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Appl Environ Microbiol. Apr 1999; 65(4): 1483–1490.
PMCID: PMC91211

Cloning and Nucleotide Sequence Analysis of gyrB of Bacillus cereus, B. thuringiensis, B. mycoides, and B. anthracis and Their Application to the Detection of B. cereus in Rice

Abstract

As 16S rRNA sequence analysis has proven inadequate for the differentiation of Bacillus cereus from closely related species, we employed the gyrase B gene (gyrB) as a molecular diagnostic marker. The gyrB genes of B. cereus JCM 2152T, Bacillus thuringiensis IAM 12077T, Bacillus mycoides ATCC 6462T, and Bacillus anthracis Pasteur #2H were cloned and sequenced. Oligonucleotide PCR primer sets were designed from within gyrB sequences of the respective bacteria for the specific amplification and differentiation of B. cereus, B. thuringiensis, and B. anthracis. The results from the amplification of gyrB sequences correlated well with results obtained with the 16S rDNA-based hybridization study but not with the results of their phenotypic characterization. Some of the reference strains of both B. cereus (three serovars) and B. thuringiensis (two serovars) were not positive in PCR amplification assays with gyrB primers. However, complete sequencing of 1.2-kb gyrB fragments of these reference strains showed that these serovars had, in fact, lower homology than their originally designated species. We developed and tested a procedure for the specific detection of the target organism in boiled rice that entailed 15 h of preenrichment followed by PCR amplification of the B. cereus-specific fragment. This method enabled us to detect an initial inoculum of 0.24 CFU of B. cereus cells per g of boiled rice food homogenate without extracting DNA. However, a simple two-step filtration step is required to remove PCR inhibitory substances.

Bacillus cereus is a gram-positive, spore-forming, facultatively anaerobic bacterium. Differentiation of B. cereus from its closely related microorganisms depends upon the absence of toxin crystals (from B. thuringiensis), hemolytic activity (from B. anthracis), and nonrhizoid growth and motility (from B. mycoides). B. cereus produces emetic toxin and enterotoxins (2, 16, 18, 28, 29, 33). Strains of B. thuringiensis are also reported to produce enterotoxins (1, 3, 14, 23), and molecular characterization revealed that the enterotoxin-encoding gene isolated from B. thuringiensis was similar to that of B. cereus (3).

The nucleotide sequence of 16S rRNAs of the B. cereus group exhibited very high levels of sequence similarity (>99%) that were consistent with the close relationships shown by previous DNA hybridization studies (37). The 16S rRNA sequences of B. mycoides and B. thuringiensis differed from each other and from the sequences of B. anthracis and B. cereus by 0 to 9 nucleotides (5). Likewise, Ash and Collins (4) reported that the 23S rRNA gene sequences of B. anthracis and an emetic strain B. cereus were found to be identical. The lesser variations noted in the spacer regions between the 16S and 23S rRNAs do not seem to be sufficient to allow the design of a species-specific oligonucleotide probe for the B. cereus-B. thuringiensis-B. anthracis group (9). Specific DNA probes based on variable region VI of 16S rRNAs of B. cereus and B. thuringiensis were designed by te Giffel et al. (39) and used in hybridization experiments, but screening of isolates with this probe from various sources and outbreaks is necessary to validate their claim. Single-strand conformation polymorphism of the PCR products did not allow species discrimination within the B. cereus group (8). Virulence factors (22, 35), restriction fragment length polymorphism (25), pulsed-field gel electrophoresis, analysis of intergenic spacer regions (20), and the arbitrary PCR (12, 21) differentiated B. anthracis from B. cereus but failed to differentiate B. cereus from B. thuringiensis. Because of the indistinguishable phenotypic and genotypic characteristics of these organisms, Ash et al. (6) proposed considering B. thuringiensis, B. mycoides, and B. anthracis as subspecies of B. cereus.

Since no universal probe is available to differentiate B. cereus from other related species, we have studied the possibility of using gyrB genes that encode the subunit B protein of DNA gyrase (topoisomerase type II) as targets of highly specific probes (50). In this study, 1.2-kb fragments of the gyrB genes of B. cereus, B. anthracis, B. thuringiensis, and B. mycoides were amplified, cloned, and sequenced. We designed suitable PCR primer sets that could amplify the gyrB fragments of B. cereus, B. anthracis, and B. thuringiensis to specifically identify the organism irrespective of its phenotypes, serotypes, and virulence factors. A protocol for the direct detection of B. cereus from boiled rice without extracting DNA is also described.

