Institute of Molecular Biology and Medicine,
University of Scranton, Scranton, Pennsylvania 18510
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TEXT |
Bacillus anthracis is a
causal agent of anthrax, a serious and often fatal infection of
livestock and humans. It is considered one of the most effective
biological weapons of mass destruction because of its highly pathogenic
nature and spore-forming capability and has attracted attention due to
its potential use as a biological warfare agent (2). This
bacterium can infect humans by cutaneous, gastrointestinal, or
respiratory routes. The standard laboratory method of identification
takes advantage of the lytic nature of the B. anthracis-specific gamma bacteriophage (9). Anthrax bacilli are often distinguished on the basis of time-consuming morphological or phenotypic characteristics, such as gram-positive staining, spore-forming capability, nonhemolytic reaction on sheep blood agar, sensitivity to penicillin, nonmotile nature, and inability to ferment salicin (11). B. anthracis is
distinguished from the other members of the closely related
Bacillus cereus group of bacteria by the presence of the
toxin-encoding pXO1 (19, 24) and capsule-encoding pXO2
plasmids (14, 23, 34). Both plasmids are needed for
virulence; thus, the absence of either plasmid results in attenuation.
B. anthracis, Bacillus thuringiensis, B. cereus, and
Bacillus mycoides, are members of the B. cereus
group of bacilli. These closely related bacteria are pathogens of
mammals (B. anthracis and B. cereus) and insects
(B. thuringiensis). The B. cereus group is one of
the most taxonomically ambiguous group of bacilli (27). In
fact, DNA-DNA hybridization (30) and pulsed-field gel
electrophoresis (15) have shown great homology among
B. anthracis, B. thuringiensis, and B. cereus. A
recent multilocus enzyme electrophoresis study has concluded that the
members of this group belong to one species (16).
Although specific assays are available for the detection of
pathogenicity-related plasmids (18, 28), chromosomal
markers in conjuction with plasmid markers should be used for complete genotyping of B. anthracis strains. Such a combined approach
will provide insight into the chromosomal backbone or genetic
background and indicate the pathogenic nature of the strain. Plasmids
are more unstable than chromosomal DNA, and isolates lacking either or
both plasmids have been found to exist in nature (33).
Also, pXO2 has been successfully transferred into other bacilli, and toxin genes, such as lef and cya, have been
expressed in heterologous systems (4, 5, 20). Thus,
naturally occurring as well as genetically modified B. anthracis strains cannot be characterized without ambiguity.
Moreover, chromosomal markers are stable targets for detection and are
important for accurate identification of B. anthracis in
outbreaks (26) as well as during the analysis of ancient
samples (C. Redmond, M. J. Pearce, R. J. Manchee, and B. P. Berdal, Letter, Nature 393:747-748).
Several chromosomal markers are currently available for B. anthracis detection, such as the vrrA gene (1,
17), Ba813 marker (25), and SG-850 marker
(10). These marker assays suffer from being time consuming
or labor intensive or having limited specificity. For instance, the
SG-850 assay involves PCR amplification of the SG-749 locus, followed
by enzymatic digestion with AluI and gel analysis. The
vrrA marker can group B. anthracis isolates into
several categories based on the number of repeat units of this
sequence, which requires post-PCR analysis (17). Recently some B. cereus and B. thuringiensis isolates have
been found to contain the Ba813 marker (26); hence its use
as a B. anthracis-specific target is questionable
(29). The 16S rRNA gene also does not provide sufficient
polymorphism to differentiate B. anthracis from closely
related bacilli (3). Thus, no absolutely specific chromosomal marker is presently available for the detection of B. anthracis.
The rpoB gene, which codes for the 
-subunit of RNA
polymerase, has served as a signature sequence for bacterial
identification as well as a locus for phylogenetic analysis
(21). Moreover, rpoB is a highly conserved
housekeeping gene, and at least one copy is present in all bacteria
because of its essential role in cellular metabolism. This gene, along
with rpoC, which encodes for the '-subunit, constitutes
the catalytic center of the pentameric bacterial RNA polymerase
(6). Due to its discriminatory power, the rpoB
gene has been used to develop probes for specific detection and
phylogenetic analysis of Coxiella burnetti, Rickettsia, and Yersinia pestis (12, 13, 22).
Bacterial strains and DNA preparation.
