Prophages and Introns
The B. anthracis genome contains 4 prophages (6th circle of Suppl Fig. 4), which make up 2.8% of the total chromosome (Table S2). Analysis of the B. cereus 10987 data indicates the presence of a number of phage genes but the locations of the insertions differ between B. anthracis and B. cereus. B. anthracis insertion sites are in genes or intergenic regions conserved with other Bacillus species (supplementary information). Prophage G+C content (Table S2) and trinucleotide composition (Fig. 2) is typical of the B. anthracis genome and the orientations of the insertions preserve the G+C skew and ORF strand bias of the genome (Fig. 2). There are no obvious virulence genes on the prophages and, apart from putative antibiotic resistance determinants and regulatory proteins, the functions of the ORFs are largely unassigned. However, several of the prophage genes encode putative membrane or secreted proteins that may affect B. anthracis interaction with external environments such as the mammalian immune system.
There are two type I introns identified in the chromosome. One, a recently reported unique interruption in the recA gene 1. The other is a phage-like type I intron interrupting ribonucleotide reductase (BA1370). The presence of introns in ribonucleotide reductase has been reported previously in Bacillus prophages 2, although BA1370 is not associated with a prophage in B. anthracis. The two type I introns share only very limited DNA sequence similarity. While the recA intron was present in the same position in B. cereus 10987, BA1370 appeared to missing from the ribonucleotide reductase gene, giving evidence for the mobility of these elements. A type II intron sequence is present on the pXO1 plasmid (pXO1-23 3).
Competence
Natural competence, the ability to import exogenous DNA through a series of programmed genetic events, is a classical feature of B. subtilis4. However, little is known about natural uptake of DNA by B. anthracis5. The B. anthracis genome contains homologs of the key B. subtilis late competence loci comC and comE but the comFB gene is missing and in the place of the comGE and comGG prepilin-like genes are short hypothetical geneGENEs lacking typical type 4 pili N-terminal methylation domains 6. Since all members of the B. subtiliscomG operon are essential for the initial binding of DNA to the cell via the ComEA receptor 7, this suggests an altered DNA processing machinery in relation to B. subtilis. This might cause a reduced competence in B. anthracis and/or different specificity of the DNA taken up by the cell.
Supplementary methods
PCR amplification, PFGE, and Southern blotting.
Bacterial genomic DNA was isolated in agarose plugs as described previously 8. Undigested total genomic DNA was resolved by PFGE in 1% SeaKem LE agarose, 0.25 x TBE pH 8.3 at 14°C, with a pulse time as stated previously 9. Genomic DNA (100 ng) was used with gene specific primers in a PCR where cycling was: 35 cycles (94C for 1 min, 57C for 1 min, 72C for 2 min) using 5 U Dynazyme (Finnzymes Oy). PCR products were purified on agarose gels using the Qiaquick Gel Extraction Kit (QIAGEN), radiolabeling PCR products was performed as described previously 10.
PCR amplicons for microarray were generated as previously described 11 based on B. anthracisAmes genome sequence data at 8X coverage.
Phylogenetic analysis of chromosomal data.
Microarray data. Phylogenetic trees were reconstructed by means of either the Neighbor-Joining (NJ) 12 or the maximum-parsimony (MP) methods. For NJ analyses, pairwise distances between bacterial strains were defined as the percentage of difference between the gene presence/absence patterns of any two strains. Diverged genes were not taken into account. MP was carried out using the Wagner method for binary data, as implemented in the MIX program of the PHYLIP package 13.
Sequence data. Potential homologs of all ORFs within the annotated genome sequence of B. subtilis 168 14 were searched using BLASTP 15. Phylogenetic trees were built using NJ. Pairwise evolutionary distances were computed according to the PAM 001 similarity matrix for protein sequences and the Ka measure for nucleotide sequences. For each of 279 genes, the phylogenies inferred from both types of sequences were identical and thus could be assumed to depict the actual evolutionary history of the gene.Putative signal peptides were identified using SignalP 16 (y-score lower limit = 0.36; S-mean lower limit = 0.54).
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