MATERIALS AND METHODS

Bacterial strains.

Microorganisms included in this study were purchased from various culture collections or were gifts from the Nagoya Public Health Research Institute and Mie University, Nagoya, Japan. Strains isolated from various aquatic environments and foods are also included. All microorganisms were grown in nutrient broth (Nissui, Tokyo, Japan) at 35°C for 24 h before use. Presumptive colonies appearing on the NGKG agar (Nissui) were picked, and biochemical characterization of the isolates was carried out as described elsewhere (28, 38, 45).

DNA isolation.

Chromosomal DNA of overnight-grown cultures was extracted by phenol-chloroform solvents and ethanol precipitation (36). The dried DNA was then dissolved in Tris-EDTA (TE) buffer (pH 7.5) and used as the DNA template. The purity of the DNA was checked by agarose gel electrophoresis, and the DNA concentration was measured with a spectrophotometer.

Cloning and sequencing of the gyrB gene.

Primers (UP-1 and UP-2r) within the known DNA sequence (50) were added to the PCR mixture at a concentration of 1 μM, and the solution was subjected to 30 cycles of PCR (43). The amplified gyrB fragments from B. cereus JCM 2152T, B. thuringiensis IAM 1077T, B. anthracis (Pasteur #2H), and B. mycoides ATCC 6462T were cloned in pGEM-ZF+ (Promega, Madison, Wis.) by conventional recombinant methods (36). Expansion of the probes was carried out as documented previously (36). After ligation of the PCR fragments into the vector, Escherichia coli cells were transformed with the ligation mixture by calcium chloride-mediated transformation. After transformation, the transformants were cultured under conditions that promote growth. Plasmids were recovered from a transformant by lysis and purification by an alkaline method. The purified intact plasmid was then utilized as a probe. The identity of the fragment was verified by sequencing from both ends by the dideoxy chain termination method with a Sequenase DNA sequencing kit (U.S. Biochemical Corp., Cleveland, Ohio) and with an ABI 373A automatic sequencer as described by the manufacturer (Perkin-Elmer Corp., Foster City, Calif.). DNA sequences were determined from both strands by extension from vector-specific (T7 and SP6 primers from pGEM-ZF+) priming sites and by primer walking.

Oligonucleotide primers.

Various oligonucleotide primers based on the gyrB nucleotide sequence data of the Bacillus species were synthesized according to the instructions of the manufacturer (Beckman, Fullerton, Calif.).

PCR assay.

Whole bacterial cells without extracting DNA were used as templates. In this case, the freshly grown cells (overnight incubation at 35°C) on nutrient agar plates were used. If the bacterial cells were grown in liquid medium, the bacterial cells were harvested by centrifugation and washed once with phosphate-buffered saline (PBS; 0.1 M, pH 7.5), and appropriate counts of bacterial cells were used. In some cases, purified DNA was used as a template for PCR amplification.

PCR assays were performed in a DNA Thermal Cycler (Perkin-Elmer). Reaction volumes of 100 μl contained 100 ng of genomic DNA, deoxynucleoside triphosphates at a concentration of 200 μM each, and primers at 1 μM each in reaction buffer (Tris-HCl, 100 mM; MgCl2, 15 mM; KCl, 500 mM; pH 8.3). The 1.2-kb gyrB gene was amplified as described elsewhere (50). The amplification of various Bacillus species-specific (B. cereus, 365 bp; B. thuringiensis, 368 bp; and B. anthracis, 245 bp) fragments was performed by using PCR for 30 cycles, each consisting of 1 min at 94°C, 1.5 min at 58°C, and 2.5 min at 72°C, with a final extension step at 72°C for 7 min. After DNA amplification, PCR fragments were analyzed by submarine gel electrophoresis, stained, and visualized under UV illumination (44). Suitable molecular size markers were included in each gel.

Hybridization with 16S rDNA probes.

