Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912Society for Applied Microbiology and Blackwell Publishing Ltd, 20057 913181328Original ArticleStable isotope probing and methyl chloride utilizationE. Borodina, M. J.
    Cox, I. R. McDonald and J. C. Murrell
    
    Environmental Microbiology (2005) 7(9), 1318–1328
    
    doi:10.1111/j.1462-2920.2005.00819.x
    
    Use of DNA-stable isotope probing and functional gene probes to investigate the diversity of methyl chloride-utilizing bacteria in soil
    Elena Borodina1, Michael J. Cox1, Ian R. McDonald2 and J. Colin Murrell1* 1 Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. 2 Department of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton, New Zealand. Summary Enrichment and isolation of methyl chloride-utilizing bacteria from various terrestrial environments, including woodland and forest soils, resulted in the identification of seven methyl chloride-utilizing strains belonging to the genus Hyphomicrobium, an Aminobacter strain TW23 and strain WG1, which grouped closely with the genus Mesorhizobium. Methyl chloride enrichment cultures were dominated by Hyphomicrobium species, indicating that these bacteria were most suited to growth under the enrichment and isolation conditions used. However, the application of culture-independent techniques such as DNA-stable isotope probing and the use of a functional gene probe targeting cmuA, which encodes the methyltransferase catalysing the first step in bacterial methyl chloride metabolism, indicated a greater diversity of methyl chloride-utilizing bacteria in the terrestrial environment, compared with the diversity of soil isolates obtained via the enrichment and isolation procedure. It also revealed the presence of as yet uncultured and potentially novel methyl chloridedegrading bacteria in soil. Introduction Emission of methyl chloride (CH3Cl) and methyl bromide (CH3Br) into the atmosphere results in transport to the stratosphere of bromine and chlorine atoms, which contribute to catalytic ozone destruction (Mellouki et al., 1992; Harper, 2000). CH3Cl is by far the most abundant volatile halocarbon in the atmosphere, with an average concentration of 500–600 pptv (parts per trillion by volume) and is responsible for around 17% of chlorine-catalysed ozone destruction (Harper and Hamilton, 2003; Harper et al., 2003). CH3Cl is mainly of natural origin and current sources identified include biomass burning, oceans, salt marshes, wood-rotting fungi, higher plants, coal combustion and other industrial emissions (Moore et al., 1996; Watling and Harper, 1998; Keene et al., 1999; Khalil et al., 1999; Lobert et al., 1999; Rhew et al., 2000; Yokouchi et al., 2002). One of the CH3Cl production processes is from Cl– during combustion of vegetation, from forest fires and slash-and-burn agriculture (Lobert et al., 1999). In the marine environment, CH3Cl is formed by the reaction of CH3I, derived from macroalgae, with Cl– in seawater (Moore et al., 1996). Current evidence indicates that the majority of the atmospheric CH3Cl burden is of direct biological origin and biosynthesis of CH3Cl has been demonstrated in a wide variety of organisms (Watling and Harper, 1998; Khalil et al., 1999; Yokouchi et al., 2002). CH3Br accounts for up to 10% of stratospheric ozone destruction and its use as a fumigant is being phased out by 2010 under amendments to the Montreal Protocol (United Nations, 1997). Natural sources of CH3Br include macroalgae, phytoplankton, fungi, higher plants and wetlands (Scarratt and Moore, 1996; Itoh et al., 1997; Gan et al., 1998; Varner et al., 1999; Rhew et al., 2000). The dominant loss process for both CH3Cl and CH3Br is via reaction with OH radicals in the troposphere. The kinetic isotope effect for reaction of CH3Cl with OH radicals is unknown (Thompson et al., 2002). Another significant sink for both methyl halides is soils, where they are degraded by methylotrophic bacteria and nitrifiers (Messmer et al., 1993; Doronina et al., 1996; Miller et al., 1997; 2001; 2004; Hines et al., 1998; Connell-Hancock et al., 1998; Coulter et al., 1999; Varner et al., 1999; Khalil and Rasmussen, 1999; Duddleston et al., 2000; McAnulla et al., 2001a; Goodwin et al., 2001; Thompson et al., 2002). Methyl halide-utilizing bacteria appear to be ubiquitous in the environment and have been isolated from a range of pristine sites including terrestrial, freshwater, estuarine and marine environments. The majority of bacteria that use methyl halides as a carbon and energy source are aerobic methylotrophs. Trotsenko and colleagues were the
    
    Received 23 November, 2004; accepted 8 March, 2005. *For correspondence. E-mail j.c.murrell@warwick.ac.uk; Tel. (+44) 24 7652 3553; Fax (+44) 24 7652 3568.
    
    © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd
    
    Stable isotope probing and methyl chloride utilization 1319 first to isolate CH3Cl-utilizing bacteria from industrially contaminated Russian soils (Doronina et al., 1996). Two strains, Hyphomicrobium chloromethanicum CM2 and Methylobacterium chloromethanicum CM4, are the most extensively characterized CH3Cl utilizers and have been used in genetic and biochemical studies to investigate CH3Cl utilization (Vannelli et al., 1998; 1999; Studer et al., 1999; 2001; 2002; Woodall, 2000; McAnulla et al., 2001b; Borodina et al., 2004). Two more facultative methylotrophs capable of growth on methyl halides were subsequently isolated from different terrestrial environments and were found to be Aminobacter species; Aminobacter strain IMB1 from agricultural soil fumigated with CH3Br in California (Miller et al., 1997; Connell-Hancock et al., 1998; Schaefer and Oremland, 1999) and Aminobacter strain CC495 from woodland soil in Northern Ireland (Coulter et al., 1999). Leisingera methylohalidivorans MB2 is a marine isolate which is also capable of growth on methyl halides (Goodwin et al., 2001; Schaefer et al., 2002). All five methyl halide utilizers described above grow on CH3Cl. Hyphomicrobium chloromethanicum CM2, L. methylohalidivorans MB2 and the two Aminobacter strains IMB1 and CC495 also grow on CH3Br. All four CH3Clutilizing terrestrial isolates were found to possess the gene cmuA, along with other CH3Cl-specific genes within their cmu (chloromethane utilization) gene clusters (Coulter et al., 1999; Vannelli et al., 1999; McAnulla et al., 2001b; Woodall et al., 2001; McDonald et al., 2002). CmuA is a methyltransferase I, which catalyses the key initial dehalogenation step in the bacterial CH3Cl oxidation pathway. This 67 kDa polypeptide is induced by CH3Cl or CH3Br and consists of a methyltransferase domain and a corrinoid-binding domain (Vannelli et al., 1998; Coulter et al., 1999; McAnulla et al., 2001b; Studer et al., 2001; Woodall et al., 2001). This unique feature was exploited for designing polymerase chain reaction (PCR) primer sets which amplify a fragment of cmuA spanning both domains (McAnulla et al., 2001a; Miller et al., 2004). These PCR primers have been used successfully to detect cmuA in CH3Cl- and CH3Br-utilizing strains, in enrichment cultures and in DNA extracted from soils and the marine environment (Woodall, 2000; McAnulla et al., 2001a; McDonald et al., 2002; Miller et al., 2004; Schäfer et al., 2005). In a study by McAnulla and colleagues (2001a), soil enrichments for CH3Cl utilizers were dominated by Hyphomicrobium species. Of eight isolates, six were identified as cluster II Hyphomicrobium species (S3, S4, MAR1, PMC, SAC1 and SAN1). Strain CMC grouped closely with CH3Cl-utilizing isolates within the Aminobacter genus and strain SAC4 was a Gram-positive isolate which grouped within Nocardioides (McAnulla et al., 2001a). In order to assess the distribution and diversity of CH3Cl degraders in the terrestrial environment, we employed both culture-dependent and culture-independent approaches. The enrichment and isolation of CH3Cl-utilizing bacteria from soil from various locations indicated the diversity and ease of cultivation of terrestrial CH3Cldegrading bacteria. A molecular ecological approach was used to analyse total diversity of CH3Cl-specific cmuA sequences in clone libraries constructed using DNA extracted directly from soils and DNA-based stable isotope probing (SIP) was applied for the first time to specifically examine the bacterial population actively utilizing CH3Cl in pristine soil samples. Stable isotope probing exploits the fact that DNA of a bacterium growing on a 13 C-enriched carbon source becomes ‘heavy’ (13Clabelled), enabling it to be resolved from the total community DNA by density gradient centrifugation. The ability to isolate DNA from bacteria actively metabolizing 13CH3Cl facilitates characterization of the microbial population involved via molecular biological analysis of 13C-labelled DNA. DNA-SIP has been used previously to characterize bacteria metabolizing methane, methanol and methyl halides in soil microcosms (Radajewski et al., 2000; 2002; Morris et al., 2002; Hutchens et al., 2004; Lin et al., 2004; Miller et al., 2004). We report here on the identification of cmuA sequences which probably represent the active populations of methyl halide utilizers that metabolize elevated CH3Cl concentrations in pristine woodland soil.
    
