A toxin-antitoxin system is essential for the stability of mosquitocidal plasmid pBsph of Lysinibacillus sphaericus

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 coil 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.