Kunnimalaiyaan Muthusamy, Vary Patricia S
Department of Biological Sciences, Northern Illinois University, De Kalb 60115, USA.
Appl Environ Microbiol. 2005 Jun;71(6):3068-76. doi: 10.1128/AEM.71.6.3068-3076.2005.
Strain QM B1551 of Bacillus megaterium contains seven compatible plasmids: two small rolling circle plasmids and five theta-replicating plasmids with cross-hybridizing replicons. To expand our understanding of these plasmids, the replicon region (6.7 kb) from pBM300 was cloned, sequenced, and functionally characterized. Sequence analysis showed that the replication protein (RepM300) was highly homologous to two other plasmid Rep proteins of the same strain but to no other known proteins. Furthermore, the location of the replication origin was within the RepM300 coding region, and the origin contained three 12-base direct repeats. Deletion analysis of the replicon confirmed the role of the Rep protein and showed that open reading frame 2 (ORF2) was required for stability. However, the protein encoded by ORF2 is entirely different from the replicon stability proteins encoded by the other two replicons. The entire plasmid was isolated from the plasmid array by integrating a spectinomycin resistance gene and transforming a plasmidless strain, PV361. Complete sequencing showed that pBM300 was 26,300 bp long, had a G+C content of 35.2%, and contained 20 ORFs, two of which encoded proteins that had no similarity to other proteins in the database. The proteins encoded by the plasmid ORFs had similarity to proteins for mobilization and transfer, an integrase, a rifampin resistance protein, a cell wall hydrolase, glutathione synthase, and a biotin carboxylase. The similarities were to several gram-positive genera and a few gram-negative genera and archaea. oriT and ssoT-like regions were detected near two mob genes. These results suggest that pBM300 is a mobilizable hybrid plasmid that confers increased metabolic and germination ability on its host. Its replicon also helps define a new plasmid family.
巨大芽孢杆菌菌株QM B1551含有七个相容质粒：两个小型滚环质粒和五个具有交叉杂交复制子的θ复制质粒。为了加深我们对这些质粒的理解，对来自pBM300的复制子区域（6.7 kb）进行了克隆、测序和功能表征。序列分析表明，复制蛋白（RepM300）与同一菌株的另外两个质粒Rep蛋白高度同源，但与其他已知蛋白无同源性。此外，复制起点位于RepM300编码区域内，该起点包含三个12碱基的直接重复序列。对复制子的缺失分析证实了Rep蛋白的作用，并表明开放阅读框2（ORF2）对稳定性是必需的。然而，ORF2编码的蛋白与其他两个复制子编码的复制子稳定性蛋白完全不同。通过整合壮观霉素抗性基因并转化无质粒菌株PV361，从质粒阵列中分离出完整的质粒。完整测序表明，pBM300长26,300 bp，G+C含量为35.2%，包含20个开放阅读框，其中两个编码的蛋白与数据库中的其他蛋白无相似性。质粒开放阅读框编码的蛋白与参与转移和转运的蛋白、一种整合酶、一种利福平抗性蛋白、一种细胞壁水解酶、谷胱甘肽合酶和一种生物素羧化酶具有相似性。这些相似性存在于几个革兰氏阳性菌属、少数革兰氏阴性菌属和古菌中。在两个mob基因附近检测到oriT和ssoT样区域。这些结果表明，pBM300是一种可移动的杂交质粒，赋予其宿主增强的代谢和萌发能力。其复制子也有助于定义一个新的质粒家族。
