Hoflack L, Seurinck J, Mahillon J
Laboratorium Genetika, Universiteit Gent, Belgium.
J Bacteriol. 1997 Aug;179(16):5000-8. doi: 10.1128/jb.179.16.5000-5008.1997.
The complete nucleotide sequence of plasmid pGI3 from Bacillus thuringiensis subsp. thuringiensis H1.1. was obtained. Although this 11,365-bp molecule contained at least 11 putative open reading frames (ORFs), extensive database searches did not reveal any homologous sequences with the exception of ORF6, which displayed similarity to the largest ORF of pSTK1, a 1,883-bp cryptic plasmid isolated from Bacillus stearothermophilus. Deletion analysis to determine the pGI3 minimal replicon revealed that ORF6 is the rep gene. Replication occurred via a single-stranded DNA (ssDNA) intermediate, as demonstrated by S1 treatment and Southern hybridization in nondenaturating conditions. Interestingly, however, no homology was found between the pGI3 (ORF6) and pSTK1 (ORF3) rep genes and those from other single-stranded DNA plasmids, nor was there any DNA similarity to the double-strand origins of replication characterized so far, indicating that pGI3 and pSTK1 form another, new family of ssDNA plasmids. PCR analysis revealed that the pGI3 rep gene is largely distributed among B. thuringiensis strains but can also be found in B. cereus and B. mycoides strains, albeit at a lower frequency. Finally, segregation experiments performed with B. subtilis and B. thuringiensis showed that the pGI3 derivatives, including the minimal replicon, were segregationally stable at temperatures suitable for B. thuringiensis growth (<43 degrees C).
获得了苏云金芽孢杆菌亚种苏云金芽孢杆菌H1.1的质粒pGI3的完整核苷酸序列。尽管这个11365碱基对的分子包含至少11个推定的开放阅读框（ORF），但广泛的数据库搜索未发现任何同源序列，除了ORF6，它与从嗜热脂肪芽孢杆菌分离的1883碱基对的隐蔽质粒pSTK1的最大ORF显示出相似性。用于确定pGI3最小复制子的缺失分析表明ORF6是复制基因。如在非变性条件下通过S1处理和Southern杂交所证明的，复制通过单链DNA（ssDNA）中间体发生。然而，有趣的是，在pGI3（ORF6）和pSTK1（ORF3）的复制基因与其他单链DNA质粒的复制基因之间未发现同源性，并且与迄今为止表征的双链复制起点也没有任何DNA相似性，这表明pGI3和pSTK1形成了另一个新的单链DNA质粒家族。PCR分析表明，pGI3复制基因在苏云金芽孢杆菌菌株中广泛分布，但在蜡样芽孢杆菌和蕈状芽孢杆菌菌株中也能发现，尽管频率较低。最后，用枯草芽孢杆菌和苏云金芽孢杆菌进行的分离实验表明，包括最小复制子在内的pGI3衍生物在适合苏云金芽孢杆菌生长的温度（<43摄氏度）下是分离稳定的。
