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KMID : 0545120010110050819
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Journal of Microbiology and Biotechnology
2001 Volume.11 No. 5 p.819 ~ p.824
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Identification of a Sequence Containing Methylated Cytidine in Corynebacterium glutamicum and Brevibacterium flavum Using Bisulfite DNA Derivatization and Sequencing
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JANG, KI-HYO
CHAMBERS, PAUL J./BRITZ, MARGARET L.
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Abstract
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The principal DNA modification systems of the amino-acid-producing bacteria Corynebacterium glutamicum AS019, Brevibacterium flavum BF4, and B. lactofermentum BL1 was investigated using two approaches; digestion of plasmid DNA isolated from these species using TseI and Fnu4HI, and sequence analysis of the putative methyltransferase target sites following the derivatization of DNA using metabisulfite treatment. The C. glutamicum and B. flavum strains showed similar digestion patterns to the two enzymes, indicating that the target for cytidine methyltransferase recognizes 5´-GCSGC-3´ (where S is either G or C). Mapping the methylated cytidine sites by bisulfite derivatization, followed by PCR amplification and sequencing, was only possible when the protocol included an additional step eliminating any underivatized DNA after PCR amplification, thereby indicating that the derivatization was not 100% efficient. This may have been due to the high G-C content of this genus. It was confirmed that C. glutamicum AS019 and B. flavum BF4 methylated the cytidine in the Gm^5CCGC sequences, yet there were no similar patterns of methylation in B. lactofermentum, which was consistent with the distinctive degradation pattern seen for the above enzymes. These findings demonstrate the successful application of a modified bisulfite derivatization method with the Corynebacterium species for determining methylation patterns, and showed that different species in the genus contain distinctive restriction and modification systems.
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