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

Abstract

In this paper we describe transformation of Clavibacter michiganensis subsp. sepedonicus, the potato ring rot 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 with 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 4.6 ؋ 10 4 CFU/g of pHN216 plasmid DNA. To demonstrate the utility of this transformation system, we cloned a ␤-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.

A family of serine proteases of Clavibacter michiganensis subsp. michiganensis: chpC plays a role in colonization of the host plant tomato

“…To confirm that the phenotype of the CMM101 chpC β mutant was not caused by some other mutation in the chromosome, a merozygote containing the inactivated and the wild‐type chpC gene was constructed. A 2.5‐kb Eco RV fragment containing the complete chpC of cmis2p456d03 was cloned into the Eco RV‐digested E. coli – Cmm shuttle vector pHN216 (Laine et al ., 1996), resulting in plasmid pIG216Cβ, which was introduced into the chpC mutant by electroporation. Plasmid DNA from neomycin‐resistant colonies was isolated and the presence of the plasmids was verified by Nco I digestion (data not shown).…”

“…Plasmid pIG216Cβ used for complementation was constructed based on the E. coli – Cmm shuttle vector pHN216 (Laine et al ., 1996). The 2.5‐kb Eco RV fragment of cmis2p0456d03 carrying the chpC gene was integrated into the vector pHN216 linearized by Eco RV.…”

Interactions between Clavibacter michiganensis and its host plants

“…sepedonicus or C. michiganensis ssp. michiganensis (Laine et al, 1996). This is now clear evidence that endo-␤-1,4-glucanases play a role in the establishment of the wilting disease in tomato and potato.…”

“…michiganensis NCPPB382 using the bacteriophage CMP1 and electroporation of DNA (Meletzus and Eichenlaub, 1991). This provided the basis for the construction of cloning vectors for C. michiganensis (Meletzus and Eichenlaub, 1991;Laine et al, 1996). As strain C. michiganensis ssp.…”

“…michiganensis NCPPB382 carries two endogenous plasmids, pCM1 (27.5 kb) and pCM2 (72 kb), these were chosen as a source of a Clavibacter-specific replicon that could be integrated into pBR325 derivatives carrying antibiotic resistance genes such as the neomycin phosphotransferase gene (nptII) of Tn5, the gentamicin acetyltransferase gene of Tn1696 or the chloramphenicol resistance gene from Corynebacterium striatum (xerosis) M82B (Tauch et al, 1998), which are generally well expressed in Gram-positive bacteria. With this strategy, two families of shuttle vectors were constructed, carrying the replicon of either pCM1 or pCM2, which were able to replicate stably in various subspecies of C. michiganensis, such as subspecies michiganensis, sepedonicus, nebraskensis and insidiosus (Meletzus and Eichenlaub, 1991;Laine et al, 1996).…”