Hybridizations were performed with 16S rDNA probes that are specific to B. cereus (TTA AGA ACT TGC TCT TAT) and B. thuringiensis (TTG AGA GCT TGC TCT CAA) as described by te Giffel et al. (39). The purified 16S fragment was transferred onto a nylon membrane (Hybond-N; Amersham) by Southern blotting. The blotted membranes were neutralized in 0.2 M Tris-HCl (pH 7.5) in 0.3 M NaCl–0.03 M sodium citrate and air dried. Prehybridization and hybridization were performed in 0.5 M sodium dodecyl sulfate and 1% bovine serum albumin. After 30 min of prehybridization at 45°C, the commercially available probe, which had been 3′ end DIG labeled (Boehringer Mannheim, Foster City, Calif.), was added, and signals were detected according to the manufacturer’s instruction with DIG DNA labeling kits (Boehringer Mannheim).

PCR assay sensitivity for the detection of artificially contaminated B. cereus in boiled rice.

A 25-g sample of boiled rice was homogenized for 1 min with a homogenizer (SH-001; Elmex, Tokyo, Japan) in 225 ml of nutrient broth to produce a uniform food homogenate for all experiments. B. cereus JCM 2152T was grown in nutrient broth overnight at 35°C and serially diluted with the food homogenate as the diluent to final concentrations ranging from 0 to 109 CFU per g. These artificially contaminated food homogenate microcosms (10 ml each) were incubated at 35°C. Subsampling (1 ml) was carried out after incubations of 0, 6, and 15 h (overnight); cells were then centrifuged (4°C; 10,000 × g for 10 min) and resuspended in 1 ml of sterile PBS.

Suitable controls such as buffer, media, PCR mixtures, and B. cereus DNA were employed to check any false-positive or false-negative reactions. Appropriate dilutions of test sample prepared at various intervals in nutrient broth were spread plated onto standard plate count agar (Nissui) for total viable counts and on NGKG agar (Nissui) for the enumeration of the B. cereus population.

To remove any PCR-inhibitory substances from food, samples drawn from the microcosms were subjected to two-step filtration as described previously (44), with some modifications. Briefly, a 400-μl sample of a sterile-PBS-washed sample was passed through a 5-μm-diameter Ultrafree filter tube (UFC3 0GV; Millipore, Bedford, Mass.) and centrifuged (4°C; 10,000 × g for 10 min). The 5-μm-diameter-tube filtrate was then passed through 0.2-μm-diameter Ultrafree centrifuge tube (SE3P009E4; Millipore) and centrifuged (4°C; 10,000 × g for 10 min) to remove bacteria. The material trapped on the 0.2-μm (pore size) filter was then resuspended in 50 μl of sterile PBS and boiled before 10 μl of supernatant was used as a template for the PCR assay.

Nucleotide sequence accession numbers.

The nucleotide sequence data reported here will appear in the GenBank nucleotide sequence database with the indicated accession numbers: B. cereus JCM 2152T (AF090330), B. thuringiensis IAM 12077T (AF090331), B. anthracis Pasteur #2H (AF090333), and B. mycoides ATCC 6462T (AF090332).

RESULTS

gyrB sequence of Bacillus species.

Complete sequences of the 1.2-kb gyrB fragments of B. cereus JCM 2152T, B. thuringiensis IAM 12077T, B. anthracis Pasteur #2H, and B. mycoides ATCC 6462T were determined and aligned (Fig. (Fig.1).1). The percentages of similarity (Table (Table1)1) and numbers of base-pair substitutions (Table (Table2)2) in gyrB nucleotide sequences and translated protein sequences for all four of these Bacillus species are given. Likewise, the percentages of similarity and numbers of base-pair substitutions in 16S rDNA nucleotide sequences are depicted in Table Table3.3. The similarity in the gyrB sequences of B. cereus and B. thuringiensis was 90.2% versus 99.6% for the 16S rDNA sequences. Alignment of the amino acid sequences of the gyrB proteins (Fig. (Fig.2)2) translated from the nucleotide sequences showed that only 2 of the 121 substitutions caused amino acid substitutions. The amino acid sequence similarity between the gyrase subunit B proteins of B. cereus and B. thuringiensis was 96.8% (Table (Table1).1). The frequency of base substitutions in the published 16S rRNA was lower than that in gyrB. For example, between the sequences of B. cereus and B. thuringiensis 121 base substitutions were observed in gyrB, while only 5 base substitutions were observed in 16S rRNA. It is interesting to note that no substitution was observed between the sequences of B. cereus ATCC 14579T and B. anthracis serotype Sterne.

