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with Jaccard distances of less than 0.55 formed a distinct cluster with all of the B. anthracis isolates (P.J. Jackson, unpublished data) while the other 4 isolates were present in less closely related clusters (Table 1).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 precipitation. 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) as template. Fifty μl PCR reactions contained 1X Perkin Elmer PCR buffer with 1.5 mM MgCl2, 0.8 mM each dNTP, 1.25 U AmpliTaq DNA polymerase (Perkin Elmer), and 45 μM of each primer. A PTC-200 Peltier Thermocycler (MJ Research, Watertown, MA) was used for 35-cycle reactions (94°C, 2 min for first cycle only; 94°C, 30 s.; 48°C, 30 s.; 72°C, 30 s). Reactions were resolved on 2% agarose gels that were stained with ethidium bromide and viewed using a UV trans-illuminator. A reaction was considered positive if the amplified fragment was abundant and was the expected size DNA fragment.
The majority of PCR products were sequenced using dye-terminator chemistry (ABI Prism FS, PE Applied Biosystems, Boston, MA). Sequencing primers were the same as those used in PCR amplification reactions. Sequencing reactions were resolved on 48 cm polyacrylamide gels (4%, 19:1 acrylamide:bisacrylamide, Bio-Rad Laboratories) using an ABI model 373 fluorescence sequencer (Applied Biosystems, Inc.). DNA sequence was analyzed using Lasergene software (DNASTAR, Inc., Madison, WI). Sequences were deposited in GenBank as accession numbers AF547271 to AF547318.
Hybridization assay
A dot-blot hybridization assay was performed using DNA from B. thuringiensis strain AWO6 as probe against PCR-amplified pXO2 ORF DNA applied to a nylon membrane. Ten ng of each pXO2 ORF fragment was denatured by adding 0.1 volume of 1 M NaOH and incubation for 5 min at 37°C. An equal volume of 20X SSC (3 M NaCl, 0.3 M sodium citrate, adjusted to pH 7.0 with 1 M HCl) was added and samples were quickly placed on ice for 2 min. The DNA was then applied to a Hybond-N+ membrane (Amersham, Arlington Heights, IL) pre-soaked in 10X SSC using a HYBRI-DOT Manifold (Life Technologies, Inc., Rockville, MD). The membrane was exposed to 1200 mjoules of ultraviolet light in a UV-STRATALINKER 1800 (STRATAGENE, LaJolla, CA) to crosslink DNA to the membrane. Total DNA extracted from B. thuringiensis AWO6 was used to synthesize probe by incorporating [α-32P]dCTP (6000 μCi/mMol) (NEN, Boston, MA) into randomly primed DNA synthesis reactions using the Megaprime DNA Labeling System (Amersham-Pharamacia Biotech, Piscataway, NJ) according to the manufacturer instructions. The membrane was incubated at 50°C in hybridization buffer (0.5 M NaHPO4, 1 mM EDTA pH 8.0, 7% SDS [28]) for 60 min, followed by hybridization with probe for 16 h at 50°C. After hybridization, the membrane was washed twice for 10 min at 30°C in 2X SSC containing 0.1% SDS and twice for 10 min at 45°C in 0.2X SSC containing 0.1% SDS. Results were viewed using a Fugi Phosphorimager.
Pulsed-field gel-electrophoresis (PFGE)
A 15 ml culture of B. thuringiensis AWO6 was grown in NB overnight at 37°C with shaking. Chloramphenicol was added at a concentration of 180 μg/ml and the culture was incubated for 60 min. Cells were incubated on ice for 10 min, then centrifuged at 2500 × g for 5 min. Cell pellets were suspended in 1 ml TE buffer that contained 2 mg/ml lysozyme and incubated for 5 min at 37°C. Lysozyme-treated cells were washed in 1 ml of Buffer NT (1 M NaCl, 50 mM Tris pH 7.5) and were suspended in Buffer NT to a final volume of 200 μl.
Agarose plugs containing bacterial cells were prepared in a 1 ml syringe by combining cells with an equal volume of 2% SeaKem Gold agarose (FMC BioProducts, Rockland, ME) melted in water. Plugs were allowed to solidify at 4°C for 2 h. Thin agarose slices (1–3 mm) containing embedded bacteria were incubated for 16 h in 500 μl Buffer NTE (100 mM NaCl, 50 mM Tris pH 7.5, 100 mM EDTA pH 8.0) containing 2% lysozyme at 37°C. The lysozyme/Buffer NTE solution was replaced with Buffer NTE that contained 2 mg/ml Proteinase K and incubated for 16 h at 50°C. Slices were then incubated in Buffer NTES (100 mM NaCl, 50 mM Tris pH 7.5, 100 mM EDTA pH 8.0, 1% SDS) for 16 h at 50°C. Before electrophoresis, slices were incubated twice for 30 min in 1.0 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma. St. Louis, MO) diluted in TE and twice in 0.5X TBE (45 mM Tris-borate (1:1), 1 mM EDTA).
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-32P]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.
