Laine M J, Nakhei H, Dreier J, Lehtilä K, Meletzus D, Eichenlaub R, Metzler M C
Department of Biology, University of Turku, Finland.
Appl Environ Microbiol. 1996 May;62(5):1500-6. doi: 10.1128/aem.62.5.1500-1506.1996.
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 x 10(4) CFU/microgram 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.
在本文中，我们描述了用质粒载体转化马铃薯环腐病菌——密执安棒杆菌密歇根亚种。所使用的三种质粒pDM100、pDM302和pDM306含有来自密执安棒杆菌密歇根亚种天然质粒pCM1的复制起点。我们通过使用密执安棒杆菌密歇根亚种另一种天然质粒pCM2的复制起点构建了两种新的克隆载体pHN205和pHN216。质粒pDM302、pHN205和pHN216在密执安棒杆菌密歇根亚种的各种菌株中无需抗生素选择就能稳定维持。我们观察到，对于单个质粒，密执安棒杆菌密歇根亚种的不同菌株表现出显著不同的转化效率。我们还发现，在含有不同抗生素抗性基因排列和复制起点的各种质粒构建体的稳定性方面，存在无法解释的菌株间差异。我们研究了许多因素对转化效率的影响。当密执安棒杆菌密歇根亚种细胞在DM琼脂平板上生长、在指数生长早期收获并新鲜（不冷冻）用于电穿孔时，可获得最佳转化效率。获得的最大转化效率为每微克pHN216质粒DNA 4.6×10⁴CFU。为了证明该转化系统的实用性，我们将密执安棒杆菌密歇根亚种的一个β-1,4-内切葡聚糖酶编码基因克隆到pHN216中。当将此构建体pHN216:C8电穿孔导入纤维素酶缺陷型突变体的感受态细胞时，它将纤维素酶的产生恢复到几乎野生型水平。
