State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China.
PLoS One. 2011 Jan 25;6(1):e16025. doi: 10.1371/journal.pone.0016025.
Bacillus thuringiensis is the most widely used bacterial bio-insecticide, and most insecticidal crystal protein-coding genes are located on plasmids. Most strains of B. thuringiensis harbor numerous diverse plasmids, although the plasmid copy numbers (PCNs) of all native plasmids in this host and the corresponding total plasmid DNA amount remains unknown. In this study, we determined the PCNs of 11 plasmids (ranging from 2 kb to 416 kb) in a sequenced B. thuringiensis subsp. kurstaki strain YBT-1520 using real-time qPCR. PCNs were found to range from 1.38 to 172, and were negatively correlated to plasmid size. The amount of total plasmid DNA (∼8.7 Mbp) was 1.62-fold greater than the amount of chromosomal DNA (∼5.4 Mbp) at the mid-exponential growth stage (OD(600) = 2.0) of the organism. Furthermore, we selected three plasmids with different sizes and replication mechanisms to determine the PCNs over the entire life cycle. We found that the PCNs dynamically shifted at different stages, reaching their maximum during the mid-exponential growth or stationary phases and remaining stable and close to their minimum after the prespore formation stage. The PCN of pBMB2062, which is the smallest plasmid (2062 bp) and has the highest PCN of those tested, varied in strain YBT-1520, HD-1, and HD-136 (172, 115, and 94, respectively). These findings provide insight into both the total plasmid DNA amount of B. thuringiensis and the strong ability of the species to harbor plasmids.
苏云金芽孢杆菌是应用最广泛的细菌性生物杀虫剂，其大多数杀虫晶体蛋白编码基因位于质粒上。大多数苏云金芽孢杆菌菌株含有许多不同的质粒，尽管该宿主中所有天然质粒的质粒拷贝数（PCN）和相应的总质粒 DNA 量仍不清楚。在这项研究中，我们使用实时 qPCR 测定了已测序的苏云金芽孢杆菌亚种 kurstaki 菌株 YBT-1520 中的 11 个质粒（大小范围为 2kb 至 416kb）的 PCN。发现 PCN 范围为 1.38 至 172，与质粒大小呈负相关。在该生物体的对数中期（OD（600）= 2.0），总质粒 DNA（约 8.7 Mbp）的量比染色体 DNA（约 5.4 Mbp）多 1.62 倍。此外，我们选择了三个大小和复制机制不同的质粒来确定整个生命周期中的 PCN。我们发现 PCN 在不同阶段动态变化，在对数中期或静止期达到最大值，并在芽孢前形成阶段后保持稳定且接近最小值。pBMB2062 的 PCN 变化最大，pBMB2062 是最小的质粒（2062bp），也是测试中 PCN 最高的质粒，在菌株 YBT-1520、HD-1 和 HD-136 中分别变化为 172、115 和 94。这些发现为苏云金芽孢杆菌的总质粒 DNA 量和该物种强烈的质粒容纳能力提供了深入的了解。
