STABLE TRANSFORMATION OF THE GRAM-POSITIVE PHYTOPATHOGENIC BACTERIUM CLAVIBACTER-MICHIGANENSIS SUBSP SEPEDONICUS WITH SEVERAL CLONING VECTORS

In this paper we describe transformation of Clavibacter michiganensis subsp, sepedonicus, the potato ring rat bacterium, with plasmid vector s, Three of the plasmids used, pDM100, pDM302, and pDM306, contain the origin of replication from pCM1, a native plasmid of C. michiganensis subsp, michiganensis, We constructed two new cloning vectors, pHN205 and pHN216, by using the origin of replication of pCM2, another native plasmid of C. michiganensis subsp, michiganensis. Plasmids pDM302, pH N205, and pHN216 were stably maintained without antibiotic selection i n various strains of C. michiganensis subsp, sepedonicus. We observed that for a single plasmid, different strains of C, michiganensis subsp , sepedonicus showed significantly different transformation efficienci es, We also found unexplained strain-to-strain differences in stabilit y,vith various plasmid constructions containing different arrangements of antibiotic resistance genes and origins of replication, We examine d the effect of a number of factors on transformation efficiency. The best transformation efficiencies were obtained when C, michiganensis s ubsp, sepedonicus cells were grown on DM agar plates, harvested during the early exponential growth phase, and used fresh (without freezing) for electroporation. The maximal transformation efficiency obtained w as 4.5 x 10(4) CFU/mu g of pHN216 plasmid DNA. To demonstrate the util ity of this transformation system, we cloned a beta-1,4-endoglucanase- encoding gene from C. michiganensis subsp. sepedonicus into pHN216. Wh en this construction, pHN216:C8, was electroporated into competent cel ls of a cellulase-deficient mutant, it restored cellulase production t o almost wild-type levels.