FIG. 1FIG. 1FIG. 1
Nucleotide sequence alignment of gyrB of various Bacillus species. Nucleotides identical to those of B. cereus are indicated by dots. Nucleotide positions for various primers appear on a black field. Primers BC1 and BC2r are B. cereus specific; primers ...
TABLE 1
Similarities among gyrB genes of various Bacillus species
TABLE 2
Base-pair substitutions in gyrB genes of various Bacillus species
TABLE 3
Base-pair substitutions and similarities in 16S rDNA nucleotide sequences for various Bacillus species
FIG. 2
Amino acid sequence alignment of the gyrB products of various Bacillus species. Amino acids identical to those of B. cereus are indicated by dots.

Designing B. cereus-, B. thuringiensis-, and B. anthracis-specific PCR primer sets.

Oligonucleotide primers that are universal and specific to various Bacillus species were synthesized based on the nucleotide sequences of gyrB (Table (Table4).4). A forward primer with a nucleotide position of 175 to 195 (BC1) and an antisense primer with a position of 519 to 539 were synthesized (BC2r). When these primers were used to generate 365-bp PCR products, B. cereus could be differentiated from B. anthracis and type strains of B. thuringiensis IAM 12077T and B. mycoides ATCC 6462T. Likewise, a 368-bp fragment specific to B. thuringiensis (made by using BC1 and BT2r) and a 245-bp amplicon specific to B. anthracis (made by using BA1 and BA2r) were amplified with the appropriate primer sets.

TABLE 4
gyrB PCR primers in the differentiation of B. cereus, B. thuringiensis, B. anthracis, and B. mycoides

Specificity of PCR primers in the detection of Bacillus species.

B. cereus, B. thuringiensis, B. anthracis, and B. mycoides strains received from various culture collections and identified as different serogroups or isolated from numerous food and environmental specimens were tested with gyrB primer sets specific for these Bacillus species.

PCR results in the differentiation of these Bacillus species for the type strains and various serogroups are given in Table Table5.5. Type strains of all four Bacillus species showed species-specific positive amplification. However, B. cereus-specific signal was not observed for some B. cereus serotypes (H5, H6, H7, H16, and H17). In addition, amplification of 365-bp fragment that is specific to B. cereus was noted for B. thuringiensis serotypes kurstaki (HD-1) and aizawai. When tested with various PCR primer sets that were established in this study, serotypes H5, H6, H7, and H17 showed a positive signal for B. anthracis, and serotype H16 exhibited an amplicon specific to B. thuringiensis.

TABLE 5
Specificity of gyrB PCR primers in the differentiation of B. cereus, B. thuringiensis, B. anthracis, and B. mycoides

The 1.2-kb gyrB nucleotide sequences of B. cereus serotypes H2, H6, and H16 and B. thuringiensis serotype kurstaki (HD-1) were determined. By comparing the gyrB nucleotide sequences of these strains along with type strain sequences, both H2 and kurstaki (HD-1) showed high similarity with B. cereus JCM 2152T (98.4%). Likewise, a high similarity value was noted between B. anthracis and H6 (97.9%), as well as between B. thuringiensis and H16 (93.0%).

A total of 104 Bacillus strains isolated from various environments, including food and clinical sources, consisting of various phenotypes, serotypes, and toxigenic properties were tested for PCR assay (Table (Table6).6). B. cereus isolates obtained from IFO and IAM culture collections were identified perfectly as B. cereus. However, among 50 B. cereus strains isolated from foods, 4 and 2% of the strains produced amplicons specific to B. thuringiensis and B. anthracis, respectively. Likewise, 6 of 20 B. cereus isolates from various environmental sources were identified as B. anthracis. Eight of ten environmental isolates that were identified as B. thuringiensis on the basis of crystal protein were recognized as B. cereus. One of nine B. mycoides isolates obtained from the ATCC showed a positive signal for B. cereus.

TABLE 6
Differentiation of B. cereus, B. thuringiensis, B. anthracis, and B. mycoides wild strains by using gyrB PCR primers

Comparison of gyrB PCR and 16S rDNA hybridization techniques in the differentiation of Bacillus species.

