DNA sequence conservation between the Bacillus anthracis pXO2 plasmid and genomic sequence from closely related bacteria

Background Complete sequencing and annotation of the 96.2 kb Bacillus anthracis plasmid, pXO2, predicted 85 open reading frames (ORFs). Bacillus cereus and Bacillus thuringiensis isolates that ranged in genomic similarity to B. anthracis, as determined by amplified fragment length polymorphism (AFLP) analysis, were examined by PCR for the presence of sequences similar to 47 pXO2 ORFs. Results The two most distantly related isolates examined, B. thuringiensis 33679 and B. thuringiensis AWO6, produced the greatest number of ORF sequences similar to pXO2; 10 detected in 33679 and 16 in AWO6. No more than two of the pXO2 ORFs were detected in any one of the remaining isolates. Dot-blot DNA hybridizations between pXO2 ORF fragments and total genomic DNA from AWO6 were consistent with the PCR assay results for this isolate and also revealed nine additional ORFs shared between these two bacteria. Sequences similar to the B. anthracis cap genes or their regulator, acpA, were not detected among any of the examined isolates. Conclusions The presence of pXO2 sequences in the other Bacillus isolates did not correlate with genomic relatedness established by AFLP analysis. The presence of pXO2 ORF sequences in other Bacillus species suggests the possibility that certain pXO2 plasmid gene functions may also be present in other closely related bacteria.


Background
Bacillus anthracis contains a 96.2 kb plasmid, pXO2, that is required to cause the disease anthrax [1]. Complete sequencing and annotation (GeneMark.hmm) of pXO2 predicted 85 open reading frames (ORFs) [Genbank accession NC_002146]. Little is known about the identity and function of pXO2 ORFs beyond the virulence genes associated with the B. anthracis capsule (dep, capACB, acpA) [2][3][4][5]. The goal of this study was to determine if many of the novel pXO2 ORFs were unique to B. anthracis, or were conserved in other closely related Bacillus cereus and Bacillus thuringiensis isolates. Conservation of plasmid sequences can provide clues about the origin of the pXO2 plasmid and about potentially conserved gene functions. Identification of ORFs that are specific to B. anthracis are potentially useful as markers for detection of the pathogen in clinical and forensic applications.
Amplified fragment length polymorphism (AFLP) analysis of over 350 B. cereus, B. thuringiensis, and B. anthracis isolates, identified several distinct isolate groups [17,18]. Eight of the B. cereus/B. thuringiensis isolates were found to be very closely related to all B. anthracis isolates and formed a distinct cluster. In the present study, B. cereus and B. thuringiensis isolates that vary in AFLP-based genomic relatedness to B. anthracis were examined for the presence of DNA sequence similar to pXO2, to determine whether portions of this plasmid are conserved in closely related Bacillus isolates, and to determine whether the conservation of pXO2 sequences correlated with genomic relatedness established by AFLP comparisons [17,18].

Results and Discussion
PCR was performed using template DNA from 11 Bacillus isolates that vary in relatedness to B. anthracis with primer sets designed to amplify DNA fragments from 47 different pXO2 ORFs. This method was chosen to detect sequences with potential similarity to pXO2 because it is rapid and the reaction products can be readily sequenced. Table 1 lists the isolates tested, their genomic relatedness to B. anthracis as determined by Jaccard distances calculated from AFLP profile comparisons [17,18], and the number of positive PCR reactions obtained for each isolate. DNA sequencing of the amplified PCR products revealed a high degree of sequence similarity to pXO2 ORFs [Genbank accession numbers AF547271-AF547318]. BLAST (blastn) e-values were 6 × 10 -13 or less for each ORF fragment detected, which corresponded to sequence similarity of 80% or greater. In a previous study, a similar approach was used to demonstrate that many of the ORFs from pXO1, the toxin-encoding plasmid of B. anthracis, were highly conserved in other isolates from the B. cereus/B. thuringiensis group [19].
The number of plasmid ORFs detected in a Bacillus isolate did not correlate directly with phylogenetic relationship to B. anthracis as determined by AFLP. The isolates most closely related to B. anthracis as determined by AFLP produced no more than two PCR products each. However, two of the more distantly related isolates, B. thuringiensis 33679 and B. thuringiensis AWO6, produced 10 and 16 positive PCR reactions, respectively. Neither of these isolates is known to be a human or animal pathogen. Table 2 lists the 47 pXO2 ORFs that were tested in the PCR assay, their putative functions or similarities to other genes (blastp), and the PCR results obtained in this experiment. Nineteen different pXO2 ORF fragments were detected among the 11 Bacillus isolates. Eight of the conserved ORFs were similar to sequences contained in public databases; 11 were unidentified. The only pXO2 ORFs found in common with the isolates most closely related to B. anthracis (Jaccard distance of 0.55 or less) were ORFs 47 and 48. These ORFs have sequence similarity to a conserved hypothetical protein found in several bacterial genera and the tetR family of transcriptional repressors, respectively.  B. thuringiensis AWO6 is a strain containing a 70 kb plasmid designated pAW63 [12,21]. This strain was derived from B. thuringiensis HD73 by curing of its crystal toxin bearing plasmid, pHT73 [12,21]. The pAW63 plasmid contains a replication complex that is classified as a member of the pAMB-1 family of theta replicating plasmids that are present in a broad range of Gram positive species [22]. Plasmid pXO2 also appears to be a pAMB1-like theta replicating plasmid [23] and elements surrounding the replication complex are present in both pXO2 and pAW63 (see pXO2 ORFs 35, 37, 38, 39 in Tables 2 and 3). ORFs 35, 37, and 38 were sufficiently conserved between pXO2 and pAW63 to allow detection by PCR or hybridization (see Tables 2 and 3).
Pulsed field gel electrophoresis was used to separate plasmid and chromosomal DNA in B. thuringiensis AWO6, and a Southern hybridization blot using a mixed pool of pXO2-derived probes (ORFs 6, 10, 50, 63, 72, 81) was performed to determine if any of the ORFs were present on the pAW63 plasmid ( Figure). A DNA fragment estimated to be 72 kb in size hybridized to the mixed pXO2 probe, which is slightly larger, but within 3% of the reported size of pAW63 (70 kb). This same PFGE protocol produced a similarly accurate measurement of the size of the B. anthracis plasmid pXO1 as determined by complete DNA sequencing [19]. The detection of sequences similar to pXO2 ORFs on pAW63 suggests that other pXO2 genes, in addition to those involved with replication, are also located on the pAW63 plasmid.

