The resolution and regeneration of a cointegrate plasmid reveals a model for plasmid evolution mediated by conjugation and oriT site‐specific recombination

Abstract

Cointegrate plasmids are useful models for the study of plasmid evolution if their evolutionary processes can be replicated under laboratory conditions. pBMB0228, a 17 706 bp native plasmid originally isolated from Bacillus thuringiensis strain YBT-1518, carries two nematicidal crystal protein genes, cry6Aa and cry55Aa. In this study, we show that pBMB0228 is in fact a cointegrate of two plasmids and contains two functional replication regions and two functional mobilization regions. Upon introduction into B. thuringiensis strain BMB171, pBMB0228 spontaneously resolves into two constituent plasmids via recombination at its oriT1 and oriT2 sites. The resolution does not require conjugation but can be promoted by conjugation. We further confirm that the resolution is mediated by oriT site-specific recombination requiring Mob02281 or Mob02282. Additionally, the two constituent plasmids of pBMB0228 are mobilizable, and can fuse back via oriT site-specific integration after entering into the same cell by conjugation. Our study confirms that native plasmid can reversibly interconvert between a cointegrate structure and its constituent plasmids. This study provides insight into the evolution of cointegrate plasmids, linking plasmid evolution with conjugation and the oriT site-specific recombination function of relaxase.

Diversity of Integrative and Conjugative Elements of Streptococcus salivarius and Their Intra- and Interspecies Transfer

“…This oriT recombination was likely mediated by the relaxase of the ICE, which is able to recognize and nick this sequence. Such oriT site-specific recombination has already been demonstrated for several canonical relaxases of plasmids (belonging to the MobF, MobP, MobC, and MobM families) (4,28,29) and has been suggested to occur in ICEs of the SXT/R391 family and for ICEclc (both encoding canonical relaxases of the MobH family) (30,31). This mechanism that enables creation of hybrid ICEs could thus also be mediated by relaxases of the noncanonical MobT family, which are related to rolling circle replication initiators and are found in ICEs of the Tn916/ICESt3/ICEBs1 family, a family of ICEs widespread in streptococci (11) and other Firmicutes (5).…”

A novel metalloproteinase virulence factor is involved in Bacillus thuringiensis pathogenesis in nematodes and insects

“…To confirm this observation, we evaluated the role of the spores when mixed with different Cry toxins against C. elegans . To prevent the cross‐contamination of crystals with spores, we used a mutant strain YBT‐1517 to collect the spores, which was constructed based on YBT‐1518 to produce spores but no crystals (Wang et al ., ). The crystals of Cry6Aa and Cry5Ba were purified from recombinant B .…”

A minireplicon of plasmid pBMB26 represents a new typical replicon in the megaplasmids of Bacillus cereus group

“…Bioinformatics analysis suggested that larger plasmid may be formed by fusion of smaller plasmids . The resolution and regeneration events of a native plasmid in B. thuringiensis also support this viewpoint .…”

“…Bioinformatics analysis suggested that larger plasmid may be formed by fusion of smaller plasmids [9]. The resolution and regeneration events of a native plasmid in B. thuringiensis also support this viewpoint [36]. For the plasmid with more than one minireplicon, whether all the minireplicons are functional for plasmid replication is another interesting topic.…”