    Results Enrichment and isolation of CH3Cl-utilizing bacteria from soil A total of 83 enrichments were set up with CH3Cl. Soil samples (topsoil) were obtained from four locations: a garden soil (Warwickshire); Tocil Wood soil (University of Warwick); Gibbet Hill Wood soil (University of Warwick) and garden soil (Riga, Latvia). Tocil Wood and Gibbet Hill Wood samples were typical woodland soils covered with leaf-litter; all sampling sites were located in the middle of England or Latvia, well away from the coast and had not been previously exposed to unnatural levels of methyl halides. Sixty enrichments were set up with two different concentrations of CH3Cl (1–2% v/v) in the headspace, different culture media (Paraccocus versutus medium or ammonium nitrate mineral salts, ANMS) and different temperatures (10∞C, 20∞C or 30∞C) to provide a range of growth conditions to facilitate enrichment of CH3Cl utilizers. Thirteen further enrichments were set up with 0.2% (v/v) CH3Br and variable culture conditions as described in Experimental procedures. Pasteurization (10 enrichment cultures) was attempted to aid in selection of possible Gram-positive spore-forming CH3Cl utilizers. Regardless of the enrichment conditions, it was observed that initial enrichments and first subcultures contained a
    
    © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1318–1328
    
    1320 E. Borodina, M. J. Cox, I. R. McDonald and J. C. Murrell mixture of cell types (ovoid cells, rods, cocci in chains or separate) with Gram-negative bacteria being present in larger numbers than Gram-positive bacteria. After further subculturing, cultures became dominated by Gramnegative prosthecate bacteria characteristic of hyphomicrobia. In a similar study, McAnulla and colleagues (2001a) also reported the predominance of Hyphomicrobium species in soil CH3Cl enrichments. Enrichments with pasteurized soil samples were dominated by Gram-negative bacteria, indicating that pasteurization was not an effective selection method for Gram-positive CH3Cl utilizers. Nineteen enrichments which actively consumed CH3Cl after two subcultures were plated out for the isolation of CH3Cl utilizers. Nine Gram-negative isolates which grew on CH3Cl were obtained from the sampling sites. Eight strains were isolated from CH3Cl enrichments and strain TW23 was isolated on CH3Br. Phylogenetic analysis of novel isolates 16S rRNA genes were amplified by PCR from each of the isolates and partially sequenced (1377 bp) (Fig. 1). All nine isolates belonged to the a-proteobacteria and grouped with the previously isolated CH3Cl utilizers. Four CH3Cl isolates originating from Tocil Wood (TW4, TW5, TW28, TW30) and one from Gibbet Hill Wood (MW1) grouped together within cluster II of the genus Hyphomicrobium, with identities of 97.5–97.7% to H. chloromethanicum CM2; thus confirming that the enrichments were dominated by Hyphomicrobium species (Fig. 1). The isolate from Latvian garden soil (LAT3) grouped in a separate cluster represented by three Hyphomicrobium strains: the dimethylsulfone utilizer, H. sulfonivorans; the CH3Cl utilizer Hyphomicrobium strain PMC, from Tocil Lake (University of Warwick) (McAnulla et al., 2001a); and strain LAT3 obtained in this study (Fig. 1). Only the isolate from Warwickshire garden soil (WG6) did not branch with any of the previously isolated CH3Cl-utilizing hyphomicrobia and was less closely related to known Hyphomicrobium species, with identities of 90.0%, 93.7% and 95.9% to H. chloromethanicum CM2, H. vulgare and Hyphomicrobium strain LAT3 respectively. Tocil Wood isolate TW23 had 99.6% identity to the previously isolated CH3Cl utilizers, Aminobacter strains IMB1 and CC495 (Fig. 1). Warwickshire garden isolate WG1 had 98.6% identity to the 16S rRNA gene of a-proteobacterium ‘Mena25/4-1’ and was also closely related to Mesorhizobium species (Fig. 1). Strain ‘Mena25/4-1’ was isolated from enrichment
    
    Hyphomicrobium strain S4 Hyphomicrobium strain S3 Hyphomicrobium strain MW1 Hyphomicrobium strain TW28 Hyphomicrobium strain TW4 Hyphomicrobium strain TW5 Hyphomicrobium chloromethanicum strain CM2 Hyphomicrobium strain TW30 Hyphomicrobium strain SAC1 Hyphomicrobium strain SAN1 Hyphomicrobium facilis Hyphomicrobium methylovorum Hyphomicrobium vulgare Pedomicrobium fusiforme Hyphomicrobium strain PMC Hyphomicrobium strain LAT3 Hyphomicrobium sulfonivorans Pedomicrobium australicum Pedomicrobium manganicum Hyphomicrobium strain WG6 Methylobacterium chloromethanicum strain CM4 Methylobacterium extorquens Methylosinus trichosporium Aminobacter aminovorans Aminobacter strain CC495 Aminobacter aganoensis Aminobacter strain IMB1 Aminobacter strain TW23 Sinorhizobium meliloti alpha proteobacterium 'Mena 25/4-1' strain WG1 Mesorhizobium tianshanense Mesorhizobium loti Mesorhizobium loti Mesorhizobium ciceri Mesorhizobium sp. 4FB11 Leisingera methylohalidivorans strain MB2 Ruegeria algicola Roseobacter gallaeciensis Paracoccus denitrificans 0.10
    
