Chloromethane utilization gene cluster from Hyphomicrobium chloromethanicum strain CM2(T) and development of functional gene probes to detect halomethane-degrading bacteria

Abstract

Hyphomicrobium chloromethanicum CM2(T), an aerobic methylotrophic member of the alpha subclass of the class proteobacteria, can grow with chloromethane as the sole carbon and energy source. H. chloromethanicum possesses an inducible enzyme system for utilization of chloromethane, in which two polypeptides (67-kDa CmuA and 35-kDa CmuB) are expressed. Previously, four genes, cmuA, cmuB, cmuC, and purU, were shown to be essential for growth of Methylobacterium chloromethanicum on chloromethane. The cmuA and cmuB genes were used as probes to identify homologs in H. chloromethanicum. A cmu gene cluster (9.5 kb) in H. chloromethanicum contained 10 open reading frames: folD (partial), pduX, orf153, orf207, orf225, cmuB, cmuC, cmuA, fmdB, and paaE (partial). CmuA from H. chloromethanicum (67 kDa) showed high identity to CmuA from M. chloromethanicum and contains an N-terminal methyltransferase domain and a C-terminal corrinoid-binding domain. CmuB from H. chloromethanicum is related to a family of methyl transfer proteins and to the CmuB methyltransferase from M. chloromethanicum. CmuC from H. chloromethanicum shows identity to CmuC from M. chloromethanicum and is a putative methyltransferase. folD codes for a methylene-tetrahydrofolate cyclohydrolase, which may be involved in the C(1) transfer pathway for carbon assimilation and CO(2) production, and paaE codes for a putative redox active protein. Molecular analyses and some preliminary biochemical data indicated that the chloromethane utilization pathway in H. chloromethanicum is similar to the corrinoid-dependent methyl transfer system in M. chloromethanicum. PCR primers were developed for successful amplification of cmuA genes from newly isolated chloromethane utilizers and enrichment cultures.

FIG. 1

Proposed pathway for chloromethane metabolism in M. chloromethanicum CM4^T. The pathway was modified from that of Vannelli et al. (45). CmuA, methyltransferase I; CmuB, methyltransferase II; MetF, putative 5,10-methylene-tetrahydrofolate reductase; FolD, putative 5,10-methylene-tetrahydrofolate dehydrogenase/5,10-methenyl-tetrahydrofolate cyclohydrolase; PurU, putative 10-formyl-tetrahydrofolate hydrolase; FDH, formate dehydrogenase; Co^I, corrinoid protein acting as the primary methyl acceptor and thought to be part of CmuA; H⁴ folate, tetrahydrofolate.

FIG. 2

SDS–8% PAGE of cell extract from H. chloromethanicum CM2^T. Lane 1, molecular mass markers; lanes 2 through 4, methanol-grown cells; lanes 5 through 7, acid-shocked cells; lanes 8 through 10, chloromethane-grown cells. Triplicate lanes contained three different batches of cells grown under the conditions indicated above.

FIG. 3

Schematic representation of the methyltransferase gene cluster in H. chloromethanicum CM2^T. Gene orientations and positions are shown. Genes shown by genetic analysis of M. chloromethanicum CM4^T to be involved in methyl chloride dehalogenation are indicated by black arrows. Genes indicated by shaded arrows are predicted to have functions needed in a likely complete chloromethane catabolism pathway. Other open reading frames are indicated by open arrows. pCAW1 is a 5-kb BamHI fragment, pCAW2 is a 5.1-kb HindIII fragment, and pCM1 is a 2-kb HindIII fragment.

FIG. 4

Alignment of the methyltransferase domain of CmuA from H. chloromethanicum CM2^T with CmuA from M. chloromethanicum CM4^T and MtbA and MtsA from M. barkeri. CmuA from H. chloromethanicum CM2^T was aligned with CmuA from M. chloromethanicum CM4^T (accession no. AJ011316) and with MtbA (U38918) and MtsA (U36337) from M. barkeri. Aligned sequences in GCG's MSF format were downloaded into the BoxShade 3.21 vs program, which highlighted similar residues (shaded boxes) and identical residues (black boxes). Asterisks indicate the putative zinc-binding motif identified in MtbA from M. barkeri (25).

FIG. 5

Alignment of the corrinoid domain of CmuA from H. chloromethanicum CM2^T with CmuA from M. chloromethanicum CM4^T, MtmC from M. barkeri, and MetH from E. coli. CmuA from H. chloromethanicum CM2^T was aligned with CmuA from M. chloromethanicum CM4^T (accession no. AJ011316), MtmC from M. barkeri (AF013713), and MetH from E. coli (P13009). Aligned sequences in GCG's MSF format were downloaded into the BoxShade 3.21 vs program, which highlighted similar residues (shaded boxes) and identical residues (black boxes). Asterisks indicate the D-x-H-x₂-G-x_41–42-SxL-x_24–28-GG motif, a motif consisting of conserved residues involved in cobalamin binding in methionine synthases and mutases (27).

FIG. 6

Alignment of CmuB from H. chloromethanicum CM2^T with CmuB from M. chloromethanicum CM4^T, MtrH from M. barkeri, and MetH from E. coli. CmuB from H. chloromethanicum CM2^T was aligned with CmuB from M. chloromethanicum CM4^T (accession no. AJ011317), M. barkeri MtrH (AJ132817), and E. coli MetH (P13009). Aligned sequences in GCG's MSF format were downloaded into the BoxShade 3.21 vs program, which highlighted similar residues (shaded boxes) and identical residues (black boxes). Asterisks indicate a sequence pattern that is present in all eight MtrH-related sequences and in all nine MetH-related sequences, G-[EA]-x₂-[TNG]-x_47–57-[LIM]-x_16–20-[DN]-[RSA]-x₈-[GA]-x_10–15-[NA]-S-x_19–24-[AL]-x_10–18-[GA]-x_9–23-G-x_6–8-D-x_19–27-[KRA]-x_8–10-[GA]-x-[HAS]-N (Swissprot release 38; EMBL release 55)(41).