The bcr1 DNA repeat element is specific to the Bacillus cereus group and exhibits mobile element characteristics

Abstract

Bacillus cereus strains ATCC 10987 and ATCC 14579 harbor an approximately 155-bp repeated element, bcr1, which is conserved in B. cereus, B. anthracis, B. thuringiensis, and B. mycoides but not in B. subtilis and B. licheniformis. In this study, we show by Southern blot hybridizations that bcr1 is present in all 54 B. cereus group strains tested but absent in 11 Bacillus strains outside the group, suggesting that bcr1 may be specific and ubiquitous to the B. cereus group. By comparative analysis of the complete genome sequences of B. cereus ATCC 10987, B. cereus ATCC 14579, and B. anthracis Ames, we show that bcr1 is exclusively present in the chromosome but absent from large plasmids carried by these strains and that the numbers of full-length bcr1 repeats for these strains are 79, 54, and 12, respectively. Numerous copies of partial bcr1 elements are also present in the three genomes (91, 128, and 53, respectively). Furthermore, the genomic localization of bcr1 is not conserved between strains with respect to chromosomal position or organization of gene neighbors, as only six full-length bcr1 loci are common to at least two of the three strains. However, the intergenic sequence surrounding a specific bcr1 repeat in one of the three strains is generally strongly conserved in the other two, even in loci where bcr1 is found exclusively in one strain. This finding indicates that bcr1 either has evolved by differential deletion from a very high number of repeats in a common ancestor to the B. cereus group or is moving around the chromosome. The identification of bcr1 repeats interrupting genes in B. cereus ATCC 10987 and ATCC 14579 and the presence of a flanking TTTAT motif in each end show that bcr1 exhibits features characteristic of a mobile element.

FIG. 1.

Southern blot of genomic DNA from a sample of B. cereus group strains hybridized with the bcr1 probe (see Table 1 for strain numbers). Species designations are as follows: Bc, B. cereus; Bt, B. thuringiensis; Bw, B. weihenstephanensis; Bct, B. cereus/B. thuringiensis (not investigated for crystal toxin formation).

FIG. 2.

Chromosomal distribution of bcr1 repeats in B. anthracis Ames, B. cereus ATCC 10987, and B. cereus ATCC 14579. Full-length (FL) (121 to 163 bp) elements are shown only in (a), while full-length and partial (P) (30 to 119 bp) elements are shown in (b). Each tick mark corresponds to a repeat in the circular representation of the chromosome of a strain; black, repeat located on the forward DNA strand (outer circle); red, repeat located on the reverse DNA strand (inner circle). Position 1 on the ruler indicates the origin of replication.

FIG. 3.

Size distribution of bcr1 elements (≥30 bp) from (a) B. anthracis Ames, (b) B. cereus ATCC 10987, and (c) B. cereus ATCC 14579.

FIG. 4.

Multiple alignment of 145 full-length bcr1 elements (≥120 bp) from B. anthracis Ames, B. cereus ATCC 10987, and B. cereus ATCC 14579. Sequences were aligned by using CLUSTALW (35) followed by manual editing with SEAVIEW (11). Consensus sequences computed with thresholds of 51, 60, 70, 80, and 90% conservation are included at the bottom of the alignment. When a threshold T is used, a residue appears in the consensus if it is present in ≥T% of the 145 bcr1 elements; otherwise, the character N is assigned. Furthermore, a plot of the conservation of each site was generated by CLUSTALX (36) and is shown below the alignment. The asterisk above the alignment indicates the single site where all 145 sequences have an identical nucleotide. Sequence lengths are given to the left of the alignment. Indels are represented by dashes. Bc denotes B. cereus.

FIG. 5.

Phylogenetic tree of 145 full-length bcr1 sequences (≥120 bp) from B. anthracis Ames, B. cereus ATCC 10987, and B. cereus ATCC 14579. The tree was based on the multiple alignment shown in Fig. 4 and was built by using the neighbor-joining method (32) applied to a matrix of pairwise distances between sequences. Distances were computed following Kimura's (19) two-parameter substitution model. Gaps (indels) were removed specifically for each pair of sequences compared. The scale bar shows the average number of nucleotide substitutions per site. bcr1 repeats are color coded based on the strain of origin: blue, B. cereus ATCC 14579; green, B. cereus ATCC 10987; red, B. anthracis Ames.

FIG. 6.

Examples of genetic organization around chromosomal bcr1 loci in B. anthracis Ames, B. cereus ATCC 10987, and B. cereus ATCC 14579. Green blocks represent full-length bcr1 elements, while all other blocks represent genes. (a) bcr1 locus conserved in all three strains (repeats Bc14579_1R, Banthracis_1R, and Bc10987_59R [see http://www.salmongenome.no/htdocs/Suppl_info_for_web_Okstadetal2004.html]); (b) bcr1 locus shared by B. anthracis Ames and B. cereus ATCC 10987 only (Banthracis_4F and Bc10987_64F, respectively); (c) bcr1 locus unique to B. cereus ATCC 10987 (Bc10987_28R); (d) bcr1 element (Bc10987_60F) inserted within a gene encoding a putative DNase of the TatD family (BCE0237) in B. cereus ATCC 10987. In each panel, the top strain is the reference. Additional genes in the bottom two strains that are not orthologous (as defined in Materials and Methods) to genes in the reference strain are coded in grey (orthologous in the bottom two strains) or black (not orthologous to any gene in the corresponding region of the other strains). Genes orthologous in all three strains are drawn in the same color. Right- and left-oriented arrows indicate that the gene or repeat is on the forward or reverse strand, respectively. Rulers are in base pairs.

FIG. 7.

Chromosomal organization of bcr1 gene neighbors and their orthologues in B. anthracis Ames, B. cereus ATCC 10987, and B. cereus ATCC 14579. Each panel shows the comparison of two strains. Red circles represent a protein-encoding ORF neighboring a full-length bcr1 element in one strain and its orthologue (as defined in Materials and Methods) in the other. Green crosses represent rRNA genes neighboring bcr1 and their orthologues. Blue triangles correspond to shared bcr1 elements in the two organisms. All genes and repeats are plotted according to their positions in the chromosomes. For any given bcr1 repeat, three upstream and three downstream genes were examined; 411, 327, and 573 orthologous pairs are displayed in (a), (b), and (c), respectively.

FIG. 8.

Histogram of the difference in distance between bcr1 gene neighbors and their orthologues in B. anthracis Ames, B. cereus ATCC 10987, and B. cereus ATCC 14579. The graph shows the distribution of the difference between two distances, the distance between the upstream and downstream genes neighboring a bcr1 element in a strain and the distance between the orthologues of these genes in another strain lacking bcr1 in the corresponding locus. The difference in distance was computed for all loci where bcr1 is present in one strain and absent in another and for all pairwise comparisons of the three strains (178 comparisons in total).