The Mechanism Of Resolution And Regeneration Of A Native Plasmid PBMB0228from Bacillus Thuringiensis YBT-1518

Plasmids are the key vectors of horizontal gene transfer. Plasmids often introduce genes associated with antibiotic resistance, virulence, detoxification, and other important processes. Therefore, an understanding of plasmid evolution is critical for understanding the acquisition of these important adaptive traits.Cointegrate plasmid formation is a special type of plasmid evolution, in which one plasmid acquires traits for host adaptation by capturing a second plasmid carrying potentially beneficial genes. Many plasmids containing two or more functional replicons have been reported, suggesting cointegrate plasmids exist extensively. If a native cointegrate plasmid has undergone a complicated history of recombination, it may no longer be possible for it to resolve into its constituent plasmids. If so, its precise evolutionary history is difficult to reconstruct. To date, the formation processes of a few cointegrate plasmids have been determined, and homologous recombination and transposon integration have been shown to be related to these processes. A few studies suggest that site-specific recombination at oriT sites may play a role in the evolution of plasmids. Analysis of native cointegrate plasmids could help elucidate this mechanism.pBMB0228is a native plasmid originally isolated from Bacillus thuringiensis strain YBT-1518. In previous study, we found this17.7kb plasmid expresses two nematocidal crystal protein genes, cry6Aa and cry55Aa. So far, pBMB0228is the first small plasmid containing ICP-coding genes. Sequence alignment shows, besides the two ICP-coding genes, pBMB0228contains two Rep-like protein genes and two Mob protein genes. Structurally, pBMB0228looks like a cointegrate plasmid. In this study, we mainly focus on the functional study and the features of the dual genetic elements.1. Functional study of the two replication regions indicated that the two replication regions were functional in B.thuringiensis. Mating experiments show that the two mobilization modules can mobilize the Mob-containing plasmids into recipient strain with the help of conjugative plasmid pAW63::Tn5401.2. pBMB0228::Erm could spontaneously resolve into its constituent plasmids in Bt strain BMB171over a course of multiple generations. Sequence analysis of the two constituent plasmid, pBMB02281and pBMB02282, revealed that they were resolved from pBMB0228at the core regions of oriT1and oriT2. While this event did not require conjugation, it could be promoted by conjugation. To determine if the two Mob proteins involved in the resolution reaction, three mob gene mutants of pBMB0228were constructed and characterized. The results suggested Mob02281or Mob02282was required for the resolution of pBMB0228. Cleavage assay in vitro showed pufied Mob02281and Mob02282can cleave supercoiled DNA containing oriT1or oriT2. Moever, the recombination assays showed Mob02281and Mob02282could mediated the recombination between oriT1and oriT2. These results showed that the resolution mechansim of pBMB0228was oriT site-specific recombination mediated by Mobs.3. Both pBMB02281and pBMB02282contain a mob gene and an oriT site, and both are mobilizable plasmids. When they enter into the same cell, they can fuse into one for generating pBMB0228. The cointegrate plasmid were formed by site-specific recombination of two oriT-containing plasmids in the recipient strain after conjugation. Because conjugation is the most general mechanism of horizontal gene transfer, and both oriT and relaxase are the two common components necessary for conjugation process, so it is easy to envision that the two functions of relaxases may play an important role in plasmid evolution. Therefore, we links the conjugation and site-specific recombination functions of relaxase with plasmid evolution and propose a model for plasmid evolution by relaxase and oriT.4. Using the mob02281gene and oriT sites of pBMB0228, and the core sequences which are necessary for recombination, a temperature-sensitive type recombination vector pRec-mob1-Ts was constructed. This plasmid is also a mobilizable plasmid, which have been successfully mobilized cry6Aa-containing plasmid into several Bacillus cereus group strains. These results suggest conjugation can overcome the transformation problem of some wild Bacillus strains. Moerover, without introduction the resistance marker gene, we successfully realized in-frame deletion of the amyE gene from the chromose of BMB171and insertion of the two crystal genes, cry5Ba2and cry2Aa9, into the amyE gene by using this vector pRec-mob1-Ts.5. The genome sequence of B. thuringiensis YBT-1518was determined and finished. The completed genome of YBT-1518is comprised of a circular chromosome (6,002,284bp) and six circular plasmids, named pBMB0228(17,706bp), pBMB0229(45,206bp), pBMB0230(49,195bp), pBMB0231(146,276bp), pBMB0232(171,593bp) and pBMB0233(240,661bp).This study provides new insights into plasmid evolution by reproducing the formation process of a cointegrate plasmid, linking both the conjugation and oriT site-specific recombination functions of relaxases with plasmid evolution, and propose a model for plasmid evolution.