Summary auto-generated
This study characterizes pAW63, a self-transmissible plasmid in Bacillus thuringiensis subsp. kurstaki HD73. The researchers isolated plasmid-cured strains containing only pAW63 or the previously characterized pHT73 to examine their individual conjugative properties. Through hybridization studies, they demonstrated that pAW63 and pHT73 possess distinct replication origins but share sequences homologous to transposon Tn4430 and insertion sequences. When tagged with tetracycline resistance transposon Tn5401, pAW63 transferred to recipients at high efficiency, with over 90% of recipients acquiring the plasmid within 2 hours. Notably, pAW63 proved 10-30 times more efficient at mobilizing the plasmid pBC16 compared to pHT73. The plasmid successfully transferred to multiple Bacillus species including B. israelensis, B. cereus, B. licheniformis, B. subtilis, and B. sphaericus, with conjugative functions expressed in all recipients. The study also demonstrated that mobilization of pBC16 required a functional mob gene, indicating the mechanism differs from aggregation-mediated conjugation systems. These findings clarify the genetic basis of plasmid transfer in B.t. kurstaki and reveal that two independent conjugative systems operate within this strain.
Key findings
- pAW63 is a second self-transmissible plasmid in B.t. kurstaki HD73 with a distinct replication origin from pHT73 but sharing transposon Tn4430 and insertion sequences
- pAW63 transfers to recipient cells with extremely high efficiency (>90% within 2 hours) and is 10-30 times more efficient at mobilizing smaller plasmids than pHT73
- pAW63 successfully transfers to multiple Bacillus species (B. israelensis, B. cereus, B. licheniformis, B. subtilis, B. sphaericus) with functional conjugative expression in all hosts
- Mobilization of plasmids by pAW63 and pHT73 requires a functional mob gene, distinguishing it from aggregation-mediated conjugation systems
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Abstract
Bacillus thuringiensis subspecies kurstaki HD73, toxic for lepidopteran larvae, contains two large self-transmissible plasmids of approximately 75 kb, pHT73 and pAW63. The conjugative plasmid pHT73 has been studied extensively and has been shown to harbour the toxin gene cry1Ac, the transposon Tn4430 and several insertion sequences. In this study it was demonstrated that the minor plasmid pAW63 is also self-transmissible and about 10-30 times more efficient in mobilizing plasmid pBC16. To facilitate direct selection for pAW63 transfer, the plasmid was tagged with the tetracycline resistance transposon Tn5401 and in intraspecies matings it was found that after 2 h, all recipients had acquired a copy of the plasmid. Mating experiments demonstrated that pAW63 could be transferred to Bacillus thuringiensis subsp. israelensis, Bacillus cereus, Bacillus licheniformis, Bacillus subtilis and Bacillus sphaericus, and that the conjugative functions were expressed in these hosts. Hybridization studies showed that the replicons of pAW63 and pHT73 were distinct from one another. Sequences homologous to transposon Tn4430 and several insertion sequences were, however, shown to reside on both plasmids.