    Fig. 1. Phylogenetic analysis of the 16S rRNA sequences of the novel methyl halide-utilizing bacterial isolates. Hyphomicrobium chloromethanicum CM2, M. chloromethanicum CM4, Aminobacter strains IMB1 and CC495 and L. methylohalidivorans MB2, and methyl halide utilizers (Hyphomicrobium strains S3, S4, MAR1, SAC1, SAN1 and PMC) from the study by McAnulla and colleagues (2001a) are also included in the phylogenetic analysis. 16S rRNA sequences from methyl halide-utilizing bacterial strains isolated in this study are indicated in bold. 1377 bp of sequence between positions 83 and 1460 (Escherichia coli numbering) were analysed using the AXML program of ARB. The available sequence of Hyphomicrobium strain TW30 was shorter than this (729 bp; between positions 398 and 1127) and was added to the tree by a parsimony method. Bootstrap values were calculated from 100 replicates using the DNAPARS program of PHYLIP through ARB; the NEIGHBOUR program of PHYLIP was also used with the Kimura 2–Parameter correction and was in agreement with the parsimony analysis. Bootstrap values of > 95% are shown as filled circles and those between 75% and 95% as unfilled circles. The scale bar indicates 10% sequence divergence. © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1318–1328
    
    Stable isotope probing and methyl chloride utilization 1321 cultures on 6-methyl-nicotinic acid (Tinschert et al., 1997) and was phylogenetically related to Rhizobium species but has not been described in detail. Strain WG1 was also 97.3% identical to the CH3Cl-utilizing Aminobacter strains IMB1 and CC495. In our study, CH3Cl-utilizing Hyphomicrobium species were obtained from four different geographical environments and the dominance and competitiveness of Hyphomicrobium species during the enrichment process was evident. This did not necessarily reflect the full diversity of CH3Cl-utilizing bacteria in natural environments as the presence of other CH3Cl degraders was likely to have been masked by the fast-growing and more easily culturable Hyphomicrobium species. Analysis of cmuA sequences from novel isolates Polymerase chain reaction primer set cmuA802FcmuA1609R (Miller et al., 2004) was used to amplify cmuA from seven CH3Cl utilizers isolated in this study and from Hyphomicrobium strains S4, SAC1 and PMC previously isolated by McAnulla and colleagues (2001a). All strains yielded a PCR product of the correct size (808 bp) which was subsequently cloned, sequenced and phylogenetically analysed at the DNA level. Partial cmuA sequence analysis (758 bp) showed that cmuA sequences from phylogenetically similar Hyphomicrobium strains (TW4, TW5, TW28, TW30 and S4) have high sequence identity (> 99%) to the cmuA sequence of H. chloromethanicum CM2. These clustered together (Fig. 2) in a similar manner to the 16S rRNA sequences of those strains (Fig. 1). The cmuA sequence from Hyphomicrobium strain SAC1 had 95% identity to the corresponding cmuA sequence from H. chloromethanicum CM2 and was placed on a separate branch away from cmuA sequences of the cluster II Hyphomicrobium group, containing H. chloromethanicum CM2. Sequences of cmuA from Hyphomicrobium strains PMC and LAT3 grouped together and formed a separate cluster distant from all the other Hyphomicrobium cmuA sequences (Fig. 2). Analysis of the cmuA sequence from the novel CH3Clutilizing Aminobacter isolate TW23 indicated 99.5% identity (at the DNA level) to the sequence from Aminobacter strain IMB1 (Fig. 2). Analysis of cmuA and 16S rRNA sequences from novel isolates indicated that cmuA tree phylogeny (Fig. 2) of the novel isolates and the extant CH3Cl-utilizing strains was generally congruent with their 16S rRNA phylogeny (Fig. 1), except for the isolate WG1, which grouped with the Mesorhizobium species on the basis of 16S rRNA sequence analysis. The sequences of cmuA from strain WG1 grouped with the cmuA sequence from Aminobacter strain CC495 (Fig. 2). Analysis of cmuA sequences from soil To overcome the bias observed towards more easily cultivated bacteria during the enrichment process, a cultureindependent approach was employed to investigate the total diversity of CH3Cl-specific gene sequences (cmuA) present in soil. It has become apparent that only a small proportion (approximately 1–10%) of bacteria in the environment can be cultured (Marchesi and Weightman, 2003). Hence, the bacteria that have been extensively studied are those that are easily isolated from the environment, and not necessarily those that are the most environmentally relevant (Watanabe and Baker, 2000; Marchesi and Weightman, 2003). In this study, the environmental cmuA gene pool was isolated by direct PCR amplification from soil DNA and compared with the cmuA
    
    Hyphomicrobium strain TW28 Hyphomicrobium strain TW5 Hyphomicrobium strain S4 Hyphomicrobium chloromethanicum strain CM2 Hyphomicrobium strain TW4 Hyphomicrobium strain TW30 Hyphomicrobium strain SAC1 Hyphomicrobium strain LAT3 Hyphomicrobium strain PMC Aminobacter strain IMB1 Aminobacter strain TW23 Marine strain 179 Ruegeria strain 198 Aminobacter strain CC495 strain WG1 Methylobacterium chloromethanicum CM4 0.10
    
    Fig. 2. Maximum-likelihood DNA tree of cmuA sequences amplified from the novel methyl halide-utilizing bacterial isolates. Sequences from previously isolated methyl halide-utilizing bacteria, H. chloromethanicum CM2; M. chloromethanicum CM4; Aminobacter strains IMB1 and CC495; Hyphomicrobium strains S4, SAC1 and PMC from the study by McAnulla and colleagues (2001a) are included in the analysis. cmuA sequences from methyl halide-utilizing bacterial strains isolated in this study are indicated in bold. 758 bp of sequence between positions 826 and 1584 (M. chloromethanicum numbering) were analysed using the AXML program of ARB. Bootstrap values were calculated from 100 replicates using the DNAPARS program of PHYLIP through ARB; the NEIGHBOUR program of PHYLIP was also used with the Kimura 2–Parameter correction and was in agreement with the parsimony analysis. Bootstrap values of > 95% are shown as filled circles and those between 75% and 95% as unfilled circles. The scale bar indicates 10% sequence divergence. © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1318–1328
    