A total of 144 B. anthracis strains from different geographical locations (Table
1), 29 B. cereus strains, 49 B. thuringiensis strains, 73 Bacillus spp.
Ba813+ strains (29), a strain each of B. mycoides, B. subtilis, and B. megaterium, and 22 unknown bacilli were used to test the specificity of the assay. Sixteen
B. anthracis strains and a total of 20 other bacilli strains
of B. cereus (n = 6), B. thuringiensis (n = 6), B. mycoides (n = 1), B. subtilis
(n = 1), and other bacilli (n = 6) were used for the determination of variable region 1 of
the rpoB gene sequence (Table
2). All strains used in this study were
analyzed for plasmid content by a multiplex PCR assay
(28). DNA was extracted by a method outlined by Schraft and Griffiths (32) with modifications as described
elsewhere (8). For preparing crude vegetative cell
lysates, a sterile toothpick was used to transfer a portion of a fresh
colony into 300 µl of distilled water. The cell suspension was boiled
at 100°C for 15 min and then centrifuged at 8,000 × g for 5 min. The supernatant was transferred to a fresh
microcentrifuge tube and stored at 
20°C until further use. For
isolating the rifampin-resistant mutant, an individual fresh colony of
the rifampin-sensitive B. anthracis 7700 was streaked out on
a brain heart infusion agar containing 25 µg of rifampin (Sigma
Chemical Co., St. Louis, Mo.) per ml and was incubated overnight at
37°C. Rifampin-resistant colonies were plated out a second time onto
a plate containing 50 µg of rifampin/ml in order to confirm this
phenotype.
Low-stringency PCR amplification and sequence analysis.
The
alignment of the amino acid sequences of the RNA polymerase
-subunits of Bacillus subtilis and Escherichia
coli permitted the identification of two conserved regions. The
conserved region found near the N terminus was RVIVSQ, spanning amino
acid residues 132 to 137 of B. subtilis and residues 143 to
148 of E. coli (6). The C terminus conserved
region was DDIDHL, and it was found at positions 399 to 404 of B. subtilis and positions 443 to 448 of E. coli.
The sequences of primer rpoB1 (5'-CGTGTTATCGTTTCCCAGC-3')
and rpoB2 (5'-AAGATGATCGATATCATCTG-3') were
derived from the two conserved regions and correspond to nucleotides
(nt) 1482 to 1500 and 2281 to 2300 of the B. subtilis rpoB
gene (GenBank accession no. L24376). The PCR reaction mixture of 50 µl consisted of 10 mM Tris-HCl (pH 8.3), 75 mM KCl, 3.5 mM
MgCl2, 0.2 mM dNTPs (Boehringer Mannheim Corp.,
Indianapolis, Ind.), 1 µM (each) primers rpoB1 and rpoB2, 0.05 U of
AmpliTaq DNA polymerase (Perkin Elmer Corp., Foster City,
Calif.)/µl, and 100 ng of DNA template. Amplification was performed
in a GeneAmp PCR System 2400 (Perkin-Elmer Corp., Norwalk, Conn.), and
the cycling conditions were as follows: initial denaturation at 94°C
for 5 min, followed by 35 cycles of 94°C for 1 min, 45°C for 1 min,
and 72°C for 1 min, with a final extension of 72°C for 7 min. The
amplicons were detected in 2% (wt/vol) SeaKem GTG agarose (FMC
Bioproducts, Rockland, Maine) with 40 mM Tris-acetate-1mM EDTA (pH
8.3) as a running buffer and visualized by ethidium bromide staining.
Low-stringency amplification of the variable region of the
rpoB gene from different Bacillus species and
strains yielded the expected amplicons with a size of 819 bp. Bands of
the expected size were excised from the gel, and the DNA was extracted
using a QIAquick Gel Extraction kit (QIAGEN Inc., Valencia, Calif.). The PCR products were cloned into vector pCR 2.1 (Invitrogen Corp., Carlsbad, Calif.) and transformed into E. coli. Recombinant
plasmids were prepared using the QIAGEN Plasmid Mini Kit. Three clones from each ligation reaction were sequenced in duplicate with the M13
forward and reverse primers using the Applied Biosystems model 373A
automated sequencer and the BigDye terminator ready reaction kit
(Perkin-Elmer Applied Biosystems). The nucleotide sequences were edited
and assembled with the Sequencing Analysis 3.0 and AutoAssembler 3.1.2 programs, respectively; translation into amino acids was accomplished
using the Sequence Navigator 3.0.1 program (Perkin-Elmer Applied
Biosystems). These 36 sequences were aligned using the Clustal W
program (32) from BioNavigator (eBioinformatics Pty Ltd:
http://www.ebioinformatics.com/), and four bases specific for B. anthracis were identified. Figure 1
