Curing of Plasmid pXO1 from Bacillus anthracis Using Plasmid Incompatibility

Abstract

The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures) can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.

Plasmid Conjugation from Proteobacteria as Evidence for the Origin of Xenologous Genes in Cyanobacteria

“…This technique is based in the fact that, when two plasmids carrying the same origin of replication coexist in a cell, they become unstable due to interactions between their replication machineries. Vectorial incompatibility (one of the plasmids is lost with higher probability than the other [24]) was previously exploited to cure native plasmids from Agrobacterium tumefaciens (27,28), Bacillus anthracis (29), or Yersinia pestis (30), among many other examples. To cure pANL, two dislodging vectors were built: pDEP21 and pDEP23.…”

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

“…In plasmid pXO1 of B. anthracis, repX and pXO1-14/pXO1-16 minireplicons were proven to independently support the replication of the full-length pXO1, and pXO1-14/pXO1-16 was more effective than repX minireplicon [20]. Based on the pXO1-14/pXO1-16 replication region, plasmid pXO1 was cured from B. anthracis using plasmid incompatibility [37]. These results suggested that pXO1-14/pXO1-16 minireplicon is predominantly used for replication of plasmid pXO1.…”

Bacillus anthracis toxins

“…The replication origin of pXO2 is classified as belonging to the pAMβ1 family of theta-replicating conjugative plasmids [45], and it is very similar to the replication origin of pAW63 identified in Bacillus thuringiensis [46]. The replication origin of pXO1 encodes two proteins that are sufficient for pXO1 replication [47,48]. This origin has attributes of theta-replicating plasmid replication origins and is conserved among Bacillus cereus group plasmids.…”

“…It has also been shown that small plasmids derived from pXO1 can displace and thereby cure B. anthracis of pXO1 [48]. Comparison of plasmid-cured variants proved that pXO1 is needed for production of toxin and pXO2 for production of capsule [20].…”