    1322 E. Borodina, M. J. Cox, I. R. McDonald and J. C. Murrell sequences amplified from the CH3Cl enrichment cultures and extant CH3Cl utilizers. Clone libraries (50 clones per library) were constructed from the DNA samples extracted from Tocil Wood soil (TWsoil), Gibbet Hill Wood soil (GHsoil) and Warwickshire garden soil (WGsoil). One cmuA library was also prepared using an enrichment culture of Warwickshire garden soil (WGenri). Restriction fragment length polymorphism (RFLP) analysis of TWsoil cmuA clones identified four operational taxonomic units (OTUs). Of these, OTU1 contained 43 clones and the remaining three OTUs had one or more clones. TWsoil cmuA sequences from OTU1, OTU3 and OTU4 grouped together within clade A, consisting entirely of the soil environmental cmuA clones obtained in this study (Fig. 3). Clade A is distinct from the cmuA sequences of Hyphomicrobium and Aminobacter strains, with identities of approximately 79.5–80.9% to the corresponding cmuA sequence from H. chloromethanicum CM2 and 78.6–87.6% to cmuA from Aminobacter strain
    
    Warwickshire Enrichment Clone 17 Warwickshire Enrichment Clone 25 Warwickshire Enrichment Clone 37 Gibbet Hill Soil Clone 4 Gibbet Hill Soil Clone 7 Tocil Wood Soil Clone 33 Warwickshire Garden Soil Clone 45 Warwickshire Garden Soil Clone 37 Warwickshire Enrichment Clone 14 Tocil Wood Soil Clone 3 Warwickshire Enrichment Clone 6 Tocil Wood Soil Clone 17 Warwickshire Enrichment Clone 42 Warwickshire Enrichment Clone 32 Warwickshire Garden Soil Clone 38 Warwickshire Garden Soil Clone 41 Gibbet Hill Soil Clone 14 Gibbet Hill Soil Clone 37 Gibbet Hill Soil Clone 16 Tocil Wood Soil Clone 38 Tocil Wood Soil Clone 25 Warwickshire Enrichment Clone 3 Gibbet Hill Soil Clone 38 Gibbet Hill Soil Clone 40 Gibbet Hill Soil Clone 9 Tocil Wood Soil Clone 10 Warwickshire Enrichment Clone 20 Warwickshire Enrichment Clone 10 Warwickshire Enrichment Clone 36 Tocil Wood Soil Clone 32 Tocil Wood Soil Clone 2 SIPGH22 SIPGH43 SIPGH7 Marine strain 179 Ruegeria strain 198 Miller MeCl1 Miller MeCl20 Miller MeCl28 Aminobacter strain IMB-1 Aminobacter strain TW23 Miller MeCl44 Miller MeCl5 Tocil Wood Soil Clone 19 Warwickshire Enrichment Clone 12 Warwickshire Enrichment Clone 5 Miller MeCl47 Tocil Wood Soil Clone 24 Tocil Wood Soil Clone 30 Miller MeCl26 Aminobacter strain CC495 strain WG1 Warwickshire Garden Soil Clone 17 Hyphomicrobium strain LAT3 Hyphomicrobium strain PMC Tocil Wood Soil Clone 36 Hyphomicrobium strain TW4 Miller MeCl41 SIPGH46 Hyphomicrobium strain TW30 Hyphomicrobium chloromethanicum strain CM2 Warwickshire Garden Soil Clone 21 Hyphomicrobium strain S4 Hyphomicrobium strain TW5 Hyphomicrobium strain TW28 SIPGH24 SIPGH33 Warwickshire Garden Soil Clone 1 Warwickshire Garden Soil Clone 10 Warwickshire Garden Soil Clone 3 Warwickshire Garden Soil Clone 33 Warwickshire Garden Soil Clone 26 Warwickshire Garden Soil Clone 25 Warwickshire Garden Soil Clone 51 SIPGH9 SIPGH1 Marine Clone PMLSW4 Marine Clone PMLSW9 SIPGH3 SIPGH26 SIPGH44 SIPGH16 Marine Clone PMLSW6 SIPGH6 SIPGH48 Hyphomicrobium strain SAC1 Methylobacterium chloromethanicum strain CM4 0.10
    
    Clade A
    
    Clade B
    
    Fig. 3. Maximum-likelihood tree of cmuA sequences (DNA) amplified from the soil DNA preparations (Tocil Wood soil, Gibbet Hill Wood soil and Warwickshire garden soil) and enrichment cultures from Warwickshire garden soil (Warwickshire Enrichment) and from cmuA sequences (DNA sequences) detected in the 13 C-DNA fraction of microcosm (Gibbet Hill Wood) exposed to 13CH3Cl. Clones obtained from the 13C-DNA fraction are prefixed with ‘SIPGH’. Agricultural soil cmuA clones from the 13 CH3Cl-labelled DNA-SIP experiment described by Miller and colleagues (2004) are prefixed with ‘Miller MeCl’. Sequences from Plymouth coastal waters methyl bromide enrichment library (Schäfer et al., 2004) are prefixed with PMLSW. Sequences (DNA) amplified from the extant methyl halide-utilizing bacteria, H. chloromethanicum CM2, M. chloromethanicum CM4, Aminobacter strains IMB1 and CC495; from methyl halide utilizers (Hyphomicrobium strains S4, SAC1 and PMC) from a study by McAnulla and colleagues (2001a); from marine strain 179 described in a study by Schäfer and colleagues (2005); and from the novel methyl halide-utilizing bacterial strains isolated in this study (Hyphomicrobium strains TW4, TW5, TW28, TW30, LAT3 and Aminobacter strains TW23 and WG1) are also included in the cmuA phylogenetic analysis. Trees were constructed in ARB in a manner identical to that of Fig. 2.
    