Specific DNA probes based on variable region VI of 16S rRNAs of B. cereus and B. thuringiensis were reported to be useful for differentiating these species (39). Type strains, serotypes, and wild-type strains that were identified as Bacillus species on the basis of biochemical and serological classifications were subjected to Southern hybridization by using 16S rDNA probes and gyrB PCR with various Bacillus species primer sets (Table (Table7).7). Among the type strains tested, the hybridization signal showed good correlation with its species by gyrB PCR except for B. mycoides ATCC 6462T, where a positive signal was noted when B. cereus-specific 16S rDNA probes were used. te Giffel et al. (39) also reported such a false-positive signal for B. mycoides. On the other hand, gyrB PCR did not produce any false-positive amplicon and was able to differentiate B. cereus from B. mycoides. In contrast, serotypes kurstaki and aizawai of B. thuringiensis were identified as B. cereus by conceding positive signals for B. cereus in both hybridization and PCR techniques. This indicated that B. thuringiensis serotype kurstaki and aizawai are, in fact, B. cereus and not B. thuringiensis. However, B. cereus serotype H16 showed a positive signal for B. cereus in hybridization experiments but showed an amplicon specific for B. thuringiensis by the gyrB PCR technique. To clarify these findings, we tried to procure H16 serotype from other culture collections, but we did not succeed in acquiring the strain. Also, B. cereus serotypes H5, H6, H7, and H17, which showed positive amplification for B. anthracis, did not yield any B. cereus-specific hybridization signal. Complete sequencing of 1.2-kb gyrB fragments of these strains supported our claim that these strains were indeed misidentified.

TABLE 7
Comparison of gyrB PCR and 16S rDNA hybridization probe techniques in the differentiation of B. cereus and B. thuringiensis

Sensitivity of the PCR primers in the detection of Bacillus species.

To evaluate the sensitivity of the PCR assay, a dilution series of genomic DNA from various Bacillus species was prepared in TE buffer, and aliquots were used as templates for PCR amplification. Aliquots containing a picogram level of genomic DNA were successfully detected after amplification with primer-specific PCR primer sets. A dilution series of freshly cultured cells of Bacillus species showed that the primer set employed in this study amplified species-specific PCR fragments when 2 to 5 CFU of bacterial cell per reaction tube was used.

Detection of B. cereus in the artificially contaminated boiled rice.

The sensitivity of the PCR assay for detecting artificially contaminated B. cereus in cooked boiled rice is presented in Table Table8.8. Absence of B. cereus-like organisms in the test sample was confirmed by both the conventional enrichment method and by PCR assay. When the food homogenate was incubated for 15 h in nutrient broth at 37°C, an initial inoculum of 0.24 CFU of B. cereus per g of food homogenate amplified the desired PCR product (Fig. (Fig.3).3). At time zero, 2.4 × 104 CFU of B. cereus per g of boiled-rice homogenate did not yield any PCR products. The detection of B. cereus directly from the food sample was possible by the combination of a 15-h enrichment period in nutrient broth and PCR assay even without DNA extraction.

TABLE 8
Influence of preenrichment incubation time and bacterial population in the amplification of PCR product specific to B. cereus
FIG. 3
Detection of B. cereus in artificially contaminated cooked rice by the BC-1 and BC-2 primers (365-bp product). B. cereus cells grown overnight in nutrient broth were serially diluted in cooked rice homogenate (see details in Materials and Methods) to ...

DISCUSSION

Enterotoxigenic B. cereus, which causes acute gastroenteritis after the consumption of contaminated foods, has been known to produce the emetic and the diarrheal toxin types (18). B. thuringiensis can be distinguished from B. cereus only by the production of toxin crystals and can be detected by simple microscopy or by hybridization with specific probes for the delta endotoxin gene (10). However, this character is plasmid borne and transmissible to B. cereus by conjugation (13, 15). B. cereus strains can grow at temperatures between 4 and 37°C (40, 42), and psychrotrophic B. cereus strains can produce enterotoxin both aerobically and anaerobically (17). Good diagnostic tools are thus required to ensure the hygienic quality of susceptible food items. Although some PCR methods are developed to detect B. cereus targeting the toxigenic properties (30), the production of B. cereus-type enterotoxins in Bacillus species, including B. thuringiensis (23), raised doubts about the validity of these diagnostic probes in specifically identifying B. cereus. Thus, identifying B. cereus irrespective of its virulence factors is necessary from the viewpoint of public health. We therefore wanted to develop a rapid and reliable method for differentiating B. cereus from B. thuringiensis.

