Lysinibacillus sphaericus C3-41 carries a large low-copy-number plasmid pBsph, which encodes binary toxin proteins. Our previous study found that the transcriptional activator TubX plays an important role in the newly identified type Ⅲ TubRZC replication/partition system in pBsph, and that a vector consisting of tubRZC and tubX is not as stable as pBsph, indicating the presence of other maintenance module(s). In this study, we identified that orf9 and orf10 are necessary for the stability of pBsph by a series of deletion and complementation experiments. Bioinformatics analysis showed that ORF9 contains a PIN domain of VapBC toxin-antitoxin (TA) system, whereas ORF10 share no significant sequence similarity to any of the characterized antitoxins in the database. Further studies revealed that orf9 and orf10 are transcribed as an operon. The overexpression of ORF9 repressed the growth of both Escherichia coli and L. sphaericus, which can be alleviated by overexpression of ORF10. The deletion of orf10 individually or orf9-10 together resulted a decrease on plasmid stability which was restored by the complementation of corresponding gene(s), suggesting that ORF10 plays an important role in plasmid stability. In addition, it was found the plasmid stability is related with the transcription level of tubRZ, and overexpression of TubRZ could neutralize the negative effect on plasmid stability caused by the deletion of orf9-orf10. Moreover, the recombinant vector containing tubRZC, tubX and orf9-10 was more stable than the ones containing only tubRZC and either tubX or orf9-10. The data indicate that the plasmid maintenance system on pBsph includes orf9-orf10 TA system.

球形芽孢杆菌C3-41携带一个大的低拷贝数质粒pBsph，该质粒编码二进制毒素蛋白。我们先前的研究发现，转录激活因子TubX在pBsph中新近鉴定的Ⅲ型TubRZC复制/分区系统中起着重要作用，并且由tubRZC和tubX组成的载体不如pBsph稳定，表明存在其他维护模块（s）。在这项研究中，我们确定了orf9和orf10通过一系列缺失和互补实验，pBsph的稳定性是必需的。生物信息学分析表明，ORF9包含VapBC毒素-抗毒素（TA）系统的PIN域，而ORF10与数据库中任何已表征的抗毒素没有明显的序列相似性。进一步的研究表明，orf9和orf10被转录为操纵子。ORF9的过表达抑制大肠杆菌和球形乳杆菌的生长，这可以通过ORF10的过表达来缓解。分别删除orf10或orf9-10一起导致质粒稳定性降低，其通过相应基因的互补而得以恢复，这表明ORF10在质粒稳定性中起重要作用。另外，发现质粒稳定性与tubRZ的转录水平有关，并且TubRZ的过表达可以中和由orf9-orf10的缺失引起的对质粒稳定性的负面影响。此外，含有tubRZC，tubX和orf9-10的重组载体比仅含有tubRZC和tubX或orf9-10的重组载体更稳定。数据表明pBsph上的质粒维持系统包括orf9-orf10 TA系统。