    Clade C
    
    Clade D
    
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    Stable isotope probing and methyl chloride utilization 1323 IMB1. Clone TWsoil36 (OTU1) grouped (86.3–86.6% identity at the DNA level) with cmuA from Hyphomicrobium strains LAT3 and PMC. TWsoil cmuA sequences from OTU2 had identities of 98.4–99.5% to cmuA from Aminobacter strain CC495 and grouped within Clade B (Fig. 3). cmuA clones from the GHsoil sample belonged to three OTUs, with OTU1 containing 46 clones. All sequenced cmuA clones from the three OTUs of the GHsoil sample grouped within Clade A (Fig. 3). Restriction fragment length polymorphism (RFLP) analysis of cmuA clones from the WGsoil sample produced eight OTUs. OTU1 and OTU2 contained 34 and 10 clones respectively. Each of the remaining six clones had a unique restriction pattern (six OTUs), and three clones (WGsoil2, 17 and 45) from these six are represented in Fig. 3. cmuA sequences analysed from OTU1 and clone WGsoil1 all grouped within Clade C and had 99.0–99.4% identity to cmuA from H. chloromethanicum CM2. Representative cmuA sequences from OTU2 and clone WGsoil45 branched with other soil environmental cmuA sequences within Clade A (Fig. 3). Clone WGsoil17 grouped with cmuA from Aminobacter (94.9% identity to cmuA from Aminobacter strain CC495). Analysis of the clones generated from the CH3Cl enrichment cultures set up with Warwickshire garden soil (WGenri) identified four OTUs, with OTU1 consisting of 44 clones. Sequences (cmuA) from OTU1, OTU3 and OTU4 all grouped within Clade A. Only clones from OTU2 branched separately within Clade B and had identities of 99.6% to cmuA from Aminobacter strain CC495. Comparison of culture-independent (cmuA gene libraries) and culture-dependent (enrichment and isolation) approaches indicated a clear difference in diversity of cmuA sequences for each soil sample. Enrichment and isolation from Tocil Wood soil yielded Hyphomicrobium isolates having cmuA sequences with high identity to cmuA from H. chloromethanicum CM2 (Fig. 2). In contrast, no cmuA clones with high identity to cmuA from H. chloromethanicum CM2 were retrieved from clone libraries made with Tocil Wood soil DNA. One clone, TWsoil36, showed 79.7% identity to cmuA from H. chloromethanicum CM2 (Fig. 3). The only strain isolated from Warwickshire garden soil, Aminobacter strain WG1, had high identity with Aminobacter strain CC495. The majority of cmuA clones from Warwickshire garden soil and enrichment libraries grouped within Clades A and B. Only three out of 100 Warwickshire garden cmuA clones (WGenri5, WGenri12 and WGsoil17) had high identity to cmuA of Aminobacter strain CC495. Most of the cmuA clones retrieved from all three locations grouped within Clade A, which represents cmuA sequences from a novel and, as yet, uncultivated group of CH3Cl utilizers. This emphasizes the advantage of culture-independent approaches to analyse diversity of bacterial populations with specific functions in natural environments. Stable isotope probing Stable isotope probing (SIP) allows characterization of the structure and function of microbial populations actively carrying out a specific metabolic process and is based on the incorporation of a 13C-labelled substrate into nucleic acids, which are then analysed by molecular techniques (Radajewski et al., 2000; 2002; Morris et al., 2002; Hutchens et al., 2004). DNA-SIP was used to analyse cmuA sequences from active bacterial populations in a Gibbet Hill Wood soil microcosm that was exposed to 13CH3Cl for a relatively short time (10 days). The application of elevated concentrations of 13CH3Cl (0.5% v/v per addition) was absolutely essential to avoid long incubation times and possibly formation of 13C-labelled metabolites from 13 CH3Cl, which could then be substrates for non-target microorganisms (cross-feeding). DNA was extracted from the microcosm (20 g of soil) that had oxidized approximately 1–2 mmol 13CH3Cl. A faint but distinct 13C-labelled DNA (13C-DNA) fraction was resolved from total community DNA (12C-DNA) by CsCl density-gradient centrifugation. 13C-DNA and 12C-DNA fractions were collected and used as templates for PCR amplification of cmuA genes, construction of clone libraries and subsequent phylogenetic analysis of representative cmuA sequences. Functional gene libraries (cmuA) Libraries of 50 cmuA clones were constructed with the cmuA802f-cmuA1609r primer set for both 13C-DNA and 12 C-DNA fractions. Restriction fragment length polymorphism (RFLP) analysis of cmuA clones from the 13C-DNA fraction identified five OTUs. OTU1, OTU2, OTU3 and OTU4 contained 19, 11, 6 and 11 clones respectively. OTU5 had one clone, SIP_GH9. Sequencing of clones of OTU1 (SIP_GH24, SIP_GH33, SIP_GH46) showed them to be closely related (99.1–99.4% identity at the DNA level) to cmuA from H. chloromethanicum CM2 (Fig. 3). The sequence of clone SIP_GH9 (OTU5) was also most similar (95.6% identical) to the cmuA sequences of cluster II Hyphomicrobium species, including H. chloromethanicum CM2, but branched separately from Clade C. Sequencing of clones of OTU2 (SIP_GH3, SIP_GH6, SIP_GH16, SIP_GH26 and SIP_GH44) and OTU3 (SIP_GH1 and SIP_GH48) identified a group of cmuA sequences most similar to sequences from a Plymouth coastal water CH3Br enrichment library (PMLSW4, PMLSW9 and PMLSW6) (Schäfer et al., 2005) grouping together within a separate clade (Clade D) (Fig. 3). This unexpected grouping of soil and marine cmuA sequences (PMLSW clones) indicated the presence of novel types of
    
    © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1318–1328
    
    1324 E. Borodina, M. J. Cox, I. R. McDonald and J. C. Murrell soil cmuA sequences which are widespread in natural environments. SIP_GH cmuA sequences from Clade D with identities of 77.6–86.6% to cmuA from H. chloromethanicum CM2 (Fig. 3) are likely to represent cmuA sequences from a functional population of CH3Cl utilizers which have yet to be cultivated. All three sequenced clones of OTU4 (SIP_GH7, SIP_GH22 and SIP_GH43) formed a distinct group containing cmuA sequences from methyl halide-utilizing marine strain 179 and Ruegeria strain 198 (Schäfer et al., 2005). Interestingly, soil clones from the 13CH3Cl-labelled DNA-SIP experiment described by Miller et al. (2004) were also closely related to the clones of OTU4 with identities of 69.5–70.4%. OTU4 had sequences that were 75.7–76.6% identical to, but clearly distinct from cmuA of Aminobacter strain IMB1 (Fig. 3). This indicated that OTU4 is represented by cmuA sequences from a novel group of CH3Cl utilizers which are active in this soil environment. Analysis of the corresponding 12C-DNA library (not shown) demonstrated a clear difference in the population of cmuA sequences retrieved from 13C-DNA. In contrast to the five OTUs detected in the 13C-DNA library, only one OTU was detected in the 12C-DNA library and this contained cmuA sequences with high identity (> 99%) to the corresponding cmuA sequence of H. chloromethanicum CM2. This indicated that the functionally active population of CH3Cl utilizers probably represents a more diverse group of organisms in comparison with the total population of CH3Cl-utilizing organisms detected by PCR, which seem to be dominated by Hyphomicrobium species. Discussion Culture-dependent methods showed that CH3Cl-utilizing Hyphomicrobium species predominated during CH3Clenrichment procedures in soil samples taken from various locations. Aminobacter strain TW23 and strain WG1 (phylogenetically related to Mesorhizobium strain ‘Mena25/41’ and Mesorhizobium tianshanense) which could utilize CH3Cl were also isolated, indicating that species of the genera Hyphomicrobium, Aminobacter and Mesorhizobium are the most easily culturable organisms under the laboratory conditions used in this study. Most of the CH3Cl-utilizing Hyphomicrobium species from this study shared high identity with the well-characterized H. chloromethanicum CM2. The novel Aminobacter strain TW23 grouped with the previously isolated CH3Cl-utilizing Aminobacter strain IMB1. Analysis of cmuA sequences from novel isolates indicated that the phylogeny of their cmuA sequences (Fig. 2) and previously isolated CH3Cl-utilizing strains was congruent with their 16S rRNA phylogeny (Fig. 1), except for strain WG1. The diversity of methyl halide utilizers within soil environmental samples was assessed by targeting functional genes (cmuA) through PCR amplification. Clone library (cmuA) analysis indicated that only a relatively small proportion of cmuA sequences with high identities to those from the isolated CH3Cl utilizers was retrieved from DNA extracted from various soils used in this study. Interestingly, most of the novel Hyphomicrobium strains were isolated from the Tocil Wood soil sample, but no cmuA clones from that location grouped within Clade C, where cmuA from the novel strains grouped. Sequences highly related (> 99% identity) to cmuA from Hyphomicrobium spp. were the dominant sequence type isolated from Warwickshire garden soil. Clones with high identities to cmuA from previously isolated and novel Aminobacter strains were obtained from Tocil Wood soil (TWsoil), Warwickshire garden soil (WGsoil) and Warwickshire garden enrichment (WGenri) libraries. Both the enrichment procedure and analysis of environmental cmuA clone libraries indicated that CH3Cl-utilizing Hyphomicrobium and Aminobacter are prevalent in natural terrestrial environments. However, the soil cmuA clones formed a group of sequences (Clade A), entirely consisting of soil cmuA sequences that potentially represent an uncultivated group of CH3Cl utilizers. In this study, a selection of clones arising from the 13CDNA SIP library represent active methyl halide utilizers that use CH3Cl at elevated concentrations. Surprisingly, analysis of the 13C-DNA-SIP library indicated the presence of active populations of methyl halide utilizers with cmuA sequences (OTU4) closely related to cmuA sequences retrieved from Plymouth coastal water enrichments and the marine methyl halide-utilizing marine strain 179 and Ruegeria strain 198 (Schäfer et al., 2005). Interestingly, OTU4 also represented cmuA sequences with high identity to those obtained from CH3Br-fumigated agricultural soil in another 13CH3Cl-SIP experiment (Miller et al., 2004), confirming that these cmuA sequences are derived from methyl halide utilizers active in the environment. Only one out of five OTUs from the 13C-DNA-SIP library contained cmuA sequences highly (> 99% identity) related to those from H. chloromethanicum CM2. Comparison of cmuA sequences detected in the 13C-DNA-SIP library and the corresponding cmuA sequences in the 12 C-DNA fraction indicated that CH3Cl-utilizing Hyphomicrobium species might not be the predominant group of functionally active CH3Cl-utilizing microorganisms in all soil environments. Surprisingly no cmuA clones from either the 13C-DNA or the 12C-DNA libraries were found within Clade A. Contrary to our SIP data, all cmuA clones from direct cloning experiment in this study grouped within Clade A. This suggests that a more detailed survey of cmuA sequences in a variety of soils, retrieved using refined PCR primer sets, will be required in order to resolve this inconsistency in data. Application of reverse transcription polymerase chain reaction (RT-PCR) and
    
    © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1318–1328
    
    Stable isotope probing and methyl chloride utilization 1325 analysis of cDNA cmuA clones would also provide data on the expression of cmuA in soils. Due to the technical difficulties in extraction of RNA from soils, this was not attempted, but such future studies should provide complementary data for this DNA-SIP study. It is likely that Hyphomicrobium species tend to be more easily cultivated and out-compete other groups of bacteria through the enrichment process. The use of enrichment or elective culture techniques that do not favour the fastgrowing hyphomicrobia will be needed to isolate from the environment the diverse range of CH3Cl utilizers which the culture-independent techniques used in this study have indicated to be present. Such techniques could include continuous chemostat enrichments poised at different dilution rates and substrate concentrations, which will possibly facilitate the selection of bacteria of diverse mmax and with high substrate affinities for CH3Cl. Most importantly, new data on environmental cmuA sequences obtained here and in a recent study by Schäfer and colleagues (2005) will enable refinement of cmuA primer sets in order to retrieve more diverse cmuA sequences present in the natural environment. Experimental procedures Cultivation experiments
    Enrichment and isolation of CH3Cl-utilizing bacteria. Environmental soil samples were collected from Tocil Wood (UK), Gibbet Hill Wood (UK), Warwickshire garden soil (UK) and Latvian garden soil. For enrichment cultures, soil samples (1 g) were slurried with either 10 ml of ANMS (ammonium nitrate mineral salts medium) (Whittenbury et al., 1970) or 10 ml of P. versutus medium (Borodina et al., 2000). The enrichment cultures were incubated at either 10∞C, 20∞C or 30∞C. For growth on CH3Cl or CH3Br, 125 ml of serum vials sealed with Teflon-coated butyl rubber stoppers (Owens Polyscience, Macclesfield, UK) were used. Growth substrate (CH3Cl or CH3Br) was added directly to the headspace through the stoppers. For the initial enrichment, either 1% or 2% (v/v) CH3Cl or 0.2% (v/v) CH3Br in the gas phase was used as sole carbon and energy source. Pasteurized samples were subjected to 80∞C for 10 min. Enrichments actively consuming CH3Cl or CH3Br were then subcultured with 2% (v/v) CH3Cl or 0.2% (v/v) CH3Br in the headspace. CH3Cl consumption was measured using gas chromatography by removing 100 ml samples from the headspace. Enrichments showing both growth and consumption of CH3Cl or CH3Br after three subcultures were serially diluted and spread onto plates of the corresponding medium and incubated with 2% (v/v) CH3Cl or 0.2% (v/v) CH3Br. Plates were incubated in sealed, gas-tight jars for 7–10 days until the colonies became visible. Individual colonies were then streaked onto plates and re-incubated and the process was repeated until pure cultures were obtained. Individual pure colonies were subsequently used to inoculate liquid media to confirm that CH3Cl isolates were genuine CH3Cloxidizers. Microscopy, uniformity of colony morphology and lack of growth on media without the growth substrate were routinely used to assess purity of cultures. Measurement of CH3Cl by gas chromatography. Samples of headspace gas (100 ml) were injected into a GCD gas chromotograph (Pye Unicam, Cambridge, UK) fitted with a Porapak Q column (Phase Separation, Deeside, UK) at 200∞C. A flame ionization detector was used to detect products and the peak areas were determined with a 3390A integrator (Hewlett-Packard, Berkshire, UK). The gas chromatograph was calibrated by using samples of known concentrations of the above gases at 30∞C. Polymerase chain reaction. Polymerase chain reaction amplifications were performed in 50 ml (total volume) of mixtures in 0.5 ml microcentrifuge tubes using a Hybaid Touchdown thermal cycling system. Each PCR mix consisted of 3 ml of MgCl2 (1.5 mM), 5 ml of PCR buffer (supplied by the manufacturer), 5 ml of dNTP (200 mM), 1 ml (0.1 mg) of each forward and reverse primers, 33.5 ml of dH2O, 0.5 ml (2.5 U) of Taq Polymerase and 1–2 ml of template DNA. After an initial denaturation step at 94∞C (5 min), 2.5 units of Taq polymerase (MBI Fermentas) were added. Amplification was carried out using 30 cycles of 94∞C for 1 min, various annealing temperatures as required for 1 min, extension at 72∞C for 1 min, and a final extension period at 72∞C for 10 min. Annealing temperatures used were 60∞C (for amplification of 16S rRNA genes) and 55∞C (for amplification of cmuA genes). Primers specific for 16S rRNA (Eubac27F/1492R; Lane, 1991) and cmuA genes (cmuA802F/cmuA1609R, Miller et al., 2004) were used in this study. DNA sequencing and analysis. DNA sequencing was performed by cycle sequencing with a Dye Terminator kit (PE Applied Biosystems, Warrington, UK). DNA sequences were analysed using a 373A automated sequencing system (PE Applied Biosystems). DNA sequences were analysed using the DNASTAR package. 1377 bp of sequence of 16S rRNA genes between positions 83 and 1460 (Escherichia coli numbering) and 758 bp of sequence of cmuA genes between positions 826 and 1584 (M. chloromethanicum numbering) were analysed using the AXML program of ARB. Identities of the 16S rRNA gene sequences were calculated using the Neighbour-Joining program of PHYLIP through ARB using the Felsenstein correction (as recommended by the ARB program during analysis of the sequences obtained in this study) over the same sequence lengths. Similarity searches were performed using the gapped BLAST (Basic Local Alignment search Tool) program (Altschul et al., 1990) against public protein and gene databases (http://www.ncbi.nlm.nih.gov). Sequences from the CH3Cl-utilizing isolates obtained in this study were deposited in GenBank under the following Accession No., for 16S rRNA: AY934487 (strain TW30), AY934488 (strain WG6), AY934489 (strain LAT3), AY934490 (strainWG1), AY934491 (strain TW5), AY934492 (strain TW4), AY934493 (strain TW28), AY934494 (strain TW23) and AY934495 (strain MW1); and for cmuA sequences: AY934496 (strain TW30), AY934497 (strain LAT3), AY934498 (strain TW28), AY934499 (strain TW4), AY934500 (strain TW5), AY934501 (strainWG1) and AY934502 (strain TW23). Environmental sequences from this study were deposited in GenBank under Accession No. AY934426–AY934486.
    
    © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1318–1328
    
    1326 E. Borodina, M. J. Cox, I. R. McDonald and J. C. Murrell Molecular ecology experiments
    DNA extraction and construction of gene libraries from soil samples. DNA was extracted from topsoil samples by a bead beating method. Soil was processed in 0.3–0.5 g of samples using a DNA extraction kit (FastPrep, Bio 101) and a Ribolyser cell disrupter (Hybaid) as described previously (Yates and Gillings, 1998). Supernatant containing DNA was separated from large particles by centrifugation for 5 min at 15 000 g. Proteins in the supernatant were precipitated with potassium acetate (7.5 M, one-sixth sample volume) and removed by centrifugation for 5 min at 15 000 g. To the supernatant, an equal volume of binding matrix (Bio 101), diluted 1:5 with 6 M guanidine isothiocyanate, was added. The tube was inverted regularly for 5 min and centrifuged for 1 min at 15 000 g, and then the supernatant was discarded. The binding matrix was washed twice by resuspension in an equal volume of wash buffer (70% ethanol, 100 mM sodium acetate) and centrifugation at 15 000 g for 1 min. The supernatant was discarded, tubes were air-dried for 30 min and the matrix was resuspended in 100 ml of TE buffer. The matrix solution containing DNA was then run on a 1% (w/v) agarose gel and DNA was purified by gel extraction with the QIAquick® Gel Extraction Kit (Qiagen) according to manufacturer’s instructions. Polymerase chain reaction amplification, cloning and sequencing. DNA extracted from each soil sample was used as a template for PCR amplification, with primers specific for cmuA (cmuA802F/cmuA1609R; Miller et al., 2004) to analyse diversity of cmuA sequences present in samples from the natural environment. Reactions (50 ml) contained 1 ml of template (approximately 5–50 ng) and were performed as described previously for each primer set. Amplicons of the correct size (808 bp) were cloned using the TOPO TA cloning kit (Invitrogen) and libraries of 50 clones were constructed for each sampling site. Plasmid inserts were screened by digestion with restriction endonucleases EcoRI and DdeI for cmuA gene libraries. DNA fragments were resolved by electrophoresis and each clone was assigned to an OTU that represented a unique RFLP pattern. At least 10% of clones within each OTU were sequenced to verify that the restriction pattern represented a single sequence type. DNA extraction, ultracentrifugation and amplification. DNA was extracted from 20 g of soil exposed to 13CH3Cl by a modified method of Zhou and colleagues (1996). The soil sample was split into 5 g aliquots and placed into separate tubes. To each tube, 13.5 ml of extraction buffer [100 mM TrisHCl (pH 8.0), 100 mM disodium EDTA (pH 8.0), 100 mM Naphosphate (pH 8.0), 1.5 M NaCl and 1% w/v CTAB] and 300 ml of Proteinase K (10 mg ml-1) were added. The tubes were subsequently shaken in a horizontal position at 225 r.p.m. for 30 min (37∞C). This was followed by addition of 3 ml of SDS (20% w/v) and incubation for 2 h at 65∞C with gentle mixing by inversion every 15–20 min. After incubation, the tubes were centrifuged for 10 min at 6800 g (Beckman Instruments, California, USA) at 20∞C. The supernatant was transferred into fresh tubes, to which an equal volume of chloroform:isoamyl alcohol was added (24:1) and the tubes were centrifuged for 10 min at 20 400 g (20∞C). Following centrifugation, the aqueous phase (upper layer) was placed into separate tubes, to which a 0.6 volume of isopropanol was added and the DNA was left to precipitate for 1 h (20∞C). The tubes were subsequently centrifuged for 20 min at 16 000 g (20∞C) and the resulting pellet was washed with 70% (v/v) ethanol. DNA was air-dried and DNA pellets from each preparation were combined and resuspended in a total volume of 2 ml of TE. The DNA sample was further subjected to CsCl-ethidium bromide gradient centrifugation to separate 13C-labelled DNA (DNA from organisms actively utilizing CH3Cl) from unlabelled 12C-DNA (non-CH3Cl utilizers or inactive CH3Cl utilizers). CsCl (2 g) and ethidium bromide (100 ml, 10 mg ml-1) were added to 2 ml of DNA solution, which was then transferred to a polyallomer quick-seal ultracentrifuge tube (13 ¥ 51 mm; Beckman Instruments, California, USA). The tubes were topped up with 1 g ml-1 CsCl solution and DNA fractions were resolved following density gradient centrifugation at 265 000 g (Beckman Vti 65 rotor) for 17 h at 20∞C (Sambrook and Russell, 2001) and visualized with UV light. DNA from both 13C-DNA and 12C-DNA fractions was withdrawn gently using a 1 ml syringe fitted with a hypodermic needle (19 gauges). Care was taken during collection of the13C-DNA fraction to avoid co-extraction of the 12C-DNA band, as described in Hutchens and colleagues (2004). CsCl from DNA extracted from both fractions was then removed by drop dialysis against 25–30 ml of TE buffer (Sambrook and Russell, 2001). The 13C-DNA and 12C-DNA fractions were used as templates for PCR amplification, with primers specific for cmuA genes (cmuA802F/cmuA1609R; Miller et al. 2004). Reactions (50 ml) contained 1 ml of DNA template (approximately 5–50 ng) and were performed as described above. Cloning and DNA sequencing. Amplification products of the correct size were cloned using the TOPO TA cloning kit (Invitrogen) and libraries of 50 clones were constructed. Plasmid inserts were screened by digestion with restriction endonucleases EcoRI and DdeI for cmuA genes from the 13CH3Cl microcosm as described above. DNA fragments were resolved by electrophoresis and each clone was assigned to an OTU that represented a unique RFLP. At least 10% of clones within each OTU were sequenced to verify that the restriction pattern represented a single sequence type.
    
