Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Identification of a Sequence Containing Methylated Cytidine in Corynebacterium glutamicum and Brevibacterium flavum Using Bisulfite DNA Derivatization and Sequencing

  • Jang, Ki-Hyo (Centre for Bioprocessing and Food Technology) ;
  • Chambers, Paul J. (Department of Biological and Food Sciences, Victoria University of Technology) ;
  • Britz, Margaret L. (School of Agriculture and Food System, Gilbert Chandler Campus, The University of Melbourne)
  • Published : 2001.10.01
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Abstract

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 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 G0C 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 geneus contain distinctive restriction and modification systems.

Keywords

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