Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes

Abstract

The Bacillus anthracis Sterne plasmid pXO1 was sequenced by random, "shotgun" cloning. A circular sequence of 181,654 bp was generated. One hundred forty-three open reading frames (ORFs) were predicted using GeneMark and GeneMark.hmm, comprising only 61% (110,817 bp) of the pXO1 DNA sequence. The overall guanine-plus-cytosine content of the plasmid is 32.5%. The most recognizable feature of the plasmid is a "pathogenicity island," defined by a 44.8-kb region that is bordered by inverted IS1627 elements at each end. This region contains the three toxin genes (cya, lef, and pagA), regulatory elements controlling the toxin genes, three germination response genes, and 19 additional ORFs. Nearly 70% of the ORFs on pXO1 do not have significant similarity to sequences available in open databases. Absent from the pXO1 sequence are homologs to genes that are typically required to drive theta replication and to maintain stability of large plasmids in Bacillus spp. Among the ORFs with a high degree of similarity to known sequences are a collection of putative transposases, resolvases, and integrases, suggesting an evolution involving lateral movement of DNA among species. Among the remaining ORFs, there are three sequences that may encode enzymes responsible for the synthesis of a polysaccharide capsule usually associated with serotype-specific virulent streptococci.

FIG. 1

Predicted pXO1 ORFs and physical map. The positions of ORFs predicted by GeneMark.hmm are illustrated as arrows along the 181,654-bp circular pXO1 plasmid. The precise start and stop for each predicted ORF and the most likely ribosome binding site are listed in features of pXO1 under GenBank accession no. AF065404. The direction of the arrows indicates the direction of transcription in each ORF relative to all the other ORFs. The gene map illustrates the position of the known toxin genes and the IS1627 elements and shows the relationship of the PAI to the remainder of the pXO1 sequence.