A PHYSICAL AND GENETIC-MAP OF THE CORYNEBACTERIUM-GLUTAMICUM ATCC-13032 CHROMOSOME

A combined physical and genetic map of the Corynebacterium glutamicum ATCC 13032 chromosome was constructed using pulsed-field gel electroph oresis (PFGE) and hybridizations with cloned gene probes. Total genomi c DNA was digested with the meganucleases (5'-ATTTAAAT-3'), PacI SwaI (5'-TTAATTAA-3') and PmeI (5'-GTTTAAAC-3') yielding 26,27, and 23 frag ments, respectively. The chromosomal restriction fragments were then s eparated by PFGE. By summing up the lengths of the fragments generated with each of the three enzymes, a genome size of 3082 +/- 20 kb was d etermined. To identify adjacent SwaI fragments, a genomic cosmid libra ry of C. glutamicum was screened for chromosomal inserts containing Sw aI sites. Southern blots of the PFGE gels were hybridized with these l inking clones to connect the SwaI fragments in their natural order. By this method, about 90% of the genome could be ordered into three cont igs. Two of the remaining gaps were closed by cross-hybridization of b lotted SwaI digests using as probes PacI and PmeI fragments isolated f rom PFGE gels. The last gap in the chromosomal map was closed by hybri dization experiments using partial SwaI digestions, thereby proving th e circularity of the chromosome. By hybridization of gene probes to Sw aI fragments separated by PFGE about 30 genes; including rRNA operons, IS element and transposon insertions were localized on the physical m ap.