Transformation of the phytopathogenic bacterium Clavibacter michiganense subsp. michiganense by electroporation and development of a cloning vector.

We constructed a cloning vector for use in the plant pathogenic bacterium Clavibacter michiganense subsp. michiganense. The vector pDM100 consists of a 3.2-kb restriction fragment of the Clavibacter plasmid pCM1 joined to a pBR325 derivative carrying the neomycin phosphotransferase of transposon Tn5 and the gentamicin acetyltransferase of Tn1696. Both antibiotic resistance genes are efficiently expressed in C. michiganense subsp. michiganense. Although polyethylene glycol-mediated transfection of spheroplasts with the DNA of the C. michiganense subsp. michiganense-specific bacteriophage CMP1 yielded about 3 x 10(3) transfectants per microgram of DNA, in transformations with plasmid DNA only a very few transformants were obtained. However, the transformation efficiency could be improved by electroporation of intact cells, giving about 2 x 10(3) transformants per microgram of plasmid DNA. Since a transformation procedure and a cloning vector are now available, pathogenicity in C. michiganense subsp. michiganense can now be analyzed genetically

Evidence for plasmid-encoded virulence factors in the phytopathogenic bacterium Clavibacter michiganensis subsp. michiganensis NCPPB382.

The tomato pathogen Clavibacter michiganensis subsp. michiganensis NCPPB382, which causes bacterial wilt, harbors two plasmids pCM1 (27.5 kb) and pCM2 (72 kb). After curing of the plasmids, bacterial derivatives were still proficient in the ability to colonize the host plant and in the production of exopolysaccharides but exhibited a reduced virulence. When one of the two plasmids is lost, there is a significant delay in the development of wilting symptoms after infection and a plasmid-free derivative is not able to induce disease symptoms. By cloning of restriction fragments of both plasmids in the plasmid-free strain CMM100, two DNA fragments which restored the virulent phenotype were identified. Further analysis suggested that a fragment of plasmid pCM1 encodes an endocellulase which is involved in the expression of the pathogenic phenotype

Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp sepedonicus with several cloning vectors

Laine MJ, Nakhei H, Dreier J, et al. Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp sepedonicus with several cloning vectors. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 1996;62(5):1500-1506.In this paper we describe transformation of Clavibacter michiganensis subsp, sepedonicus, the potato ring rat bacterium, with plasmid vectors, 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, pHN205, and pHN216 were stably maintained without antibiotic selection in 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 efficiencies, We also found unexplained strain-to-strain differences in stability,vith various plasmid constructions containing different arrangements of antibiotic resistance genes and origins of replication, We examined the effect of a number of factors on transformation efficiency. The best transformation efficiencies were obtained when C, michiganensis subsp, 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 was 4.5 x 10(4) CFU/mu g of pHN216 plasmid DNA. To demonstrate the utility of this transformation system, we cloned a beta-1,4-endoglucanase-encoding gene from C. michiganensis subsp. sepedonicus into pHN216. When this construction, pHN216:C8, was electroporated into competent cells of a cellulase-deficient mutant, it restored cellulase production to almost wild-type levels