Chloromethane Utilization Gene Cluster fromHyphomicrobium chloromethanicum Strain CM2T and Development of Functional Gene Probes To Detect Halomethane-Degrading Bacteria

摘要

Hyphomicrobium chloromethanicum CM2T, an aerobic methylotrophic member of the α subclass of the classproteobacteria, 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, andpurU, were shown to be essential for growth ofMethylobacterium chloromethanicum on chloromethane. ThecmuA and cmuB genes were used as probes to identify homologs in H. chloromethanicum. A cmugene 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 fromM. 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 fromM. 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 C1 transfer pathway for carbon assimilation and CO2 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.