Two independent replicons can support replication of the anthrax toxin-encoding plasmid pXO1 of Bacillus anthracis

Abstract

The large pXO1 plasmid (181.6kb) of Bacillus anthracis encodes the anthrax toxin proteins. Previous studies have shown that two separate regions of pXO1 can support replication of pXO1 miniplasmids when introduced into plasmid-less strains of this organism. No information is currently available on the ability of the above two replicons, termed RepX and ORFs 14/16 replicons, to support replication of the full-length pXO1 plasmid. We generated mutants of the full-length pXO1 plasmid in which either the RepX or the ORFs 14/16 replicon was inactivated by TargeTron insertional mutagenesis. Plasmid pXO1 derivatives containing only the RepX or the ORFs 14/16 replicon were able to replicate when introduced into a plasmid-less B. anthracis strain. Plasmid copy number analysis showed that the ORFs 14/16 replicon is more efficient than the RepX replicon. Our studies demonstrate that both the RepX and ORFs 14/16 replicons can independently support the replication of the full-length pXO1 plasmid.

Fig. 1

Schematic diagram for TargeTron plasmid construction for B. anthracis. The plasmids were generated as described under materials and methods. The mutated 350 bp intron PCR product used to retarget the intron into the repX and ORF 16 sequences is also indicated.

Fig. 2

Confirmation of the insertional inactivation of the repX gene and ORF 16 in the pXO1 plasmid by PCR and agarose gel electrophoresis. The diagram on the right shows the schematic representation of the insertion of the TargeTron vector into the repX or ORF 16 sequences in pXO1 and the sizes of the expected bands generated by PCR (shown by arrows).

Fig. 3

Agarose gel analysis of DNA isolated from B. anthracis strains containing wild-type pXO1 or its repX- and ORF 16-negative mutants. DNA bands corresponding to the chromosome and pXO1 plasmid are indicated.

Fig. 4

RT-PCR analysis of repX and ORF 16 expression in B. anthracis strains containing either wild-type pXO1, or its repX- and ORF 16-negative mutants. The various amplified cDNA products and their sizes are indicated.