Proc. Natl. Acad. Sci. USA Vol. 96, pp. 4615–4620, April 1999 Microbiology A corrinoid-dependent catabolic pathway for growth of a Methylobacterium strain with chloromethane TODD VANNELLI,MICHAEL MESSMER,ALEX STUDER,STE´PHANE VUILLEUMIER, AND THOMAS LEISINGER Mikrobiologisches Institut, Swiss Federal Institute of Technology, ETH–Zentrum, CH-8092 Zurich, Switzerland Edited by JoAnne Stubbe, Massachusetts Institute of Technology, Cambridge, MA, February 5, 1999 (received for review November 9, 1998) ABSTRACT Methylobacterium sp. strain CM4, an aerobic was also reported (10). Microbial metabolism of monoha- methylotrophic a-proteobacterium, is able to grow with chlo- lomethanes includes oxidative (11) and hydrolytic (12) co- romethane as a carbon and energy source. Mutants of this metabolic processes, as well as mineralization by methylotro- strain that still grew with methanol, methylamine, or formate, phic bacteria that use chloromethane as a growth substrate. but were unable to grow with chloromethane, were previously The homoacetogenic bacterium Acetobacterium dehalogenans obtained by miniTn5 mutagenesis. The transposon insertion (13) is the only known strictly anaerobic representative of the sites in six of these mutants mapped to two distinct DNA latter group (14). Anoxic dehalogenation of chloromethane by fragments. The sequences of these fragments, which extended this organism was shown to be catalyzed by enzymes that over more than 17 kb, were determined. Sequence analysis, transfer the methyl group of chloromethane by means of a mutant properties, and measurements of enzyme activity in corrinoid protein to H4folate to yield chloride and CH3- cell-free extracts allowed the definition of a multistep pathway H4folate, an intermediate of the acetyl-CoA pathway (15). In for the conversion of chloromethane to formate. The methyl contrast, the reactions by which some recently isolated strictly group of chloromethane is first transferred by the protein aerobic methylotrophic bacteria (8) use chloromethane as a CmuA (cmu: chloromethane utilization) to a corrinoid pro- growth substrate have yet to be elucidated. The physiological tein, from where it is transferred to H folate by CmuB. Both 4 properties of the wild-type and of chloromethane utilization- CmuA and CmuB display sequence similarity to methyltrans- ferases of methanogenic archaea. In its C-terminal part, negative mutants of a representative strain, Methylobacterium CmuA is also very similar to corrinoid-binding proteins, sp. CM4, led us to propose that this organism metabolizes indicating that it is a bifunctional protein consisting of two chloromethane by initial dehalogenation by means of a methyl domains that are expressed as separate polypeptides in methyl transfer reaction (16). transfer systems of methanogens. The methyl group derived Here we report on the sequence of two large DNA frag- from chloromethane is then processed by means of pterine- ments containing at least four genes essential for chlorometh- linked intermediates to formate by a pathway that appears to ane metabolism in strain CM4. We present experimental be distinct from those already described in Methylobacterium. evidence that this aerobic bacterium is able to catalyze transfer Remarkable features of this pathway for the catabolism of of the methyl moiety of chloromethane to H4folate by means chloromethane thus include the involvement of a corrinoid- of a corrinoid intermediate to yield CH3-H4folate, a key dependent methyltransferase system for dehalogenation in an intermediate of methylotrophic metabolism. aerobe and a set of enzymes specifically involved in funneling the C1 moiety derived from chloromethane into central me- MATERIALS AND METHODS tabolism. Materials. Reagents for molecular biology were obtained Attention has been focused on chloromethane and bro- from Fermentas (Vilnius, Lithuania) and Boehringer Mann- momethane because of their role as sources of stratospheric heim. (6S)-5,6,7,8-tetrahydrofolic acid trihydrochloride chlorine and bromine, the primary agents of ozone destruc- (H4folate) and (6S)-5-methyltetrahydrofolate (CH3-H4folate) tion. Chloromethane (CH3Cl) is the most abundant halocar- were purchased from Schircks Laboratorium (Jona, Switzer- bon in the atmosphere and is responsible for 15–20% of land). ATP, S-adenosyl methionine, and methylcobalamin chlorine-catalyzed ozone destruction in the stratosphere (1). It were from Sigma, and NADPH:FMN oxidoreductase was is released at an estimated global rate of 3.5–5 3 106 tons per from Boehringer Mannheim. All other chemicals were reagent year, primarily from natural sources, and less than 1% of the grade or better and were purchased from Fluka. global chloromethane flux is caused by industrial production of Bacterial