As 16S rDNA sequences have very high similarity between B. cereus and B. thuringiensis, we were looking for appropriate molecular taxonomic markers. Yamamoto and Harayama (50) suggested that genes that are not spread horizontally among different bacterial species may be used to trace the evolutionary record of host bacteria. Based on the information that the average substitution rate for 16S rRNA is 1% per 50 million years and that the rate for synonymous sites of protein-coding DNA is 0.7 to 0.8% per million years (31), Yamamoto and Harayama (50) proposed the gyrB gene as a substitute for 16S rRNA as a molecular taxonomic marker for bacterial species. The gyrB gene is essential for DNA replication, a housekeeping activity; it is also single copied and has conserved regions for the development of PCR primers. We have designed PCR primers by exploiting differences in gyrB genes to differentiate V. parahaemolyticus from V. alginolyticus, a pair that exhibited 99.7% similarity in their 16S rDNA nucleotide sequences (43). Likewise, the similarity of 1.2-kb gyrB sequences of B. cereus and B. thuringiensis was not high (90.7%); thus, we were able to design a suitable PCR primer set for differentiating these two organisms by amplifying a B. cereus-specific 365-bp amplicon. The type strains of both B. cereus and B. thuringiensis responded specifically with the developed PCR primer set by giving a positive signal for B. cereus and a negative signal for B. thuringiensis. Also, these PCR primers, which are specific for B. cereus, did not yield any amplicon for the B. anthracis strain and the B. mycoides type strain.

The gyrB PCR primer set designed in this study detected 2 to 5 CFU of B. cereus cells per reaction tube or correspondingly low levels (10 pg) of extracted DNA. The sensitivity accords with that described for PCR with other bacteria, being between 1 and 20 CFU (27, 32, 41, 47) or between 1 and 100 pg for DNA extracted from the bacterial population (24, 49). Increased sensitivity may be achieved by Southern blot analyses as reported earlier (7). The gyrB primer set recognized all strains identified as B. cereus and its group by conventional methods, but it did not recognize the other bacteria tested. In addition, some serotypes that were designated in culture collections as B. cereus or B. thuringiensis were differentiated and their species were identified. Specialized laboratories use elaborate techniques for confirming the B. cereus group and their strain identity, techniques such as toxin antigen detection, detection of virulent plasmids, the use of crystal protein, etc. Since the gyrB PCR result is in agreement with 16S rDNA-based hybridization probe technique, this simple PCR method is thus a powerful tool for the confirmation and differentiation of the B. cereus species in clinical, food, and environmental samples.

Two major limitations to using PCR as a diagnostic tool are that false-positive reactions can occur from DNA contamination and that false-negative reactions can occur from a number of substances found in samples that inhibit PCR (44, 47). We have included suitable negative and positive controls to overcome these limitations when and where necessary. Sensitivity of detection in food samples has, however, been low because only small samples (10 to 100 μl) can be analyzed, since many sorts of food contain substances that are PCR inhibitory. Such PCR-inhibitory substances were reported in many clinical samples, such as urine, blood, sputum, fecal specimens, food, and environmental samples (26, 46, 48). However, with bacteria in food, the sensitivity of the PCR was far lower than that with bacteria in saline. With bacteria in food, the lower detection limit was higher than the number of CFU per unit volume of food, a result which is usually found with processed food. Previous studies suggested the extraction of DNA (27) or the application of chemicals (11). A simple two-step filtration procedure that we reported previously successfully removed PCR-inhibitory products during this study (44). However, a simple preenrichment in a nonspecific medium was necessary to permit the proliferation of target bacteria. A similar phenomenon was documented for the detection of V. parahaemolyticus from shrimp (43).

The findings reported here describe a rapid, sensitive, specific, and reliable method for the detection of B. cereus in boiled rice. The fact that this technique allows detection of the genetic potential and permits differentiation from related species may make it useful as both a screening test and a confirmatory test. The data provided from this test could yield additional information that will be useful to epidemiological studies.

ACKNOWLEDGMENTS

We are grateful to M. Satake for encouragement, I. Uchida for providing DNA of B. anthracis, and I. Sugahara for various Bacillus strains. We also thank Y. Kamijoh, T. Kurusu, K. Hanai, and Y. Hara for their technical assistance.

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