Conclusions
The presence of pXO2 ORF sequences in 11 Bacillus isolates did not correlate with their genomic relatedness to B. anthracis as determined by AFLP comparisons. A similar observation was made in previous work that examined the conservation of the B. anthracis plasmid pXO1 among closely related bacteria [19].
This study explored the extent of sequence conservation between pXO2 ORFs and total DNA from other Bacillus isolates, and detected similar sequences that may be located on the chromosome or any of several plasmids in each isolate. The two isolates with the most sequence conservation with pXO2 ORFs, B. thuringiensis isolates 33679 and AWO6, are known to contain large plasmids [12,19]. Four ORFs with high sequence similarity to B. thuringiensis AWO6 plasmid pAW63 were detected [22], and a mixed pXO2 ORF probe hybridized with a PFGE fragment similar in size to pAW63. The presence of considerable sequence conservation in more distantly related isolates rather than among close relatives, combined with the observations stated above, is a pattern consistent with the potential plasmid location of these sequences. Comparative sequence analysis of these large plasmids with pXO2 could determine if the observed sequence conservation was located on these plasmids.

Bacterial isolates and DNA isolation
The genomes of the 11 Bacillus isolates selected for study were found by AFLP analysis to vary in relatedness to B. anthracis. Isolates with Jaccard distances of less than 0.55 formed a distinct cluster with all of the B. anthracis isolates (P.J. Jackson, unpublished data) while the other 4 isolates were present in less closely related clusters (Table 1).
Bacteria were grown in Nutrient Broth (NB; DIFCO Laboratories, Franklin Lakes, NJ) or on NB agar plates at 28°C. Total DNA (including chromosomal and plasmid DNA) was extracted as described by Robertson et al. [24] with slight modifications. Cultures grown for 16 h in Nutrient Broth were centrifuged into a pellet, washed in TE (10 mM Tris pH 7.5/1 mM EDTA pH 8.0), and suspended in 10% sucrose. Cells were incubated at 37°C in lysozyme solution (5 mg/ml lysozyme, 50 mM Tris pH 7.5, 10 mM EDTA pH 8.0), followed by addition of 20% SDS containing 0.3% beta-mercaptoethanol. A potassium acetate precipitation was performed to further clarify lysed cells [25]. DNA was purified by organic extraction and ethanol pre-cipitation. Purified DNA was quantified by UV spectrophotometry. DNA from a B. anthracis isolate 91-213C-1 provided by P.J. Jackson was included as a positive control.

pXO2 PCR primer sets
Oligonucleotide primer sets were identified for 47 pXO2 ORFs. PCR primer sets were typically positioned 20 to 50 bases from ORF termini unless A/T richness of the DNA sequence prohibited the design of primers in that region. Primer sequences are located at http://bdiv.lanl.gov/databases/databases.html. The remaining 38 pXO2 ORFs were not included in the present survey due to sub-optimal A/ T richness, amplicon size, and thermodynamic characteristics of the candidate primer sets.

PCR assays and amplicon sequencing
PCR assays to detect each of the 47 individual pXO2 ORFs were conducted using DNA from each bacterial isolate (Table 1)  The majority of PCR products were sequenced using dyeterminator chemistry (ABI Prism FS, PE Applied Biosystems, Boston, MA). Sequencing primers were the same as those used in PCR amplification reactions. Sequencing re-

Figure 1
Pulsed field gel electrophoresis of DNA from B. thuringiensis AWO6. Treatment of agarose slices linearized the plasmid DNA and allowed for plasmid size determination using a concatomerized bacteriophage lambda standard (New England BioLabs, Beverly, MA) (5). DNA from agarose slices was resolved on a gel of 1% SeaKem Gold agarose melted in 0.5X TBE. Electrophoresis conditions were 175 V in 0.5X TBE at 6°C for 21 h in a CHEF-DR II Pulsed Field Electrophoresis System (BIORAD, Hercules, CA) with a field switch ramp of 5 to 40 s. Gels were stained with ethidium bromide and viewed using a UV trans-illuminator.

Southern hybridization
The pulsed field gel was sequentially soaked in 0.25 N HCl for 30 min; 3 M NaCl, 0.4 M NaOH for 60 min; and 0.5X TBE for 15 min. Electro-transfer of the DNA to a nylon membrane was performed using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA) according to the manufacturer instructions. DNA was crosslinked to the membrane by exposure to 1200 mjoules of ultraviolet light in a UV-STRATALINKER 1800 (STRATAGENE, LaJolla, CA). The membrane containing B. thuringiensis AWO6 DNA was hybridized using a [α-32 P]dCTP-labeled probe prepared from a mixture of six PCR-amplified pXO2 ORF fragments (pXO2 ORFs 6, 10, 50, 63, 72, 81). Care was taken to avoid the IS elements present on the plasmid. Probe synthesis, hybridization conditions, and wash regimen were performed as described above for hybridization reactions. Results were viewed using a Fugi Phosphorimager.