromethane dissimilation (source of energy, in red) and assimilation (source of carbon for biomass, in blue) in cultivated strains and mixed cultures. Bacteria cultivated under aerobic (A to C) and anaerobic (D) conditions. Abbreviations: FADH, formaldehyde dehydrogenase; H 4 F, tetrahydrofolate; MDH, methanol dehydrogenase; pMMO, particulate methane monooxygenase; RuMP, ribulose monophosphate pathway. (A) The cmu pathway characterized in details in M. extorquens CM4 and Hyphomicrobium strains, adapted from

Table 8 .

 8

	2 Degradation of chloromethane and its isotopic enrichment factor for carbon (ε C ) and hydrogen (ε H ) of pure cultures and environmental samples
	Stable isotope enrichment factor for chloromethane upon bacterial degradation a
	Test condition	ε C (‰)	ε H (‰)	Reference

Pure bacterial cultures (cmu pathway)

	Aminobacter ciceronei IMB1	Resting cells, 26°C	-47 ± 4	Nd b	Miller et al. (2001)
	Aminobacter lissarensis CC495	Resting cells, 26°C	-42 ± 2	Nd	Miller et al. (2001)
	Methylorubrum extorquens CM4	Dividing cells, 30°C	-42	-39	Nadalig et al. (2014)
		Resting cells, 30°C	-41 ± 5	-29 ± 6	Nadalig et al. (2013)
	Hyphomicrobium sp. MC1	Dividing cells, 30°C	-54	-51	Nadalig et al. (2014)
		Resting cells, 30°C	-38 ± 3	-27 ± 10	Nadalig et al. (2013)
	Pure bacterial cultures (cmu-independent pathway)
	Leisingera methylohalidovorans MB2	Dividing cells, 30°C	-76	0	Nadalig et al. (2014)
		Resting cells, 26°C	-44 ± 4	Nd	Miller et al. (2001)
	Environmental samples
	Phyllosphere (fern: Cyathea cooperi)	Microcosm, 20-30°C	-43 ± 12	-8 ± 19	Jaeger et al. (2018b)
	Soils (agricultural, grassland, forest soils)	Microcosm, 20-30°C	-38 to -11 ± 3 -50 ± 19	Jaeger et al. (2018a)

a Increased carbon isotope fractionation compared with hydrogen isotope fractionation most likely results from the primary isotope effect in cleavage of the carbon-halogen bound during chloromethane dehalogenation, as previously suggested

[START_REF] Elsner | A new concept linking observable stable isotope fractionation to transformation pathways of organic pollutants[END_REF]

); b Nd, not determined. UNCORRECTED PROOF Date: 12:26 Friday 15 March 2019 File: Methylotrophs 3P Bacterial Chloromethane Degradation | 159

Table 8 .3

 8 Occurrence of cmuA sequences in metagenomes available from public databases

	Database	Environment	Metagenome numbers	Total size (Mb) a cmuA hit b
	IMG	Engineered	2332	254,327
		Bioreactor	108	35,565	0
		Bioremediation	68	12,879	0
		Biotransformation	24	5411	0
		Build environment	1231	31,275	0
		Food production	3	6,600	0
		Lab enrichment	218	11,002	0
		Lab synthesis	4	401	0
		Modelled	65	2804	0
		Solid waste	69	22,575	0
		Wastewater	542	132,349	0
		Aquatic	9322	1,968,340
		Freshwater	3912	755,498	0
		Marine	3034	1,049,257	1 (wetland)
		Non-marine saline and alkaline	702	86,625	0
		Sediment	271	34,754	0
		Thermal spring	1403	42,206	0
		Terrestrial	2915	1,508,938
		Deep subsurface	183	13,202	0
		Geologic	4	170	0
		Oil reservoir	3	283	0
		Peat	56	10,558	0
		Plant litter	47	2663	0
		Rock-dwelling	8	3220	0
		Soil	2609	1,481,714	0
		Volcanic	5	26	0
		Host-associated	4106	876,365
		Algae	64	44,092	0
		Animal	25	6426	0
		Annelida	91	43,120	0
		Arthropoda	147	73,213	0
		Birds	18	5505	0
		Cnidaria	36	723	0
		Fish	4	489	0
		Fungi	102	19,978	0
		Human	2489	139,540	0
		Insecta	34	3153	0
		Invertebrate	15	8056	0
		Mammal	267	104,135	0
		Microbial	24	2132	0

Date: 12:26 Friday 15 March 2019 File: Methylotrophs 3P Bacterial Chloromethane Degradation | 163

  Total of 4,783,777 Mb publicly available in May 2018; b Blast searches were performed using a cmuA consensus template from 122 nucleic acid sequences downloaded from GenBank. Hits displaying an E-value lower than 10 -5 were kept, and the corresponding proteins aligned with characterized CmuA sequences to only retain members closely related to one of three clades shown in Figure8.6B.

	Table 8.3 Continued
	Database	Environment	Metagenome numbers	Total size (Mb) a cmuA hit b
		Mollusca	11	3247	0
		Plants	761	581,931	3 (Arabidopsis thaliana)
		Porifera	10	1642	0
		Tunicates	8	3377	0
	NCBI	Diverse	Metagenome BLAST database
		Marine		81,542	7 (sediment)
		Freshwater, hydrothermal vent, human gut		94,265	0
	UNCORRECTED PROOF

a Date: 12:26 Friday 15 March 2019 File: Methylotrophs 3P

  . Sequences of experimentally validated chloromethanedegraders are written in bold. Sequences marked with a 13 C icon result from DNA-SIP ( 13 CH 3 Cl) studies in soil microcosms from a deciduous German forest dominated by beech (16S RNA: LT674486.1 to LT674490.1; CmuA: LT674491.1 to LT674498.1 in[START_REF] Chaignaud | Methanol consumption drives the bacterial chloromethane sink in a forest soil[END_REF]), clone libraries from Californian agricultural soils (16S RNA: AY439185.1 to AY439194.1; CmuA: AY439202 to AY439204 in[START_REF] Miller | Degradation of methyl bromide and methyl chloride in soil microcosms: Use of stable C isotope fractionation and stable isotope probing to identify reactions and the responsible microorganisms[END_REF]), and CmuA from clone libraries from English woodland soil (AY934428 to AY934484)

Date: 12:26 Friday 15 March 2019 File: Methylotrophs 3P