CLONING AND CHARACTERIZATION OF A DNA REGION ENCODING A STRESS-SENSITIVE RESTRICTION SYSTEM FROM CORYNEBACTERIUM-GLUTAMICUM ATCC-13032 AND ANALYSIS OF ITS ROLE IN INTERGENERIC CONJUGATION WITH ESCHERICHIA-COLI

RP4-mediated transfer of mobilizable plasmids in intergeneric conjugat ion of Escherichia coli donors with Corynebacterium glutamicum ATCC 13 032 is severely affected by a restriction system in the recipient that can be inactivated by a variety of exogenous stress factors. In this study a rapid test procedure based on intergeneric conjugal plasmid tr ansfer that permitted the distinction between restriction-negative and restriction-positive C. glutamicum clones was developed. By using thi s procedure, clones of the restriction-deficient mutant strain C. glut amicum RM3 harboring a plasmid library of the wild-type chromosome wer e checked for their restriction properties. A complemented clone with a restriction-positive phenotype was isolated and found to contain a p lasmid with a 7-kb insertion originating from the wild-type chromosome . This plasmid, termed pRES806, is able to complement the restriction- deficient phenotype of different C. glutamicum mutants. Sequence analy sis revealed the presence of two open reading frames (orf1 and orf2) o n the complementing DNA fragment. The region comprising orf1 and orf2 displayed a strikingly low G+C content and was present exclusively in C. glutamicum strains. Gene disruption experiments with the wild type proved that orf1 is essential for complementation, but inactivation of orf2 also resulted in a small but significant increase in fertility. These results were confirmed by infection assays with the bacteriophag e CL31 from Corynebacterium lilium ATCC 15990.