TRANSFORMATION OF BACILLUS-STEAROTHERMOPHILUS WITH PLASMID DNA AND CHARACTERIZATION OF SHUTTLE VECTOR PLASMIDS BETWEEN BACILLUS-STEAROTHERMOPHILUS AND BACILLUS-SUBTILIS
TRANSFORMATION OF BACILLUS-STEAROTHERMOPHILUS WITH PLASMID DNA AND CHARACTERIZATION OF SHUTTLE VECTOR PLASMIDS BETWEEN BACILLUS-STEAROTHERMOPHILUS AND BACILLUS-SUBTILIS
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DOI：
10.1128/jb.149.3.824-830.1982
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发表时间：
1982-01-01
影响因子：
3.2
通讯作者：
AIBA, S
中科院分区：
文献类型：
--
作者：
IMANAKA, T;FUJII, M;AIBA, S
用pUB110(卡那霉素抗性，Kmr)、pTb19(Kmr和四环素抗性[Tcr])及其衍生物pTb90(KmrTcr)，在48℃的聚乙二醇存在下，用原生质体程序转化嗜热脂肪杆菌B.stearthermophilus IFO 12980(ATCC 12980)。PUB110、pTB19和pTB90的每个再生剂的转化频率为5.9倍。10-3，5.5倍。10-3和2.0倍。10-1。其中pTB90是新衍生的，并构建了限制性内切酶酶切图谱。当添加四环素(5微克/毫升)时，嗜热脂肪芽孢杆菌中pTB90的拷贝数比添加卡那霉素(5微克/毫升)时高出约4倍。从嗜热脂肪芽孢杆菌中提取的质粒也可以转化枯草芽孢杆菌，反之亦然。因此，pUB110、pTB19和pTB90可作为嗜热脂肪芽孢杆菌和枯草杆菌之间的穿梭载体。PTB19在枯草芽孢杆菌和嗜热脂肪芽孢杆菌中的复制要求似乎不同，因为pTB19的一些缺失质粒(pTB51、pTB52和pTB53)只能在枯草杆菌中复制，而另一种缺失质粒pTB92只能在嗜热脂肪杆菌中复制。表达载体pTB19和pTB90在嗜热脂肪芽孢杆菌中的表达可达65℃。C，而pUB110在同一菌株中的表达高达55度。C。
A thermophilic bacterium B. stearothermophilus IFO 12550 (ATCC 12980) was transformed by plasmid pUB110 (kanamycin resistance, Kmr), plasmid pTB19 (Kmr and tetracycline resistance [Tcr]), and by the pTB19 derivative, plasmid pTB90 (KmrTcr), by the protoplast procedure in the presence of polyethylene glycol at 48.degree. C. The transformation frequencies per regenerant for pUB110, pTB19 and pTB90 were 5.9 .times. 10-3, 5.5 .times. 10-3 and 2.0 .times. 10-1, respectively. Among these plasmids, pTB90 was newly derived, and the restriction endonuclease cleavage map was constructed. When tetracycline (5 .mu.g/ml) was added into the culture medium, the copy number of pTB90 in B. stearothermophilus was about 4-fold higher than that when kanamycin (5 .mu.g/ml) was added instead of tetracycline. B. subtilis could also be transformed with the plasmids extracted from B. stearothermophilus and vice versa. Accordingly, pUB110, pTB19 and pTB90 served as shuttle vectors between B. stearothermophilus and B. subtilis. The requirements for replication of pTB19 in B. subtilis and B. stearothermophilus appear to be different, because some deletion plasmids (pTB51, pTB52 and pTB53) derived from pTB19 could replicate only in B. subtilis, whereas another deletion plasmid pTB92 could replicate solely in B. stearothermophilus. Plasmids pTB19 and pTB90 could be maintained and expressed in B. stearothermophilus up to 65.degree. C, whereas the expression of pUB110 in the same strain was up to 55.degree. C.