Chloromethane-dependent expression of the cmu gene cluster of Hyphomicrobium chloromethanicum

The methylotrophic bacterium Hyphomicrobium chloromethanicum CM2 can utilize chloromethane (CH3Cl) as the sole carbon and energy source. Previously genes cmuB, cmuC, cmuA, and folD were shown to be essential for the growth of Methylobacterium chloromethanicum on CH3Cl. These CH3Cl-specific genes were subsequently detected in H. chloromethanicum. Transposon and marker exchange mutagenesis studies were carried out to identify the genes essential for CH3Cl metabolism in H. chloromethanicum. New developments in genetic manipulation of Hyphomicrobium are presented in this study. An electroporation protocol has been optimized and successfully applied for transformation of mutagenesis plasmids into H. chloromethanicum to generate stable CH3Cl-negative mutants. Both transposon and marker exchange mutageneses were highly applicable for genetic analysis of Hyphomicrobium. A reliable and reproducible selection procedure for screening of CH3Cl utilization-negative mutants has also been developed. Mutational inactivation of cmuB, cmuC, or hutI resulted in strains that were unable to utilize CH3Cl or to express the CH3Cl-dependent polypeptide CmuA. Reverse transcription-PCR analysis indicated that cmuB, cmuC, cmuA, fmdB, paaE, hutI, and metF formed a single cmuBCA-metF operon and were coregulated and coexpressed in H. chloromethanicum. This finding led to the conclusion that, in cmuB and cmuC mutants, impaired expression of cmuA was likely to be due to a polar effect of the defective gene (cmuB or cmuC) located upstream (5') of cmuA. The detrimental effect of mutation in hutI on the upstream (5')-located cmuA is not clear but indicated that all the genes located within the cmuBCA-metF operon are coordinately expressed. Expression of the cmuBCA-metF transcript was also shown to be strictly CH3Cl inducible and was not repressed by the alternative C-1 substrate methanol. Sequence analysis of a transposon mutant (D20) led to the discovery of the previously undetected hutI and metF genes located 3' of the paaE gene in H. chloromethanicum. MetF, a putative methylene-tetrahydrofolate reductase, had 27% identity to MetF from M. chloromethanicum. Mutational and transcriptional analysis data indicated that, in H. chloromethanicum, CH3Cl is metabolized via a corrinoid-specific (cmuA) and tetrahydrofolate-dependent (metF, purU, folD) methyltransfer system.