shows the alignment and nucleotide differences of 10 representative
strains, including 3 strains of B. anthracis, 2 strains of
B. cereus, 2 strains of B. thuringiensis, 2 Ba813+ strains of Bacillus sp.
(29), and a single strain of B. subtilis. Thus,
nucleotides C (position 42), T (position 84), C (position 108), and A
(position 174) are unique to B. anthracis, with the exception of Ba813_11 (Fig. 1). The region of the rpoB gene
described in this study appears to be the only region within the
rpoB gene that shows variation among different species of
bacteria (6).
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FIG. 1.
Alignment of the nucleotide sequences from 10 representative Bacillus strains using Clustal W
(32). The strains are the following: Bc 27877, Bacillus cereus 27877; Bc 23261, Bacillus cereus
23261; Bt 35646, Bacillus thuringiensis 35646; Ba Vollum,
Bacillus anthracis Vollum; Ba A74, Bacillus
anthracis A74; Ba Sterne, Bacillus anthracis Sterne;
Ba813_11, Bacillus sp. strain Ba813_11; Ba813_12,
Bacillus sp. strain Ba813_12; Bt B8, Bacillus
thuringiensis BtB8; Bs 6051, Bacillus subtilis 6051. The locations of primers and probes are shown (F, fluorescein; Cy5,
cyanine 5; P, phosphate group); the presence of an asterisk denotes a
mismatch, a dash indicates identity with the consensus sequence, and
nucleotide letters indicate positions showing polymorphism.
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Nucleotide sequence accession number.
The nucleotide sequences
of the portion of the rpoB gene (variable region 1)
described in this study were submitted to GenBank, and the accession
numbers are listed in Table 2.
The translation of the nucleotide sequences showed that four bases
specific for B. anthracis were in the third positions of the
codons and did not change the amino acid sequence. The positions of the
amino acids in the -subunit were alanine at 251, tyrosine at 265, tyrosine at 273, and valine at 295. Thus, although there are
differences in the nucleotide sequences, no differences were found in
the primary sequence of the RpoB proteins for B. anthracis, B. cereus, and B. thuringiensis.
FRET-PCR assay.
The primers rpoBF1 and rpoBR1 (Table 3;
Fig. 1) were selected for high-stringency
PCR amplification using Oligo 6 software (National Biosciences Inc.,
Plymouth, Minn.). The probes BaP1 (3' end labeled with Fluorescein) and
BaP2 (5' end labeled with Cy5 and 3' blocked with a phosphate group)
were placed 1 bp apart within the PCR product (Fig. 1) and had
Tms (7) at least 10°C higher than
those of the amplification primers (Table 3).
The PCR mixture (10 µl) consisted of 10 mM Tris-HCl (pH 8.3), 50 mM
KCl, 2.5 mM MgCl2, 0.2 mM dNTPs, 250 µg of bovine serum albumin/ml (Roche Molecular Biochemicals, Indianapolis, In), 1 µM
(each) primers rpoBF1 and rpoBR1, 0.2 µM probe BaP1, 0.4 µM probe
BaP2, 0.8U of DNA polymerase KlenTaq1 (Ab Peptides, St. Louis, Mo.), and 50 ng of DNA template or 2 µl of crude vegetative cell lysate. The amplification was performed on a Light-Cycler (Idaho
Technology, Idaho Falls, Idaho), which is a rapid, forced-air thermocycler with an integrated fluorimeter for real-time monitoring of
PCR reactions (35). The amplification was accomplished by initial denaturation at 95°C for 30 s, followed by 35 cycles of 95°C for 0 s, 63°C for 15 s, and 72°C for 5 s.
Once the capillaries were placed in the thermocycler, amplification
could be completed in less than 30 min. Detection of the amplification
products is accomplished by hybridization of a pair of probes to the
amplicons as they are formed, resulting in a fluorescence resonance
energy transfer (FRET) (35). Fluorescence was measured
once every cycle at the annealing step using the F2/F1 filter to
monitor amplification in real time. F1 corresponds to the baseline
fluorescein fluorescence, while F2 indicates FRET from fluorescein to
Cy5, resulting in the ratio of Cy5/fluorescein fluorescence (F2/F1).