Strains. Bacterial strains in this study included the compound (reviewed in ref. 1). For bromomethane, cur- Methylobacterium sp. strain CM4, which grows with chlo- rent estimates of ocean emission and natural formation during romethane (8), and miniTn5 (17) insertion mutants, whose 3 4 combustion of vegetation fall in the range of 8 10 tons per phenotypes have been described (16). Escherichia coli K12 year, slightly more than the amount annually emitted by the use strain DH5a (GIBCO/BRL Life Technologies) was used as a of this compound in soil fumigation (2). On a molar basis, host in DNA work. bromine is 40–100 times more effective than chlorine in DNA Manipulations. Preparation of genomic DNA, restric- depleting ozone (3). Thus, chloromethane and bromomethane tion enzyme digestions, ligations, and transformations were contribute about equally to an estimated 40% of the total performed by using standard procedures (18). Plasmid pBlue- global loss of stratospheric ozone. script-KSII(1) (Stratagene) was used for cloning. DNA frag- Green plants (4) and soil bacteria (5–9) represent terrestrial ments from mutants of strain CM4 containing a miniTn5 sinks for chloromethane and bromomethane. Evidence for insertion were cloned by selection of transformants for kana- bacterial degradation of halogenated methanes in seawater mycin resistance (25 mg/ml). The publication costs of this article were defrayed in part by page charge This paper was submitted directly (Track II) to the Proceedings office. payment. This article must therefore be hereby marked ‘‘advertisement’’ in Data deposition: The sequences reported in this paper have been accordance with 18 U.S.C. §1734 solely to indicate this fact. deposited in the GenBank database (accession nos. AJ011316 and PNAS is available online at www.pnas.org. AJ011317). 4615 Downloaded by guest on October 1, 2021 4616 Microbiology: Vannelli et al. Proc. Natl. Acad. Sci. USA 96 (1999) Sequence Analysis. The cloned genomic DNA was se- were still able to grow with methanol, methylamine, or for- quenced on both strands by using PCR methods with fluores- mate. Conversely, 73 transposon mutants defective in the cent dideoxynucleotide terminators and an ABI-Prism auto- utilization of methanol, methylamine, methanol plus methyl- matic sequencer (Perkin–Elmer). The precise site of insertion amine, or formate could still grow with chloromethane (16). of the minitransposon was determined for all mutants. The This suggested that chloromethane was metabolized in Methy- sequences of cluster I (9,658 nt, accession no. AJ011316) and lobacterium sp. CM4 by reactions different from those involved cluster II (8,457 nt, accession no. AJ011317) were assembled in the metabolism of methanol and methylamine. The genes from sequence fragments obtained from DNA cloned from the whose insertional inactivation caused loss of the ability to grow different mutants with the GCG sequence analysis package with chloromethane were isolated by selection of the kana- (Version 8.1, Genetics Computer Group, Madison, WI). Sim- mycin resistance gene present on the minitransposon (17). The DNA fragments carrying a transposon insertion were se- ilarity searches were performed by using gapped BLAST and 2 PSI–BLAST programs (19) against public protein and gene quenced from all Cmu mutants. Transposon insertion mu- databases. tants, arbitrarily labeled in order of their detection (16), were N-Terminal Sequencing. The 67-kDa protein induced dur- found in four apparently unlinked DNA regions, which were ing growth with chloromethane (16) was partially purified, and termed cluster I (mutants 30F5, 38G12, 22B3, and 38A10), cluster II (mutants 19D10 and 36D3), cluster III (mutant the N-terminal sequence of the corresponding protein band on 2 SDS/PAGE was determined by Edman degradation using an 11G7), and cluster IV (mutant 27B11). The Cmu mutant Applied Biosystems 476A automatic sequencer. 27C10 was not analyzed in detail because it appeared to carry Preparation of Cell-Free Extract. Methylobacterium sp. a partial duplication of the transposon. A schematic represen- tation of the 14 ORFs identified in the DNA sequences of strain CM4 and mutants were grown as described (16). Bac- clusters I and II is shown in Fig. 1. teria were harvested at an OD of 0.5 to 0.7 (10,000 3 g for 600 Most of the encoded polypeptides displayed significant 15 min) and resuspended (1 g wet cells per ml) in 50 mM sequence identity to proteins of known functions (Table 1). Tris-SO4 buffer (pH 7.2) containing 5 mM DTT. Cells were With respect to their possible role in metabolism, the proteins disrupted by two passages through a French pressure cell (120 encoded in DNA clusters I and II fell into four groups: MPa, 4°C), and DNaseI (50 mg/ml final) was added