The cglIM gene of the coryneform soil bacterium Corynebacterium glutamicum ATCC 13032 has been cloned and characterized. The coding region comprises 1092 nucleotides and specifies a protein of 363 amino acid residues with a deduced Mr of 40700. The amino acid sequence showed striking similarities to methyltransferase enzymes generating 5-methylcytosine residues, especially to M x NgoVII from Neisseria gonorrhoeae recognizing the sequence GCSGC. The cglIM gene is organized in an unusual operon which contains, in addition, two genes encoding stress-sensitive restriction enzymes. Using PCR techniques the entire gene including the promoter region was amplified from the wild-type chromosome and cloned in Escherichia coli. Expression of the cglIM gene in E. coli under the control of its own promoter conferred the C. glutamicum-specific methylation pattern to co-resident shuttle plasmids and led to a 260-fold increase in the transformation rate of C. glutamicum. In addition, the methylation pattern produced by this methyltransferase enzyme is responsible for the sensitivity of DNA from C. glutamicum to the modified cytosine restriction (Mcr) system of E. coli.