The increase in fluorescence is proportional to the amount of PCR
product generated (7, 35) and is displayed on the computer
screen in the real-time mode. The reactions showing an increase in
fluorescence by a minimum of 0.05 fluorescence units (y axis) were
scored as positive amplification reactions. The PCR products were also
visualized by 2% (wt/vol) gel electrophoresis.
The FRET assay was performed on 144 B. anthracis strains,
harboring any combination of the two plasmids and isolated from different geographical locations (Table 1). All these strains tested
positive in the FRET assay, since they displayed an increase in
fluorescence as well as the presence of the expected PCR product by
agarose gel analysis. Another 175 closely related strains, including
the B. cereus group and Ba813+ strains, were
tested as negative controls to check the specificity of the assay. All
related strains, with the exception of Ba813_11, were scored as
negative because they did not exhibit an increase in fluorescence.
Figure 2 shows the results of the
FRET-PCR assay and the electrophoresis of the PCR amplicons using
genomic DNA samples from representative strains. B. cereus, B. thuringiensis, Bacillus sp. strain Ba813_12, and B. subtilis did not show amplification. The rpoB FRET-PCR
assay is extremely specific for B. anthracis because of the
high-stringency PCR conditions coupled with the unique nature of the
primers and probes. This specificity occurs at two different levels.
The first is at the primer level, as seen in the case of
Bacillus sp. strain Ba813_12. In this instance PCR products
were not generated due to single base-pair difference at 3' end of both
primers, and as a result an increase in fluorescence was not observed
in spite of 100% homology of the probe region with the B. anthracis sequence. The second level of specificity is at the
probe level, since 100% base-pairing of probes with target sequence is
required for FRET to occur. A single base-pair mismatch between either
of the probe sequences with the target region stops the FRET process,
indicating a negative result (unpublished data).
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FIG. 2.
Results of the FRET-PCR assay using genomic DNA. (A)
Fluorescence ratio (F2/F1) is plotted against the number of PCR cycles.
The samples are the following: 1,  , negative control (no DNA); 2,  , Bacillus anthracis A74; 3,  , Bacillus
anthracis Sterne; 4,  ,
Bacillus sp. strain Ba813_11; 5, +, Bacillus
anthracis Vollum; 6,  , Bacillus cereus 27877; 7,  , Bacillus cereus 23261; 8, , Bacillus sp.
strain Ba813_12; 9,  , Bacillus thuringiensis BtB8. (B)
Gel electrophoresis of the PCR products. Lanes, M: 100 bp DNA ladder;
1, negative control (no DNA); samples 2 to 9 are the same as in panel
A.
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The amplicon derived from Bacillus sp. strain Ba813_11 was
sequenced, and it was found to have a nucleotide sequence identical to
that of B. anthracis (Fig. 1). Consequently, the FRET-PCR
assay reported here cannot distinguish Bacillus sp. strain
Ba813_11 from B. anthracis strains. The remaining 71 Bacillus spp. Ba813+ strains have sequence
identical to that of Bacillus sp. strain Ba813_12 in primer
and probe binding regions, and Bacillus sp. strain Ba813_11
appears to be an exception. This strain, Bacillus sp. strain
Ba813 (9594/3), was isolated from a station effluent in the Alps in
1997 and was designated a transitional strain because it could not be
assigned to a particular species (26, 29). According to
the SG-749 locus signature, Bacillus sp. strain Ba813_11 belongs to the B. cereus group (data not shown) and was
shown to contain the Ba813+ marker (26). In
contrast to the phenotypic characteristics of B. anthracis,
the Bacillus sp. strain Ba813_11 is hemolytic, motile, and
resistant to penicillin, although it has an rpoB variable region 1 identical to that of B. anthracis.
The assay was not affected by the presence of exogenously added
E. coli or mixed Bacillus species DNA (25 ng of
B. anthracis DNA + 1,000 ng of exogenously added DNA)
representing a mixed microbial community at the ratio of 1:40 (data not
shown). The sensitivity of the FRET-PCR assay was examined using
different concentrations of exogenously added DNA. Positive
fluorescence signals and amplification, as shown by gel
electrophoresis, were noticed even when as little as 1 pg of pure
genomic DNA was used.
