Bacillus anthracis virulent plasmid pX02 genes found in large plasmids of two other Bacillus species

Abstract

In order to cause the disease anthrax, Bacillus anthracis requires two plasmids, pX01 and pX02, which carry toxin and capsule genes, respectively, that are used as genetic targets in the laboratory detection of the bacterium. Clinical, forensic, and environmental samples that test positive by PCR protocols established by the Centers for Disease Control and Prevention for B. anthracis are considered to be potentially B. anthracis until confirmed by culture and a secondary battery of tests. We report the presence of 10 genes (acpA, capA, capB, capC, capR, capD, IS1627, ORF 48, ORF 61, and repA) and the sequence for the capsule promoter normally found on pX02 in Bacillus circulans and a Bacillus species closely related to Bacillus luciferensis. Tests revealed these sequences to be present on a large plasmid in each isolate. The 11 sequences consistently matched to B. anthracis plasmid pX02, GenBank accession numbers AF188935.1, AE011191.1, and AE017335.3. The percent nucleotide identities for capD and the capsule promoter were 99.9% and 99.7%, respectively, and for the remaining nine genes, the nucleotide identity was 100% for both isolates. The presence of these genes, which are usually associated with the pX02 plasmid, in two soil Bacillus species unrelated to B. anthracis alerts us to the necessity of identifying additional sequences that will signal the presence of B. anthracis in clinical, forensic, and environmental samples.

FIG. 1.

Map of oligonucleotide primers and probes for selected genes and ORFs on pX02. The genes were selected because they have not been previously described in the genomes or plasmids of Bacillus species other than B. anthracis, B. cereus, and B. thuringiensis.

FIG. 2.

Restriction enzyme-digested plasmid DNA on 0.7% agarose gels after electrophoresis. (A) B. anthracis CBD 63; (B) CBD 118; (C) CBD 119. Lanes 1, 8, and 15, HindIII-digested Lambda DNA marker; lanes 2, 9, and 16, plasmid DNA digested with EcoNI; lanes 3, 10, and 17, plasmid DNA digested with KpnI; lanes 4, 11, and 18, plasmid DNA digested with MluI; lanes 5, 12, and 19, plasmid DNA digested with NotI; lanes 6, 13, and 20, plasmid DNA digested with PvuI; lanes 7, 14, and 21, plasmid DNA digested with XhoI.

FIG. 3.

Hybridization of Southern blotted membranes of digested plasmid DNA with capC oligonucleotide probe. (A) B. anthracis CBD 63; (B) CBD 118; (C) CBD 119. Lanes 1, 7, and 13, plasmid DNA digested with EcoNI; lanes 2, 8, and 14, plasmid DNA digested with KpnI; lanes 3, 9, and 15, plasmid DNA digested with MluI; lanes 4, 10, and 16, plasmid DNA digested with NotI; lanes 5, 11, and 17, plasmid DNA digested with PvuI; lanes 6, 12, and 18, plasmid DNA digested with XhoI.

FIG. 4.

Partial sequence of the capD gene showing a single base pair difference. At position 53183 (based upon the GenBank sequence of NC_002146.1), CBD 118 and 119 match four of the six sequences of the pX02 of B. anthracis but are different from the other two GenBank sequences (A→G) (AF0188935.1 and NC_002146.1). CBD 63 is B. anthracis Pasteur strain, CBD 118 is B. circulans, CBD 119 is a Bacillus sp., and CBD 131 is B. anthracis. Strains designated AF188935.1 (B. anthracis), AE011191.1 (B. anthracis A2012), AE017335.3 (B. anthracis Ames), and NC_002146.1 (B. anthracis Pasteur) are the sequences available in GenBank for the B. anthracis pX02 plasmid.

FIG. 5.

Two partial sequences of the capsule operon promoter showing a single base pair difference at each location. At position 57837 (based upon the GenBank sequence of NC_002146.1), CBD 118 and 119 match four of the six sequences of the pX02 of B. anthracis and differ from the other two sequences (A→G). At position 58137 (based upon the GenBank sequence of NC_002146.1 CBD 118 and 119 have a cysteine (C) matching CBD 63 and 131 instead of a thymine (T) as the other sequences from GenBank have. CBD 63 is B. anthracis Pasteur strain, CBD 118 is B. circulans, CBD 119 is a Bacillus sp., and CBD 131 is B. anthracis. AF188935 (B. anthracis), AE011191.1 (B. anthracis A2012), AE017335 (B. anthracis Ames), and NC_002146.1 (B. anthracis Pasteur) are the sequences available on GenBank for the B. anthracis pX02 plasmid.