    Stable isotope probing (SIP)
    C-enriched carbon source culture. A microcosm experiment was used to characterize the active CH3Cl-utilizing communities from a Gibbet Hill Wood soil sample (0–5 cm depth sample). The microcosms consisted of fresh topsoil (20 g) in a 2-litre crimp-top serum bottle, sealed with a butyl rubber stopper, and initially injected with 0.5% (v/v) 13CH3Cl (0.45 mmol 13CH3Cl). Microcosms were incubated at 25∞C and headspace 13CH3Cl concentrations were determined every 2 days by gas chromatography. Once the initial dose of 13CH3Cl had been consumed (7–10 days), the bottles were flushed with air to remove any 13CO2 produced and to ensure that the microcosm remained aerobic. Ten-millilitre (0.45 mmol 13CH3Cl) aliquots of CH3Cl were regularly added until a total of 1–2 mmol 13CH3Cl was consumed after approximately 10 days.
    13
    
    © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1318–1328
    
    Stable isotope probing and methyl chloride utilization 1327 Acknowledgements
    We acknowledge financial support from the Natural Environment Research Council (NER/A/S/2000/00423). This work was also supported by EU 5th Framework Programme (Grant No. QLK3-CT-2000-01528). We thank Hendrik Schäfer for advice on phylogenetic analysis and critical review of the manuscript and Don Kelly for his comments on this work. tion of low concentrations of methyl bromide by soil bacteria. Appl Environ Microbiol 64: 1864–1870. Hutchens, E., Radajewski, S., Dumont, M.G., McDonald, I.R., and Murrell, J.C. (2004) Analysis of methanotrophic bacteria in Movile Cave by stable isotope probing. Environ Microbiol 6: 111–120. Itoh, N., Tsujita, M., Ando, T., Histomi, G., and Higashi, T. (1997) Formation and emission of monohalomethanes from marine algae. Phytochemistry 45: 67–73. Keene, W.C., Khalil, M.A.K., Erickson, D.J., McCulloch, A., Graedel, T.E., Lobert, J.M., et al. (1999) Composite global emissions of reactive chlorine from anthropogenic and natural sources: reactive chlorine emissions inventory. J Geophys Res 104: 8429–8440. Khalil, M.A.K., and Rasmussen, R.A. (1999) Atmospheric methyl chloride. Atmosph Environ 33: 1305–1321. Khalil, M.A.K., Moore, R.M., Harper, D.B., Lobert, J.M., Erickson, D.J., Koropalov, V., et al. (1999) Natural emissions of chlorine-containing gases: reactive chlorine emissions inventory. J Geophys Res 104: 8333–8346. Lane, D.J. (1991) 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics. Stackebrandt, E., and Goodfellow, M. (eds). Chichester, UK: J. Wiley and Sons, pp. 115–175. Lin, J., Radajewski, S., Eshinimaev, B.T., Trotsenko, Y.A., McDonald, I.R., and Murrell, J.C. (2004) Molecular diversity of methanotrophs in Transbaikal soda lake sediments and identification of potentially active populations by stable isotope probing. Environ Microbiol 6: 1049–1060. Lobert, J.M., Keene, W.C., Logan, J.A., and Yevich. R. (1999) Global chlorine emissions from biomass burning: reactive chlorine emissions inventory. J Geophys Res 104: 8373– 8389. McAnulla, C., McDonald, I.R., and Murrell, J.C. (2001a) Methyl chloride utilising bacteria are ubiquitous in the natural environment. FEMS Microbiol Lett 201: 151–155. McAnulla, C., Woodall, C.A., McDonald, I.R., Studer, A., Vuilleumier, S., Leisinger, T., and Murrell, J.C. (2001b) Methyl chloride utilization gene cluster from Hyphomicrobium chloromethanicum strain CM2T and development of functional gene probes to detect halomethane-degrading bacteria. Appl Environ Microbiol 67: 307–316. McDonald, I.R., Warner, K.L., McAnulla, C., Woodall, C.A., Oremland, R.S., and Murrell, J.C. (2002) A review of bacterial methyl halide degradation: biochemistry, genetics and molecular ecology. Environ Microbiol 4: 193–203. Marchesi, J.R., and Weightman, A.J. (2003) Comparing the dehalogenase gene pool in cultivated a-halocarboxylic acid-degrading bacteria with the environmental metagene pool. Appl Environ Microbiol 69: 4375–4382. Mellouki, A., Talukar, R.K., Schmoltner, A.M., Gierzak, T., Mills, M.J., Solomon, S., and Ravishankara, A.R. (1992) Atmospheric lifetimes and ozone depletion potentials of methyl bromide (CH3Br) and dimethyl bromide (CH2Br2). Geophys Res Lett 19: 2059–2062. Messmer, M., Wohlfarth, G., and Diekert, G. (1993) Methyl chloride metabolism of the strictly anaerobic methyl chloride-utilizing homoacetogen strain MC. Arch Microbiol 160: 383–387. Miller, L.G., Connel, T.L., Guidette, J.R., and Oremland, R.S. (1997) Bacterial oxidation of methyl bromide in fumi-
    
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