PLASMID PGA1 FROM CORYNEBACTERIUM-GLUTAMICUM CODES FOR A GENE-PRODUCTTHAT POSITIVELY INFLUENCES PLASMID COPY NUMBER

The complete nucleotide sequence (4,826 bp) of the cryptic plasmid pGA 1 from Corynebacterium glutamicum was determined, DNA sequence analysi s revealed four putative coding regions (open reading frame A [ORFA], ORFA2, ORFB, and ORFC). ORFC was identified as a rep gene coding for a n initiator of plasmid replication (Rep) according to the high level o f homology of its deduced amino acid sequence with the Rep proteins of plasmids pSR1 (from C. glutamicum) and pNG2 (from Corynebacterium dip htheriae). This function was confirmed by deletion mapping of the mini mal replicon of pGA1 (1.7 kb) which contains only ORFC. Deletion deriv atives of pGA1 devoid of ORFA exhibited significant decreases in the c opy number in C. glutamicum cells and displayed segregational instabil ity. Introduction of ORFA in trans into the cells harboring these dele tion plasmids dramatically increased their copy number and segregation al stability. The ORFA gene product thus positively influences plasmid copy number, This is the first report on such activity associated wit h a nonintegrating bacterial plasmid. The related plasmids pGA1, pSR1, and pNG2 lacking significant homology with any other plasmid seem to be representatives of a new group of plasmids replicating in the rolli ng-circle mode.