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Pulsed-field gel electrophoresis analysis of the genome of amino acid producing corynebacteria: chromosome sizes and diversity of restriction patterns.

A large number of species of corynebacteria are known to be amino acid producers, including members of the genera Corynebacterium and Brevibacterium. Pulsed-field gel electrophoresis (PFGE) of DNA fragments obtained by using endonucleases which recognize AT-rich hexanucleotide or octanucleotide sequences produces a discrete pattern of bands useful for fingerprinting and physical mapping of the chromosome. Using Pacl and Swal endonucleases the genome of Brevibacterium lactofermentum ATCC 13869 (genome size 3052 kb) was consistently cut into 26 and 20 bands, respectively, and the genome of Corynebacterium glutamicum ATCC 13032 (2987 kb) yielded 27 and 26 fragments, respectively. The pattern of restriction fragments was identical for related strains (B. lactofermentum ATCC 13869, B. lactofermentum BLO, B. lactofermentum R31) but different from the pattern of fragments of other soil isolates of the same species (B. lactofermentum DSM 20412) or from closely related organisms such as C. glutamicum; the different pattern of restriction fragments may be used to differentiate taxonomically related species. Brevibacterium linens showed a different behaviour, due to its high G+C content; its genome (3105 kb) was resolved into 8 or 15 fragments, respectively, by digestion with the hexanucleotide-recognizing endonucleases DraI and AseI. PFGE of DNA fragments obtained using these enzymes is a powerful technique for quick resolution of the corynebacteria genome into a small number of large fragments.