The FRET-PCR assay was also performed on crude vegetative cell lysates
from B. anthracis and related bacilli. Figure
3 shows the results of the assay on
selected strains. Only B. anthracis displayed an increase in
fluorescence and the presence of the expected amplification product.
The increase in fluorescence was observed after 22 to 30 cycles. The
magnitude of increase in fluorescence is dependent on the quantity of
template DNA or the copy number of the gene target in the reaction
(35). It should be noted that the DNA amount was not
normalized in the different cell lysates because the number of
vegetative cells used in different samples was not identical. Thus, the
assay can be directly used on freshly grown cultures for rapid
identification of B. anthracis strains in less than 1 h. A rifampin-resistant colony exhibited positive results when tested
by the FRET-PCR assay because the position of the mutation was found to
be outside the rpoB target region, as is the case with
B. subtilis (6). However, if new hotspots are
found in the primer and/or probe binding sites, it may not be possible
to use the assay for rifampin-resistant B. anthracis strains.
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FIG. 3.
Results of the FRET-PCR assay using crude vegetative
cell lysates. The fluorescence ratio (F2/F1) is plotted against the
number of PCR cycles. The sample are the following: , negative
control (no DNA);  , Bacillus anthracis AC3; ,
Bacillus anthracis 7702; ×, Bacillus anthracis
 UM-2311; , Bacillus
anthracis A74; , Bacillus anthracis 0074; +,
Bacillus anthracis Texas 0077; , Bacillus
anthracis ANR-1099; , Bacillus anthracis 7700;
, Bacillus anthracis A58; , Bacillus cereus
14579;  , Bacillus thuringiensis 10792.
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The FRET-PCR assay was able to clearly identify and distinguish
B. anthracis from other closely related bacilli, signifying that the target of this assay is conserved in all strains of B. anthracis used in this study and that the detection of B. anthracis is independent of the plasmid content. These strains
have been isolated from a wide variety of geographic locations, which
gives us a reason to believe that this chromosomal marker will continue to be specific to B. anthracis even on further
investigation. Extensive testing of strains of the B. cereus
group has shown that the FRET-PCR assay is virtually free of
cross-reactivity (99.4% specificity), with the exception of the case
of Bacillus sp. strain Ba813_11. This assay can be used on
endospore suspensions if PCR-amplifiable DNA is released from the spores.
The FRET-PCR assay has several advantages over standard molecular
identification techniques. The amplification is monitored in real time,
and reactions can be scored as positive or negative without
time-consuming routine gel analysis. Moreover, the assay is rapid and
highly sensitive when extracted DNA is used as a template for PCR.
Using a DNA intercalating fluorescent dye such as SYBR Gold, the
specificity of the reaction is evaluated at the end of the PCR
amplification (18). The presence of contaminating DNA does
not affect the results of the assay, and hence it can be applied for
detection of B. anthracis in epidemiological studies and
suspected bioterrorist attacks and when analyzing ancient samples.
Recent reevaluation of one of the B. anthracis strains (Zimbabwe) that was originally determined to be rpoB FRET
positive has confirmed that it is in fact rpoB FRET
negative. Moreover, using a newly described technique known as
long-range repetitive-element polymorphism-PCR (8), we
now have strong evidence suggesting that this strain needs to be
regarded as a potential transitional B. anthracis strain. We
are presently exploring this possibility, and in the meantime we have
removed any mention of this particular strain from this report.
Y.Q. was supported by research grant no. DE-FG02-98-ER62592 from
the Department of Energy.
We thank G. Bolus, M. L. Ferguson, and T. Horn for their valuable
technical assistance for the sequencing. The assistance of M. A. Wagner and M. Brumlik in critically reading the manuscript and Valerie
Taylor and R. Spalletta in editing the manuscript is greatly
appreciated. We also thank W. Beyer, R. Böhm, T. N. Brahmbhatt, J. Burans, A. Cataldi, J. Ezzel, Z. Liu, M. Mock, C. L. Turnbough, J. Vaissaire, and R. J. Zabransky for kindly offering Bacillus strains.
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Abstract
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Present address: Biomedical Engineering Graduate Program,
University of Texas, Southwestern Medical Center at Dallas, Dallas, TX
75390-9178.
