Anthrax (Bacillus anthracis), Molecular Biology of
313
is the primary characteristic used to dis-
tinguish
B. anthracis
from related bacilli.
However, it has been demonstrated ex-
perimentally that plasmids can be trans-
ferred between
B. cereus, B. thuringiensis
,
and
B. anthracis
. Further, there are nat-
urally occurring isolates of plasmidless
B. anthracis
, so a method to distinguish
chromosomal DNA is important. Because
the genomes of
B. anthracis
and
B. cereus
are
estimated
to
share
98%
DNA
se-
quence homology, identi±cation of unique
diagnostic markers will be important for
the de±nitive identi±cation of the related
strains. In the following section, several
genetic sequences that have been pur-
ported to be unique to
B. anthracis
will
be discussed, as will their usefulness in
the identi±cation of this species.
Ba813 was the ±rst locus that was pro-
posed as a unique marker for
B. anthracis
.
A PCR assay for the detection of this
chromosomal DNA
fragment was pro-
posed
as
a
reliable
method
to
detect
this species. Subsequent studies found
the
Ba813
marker
in
other
bacilli,
so
this marker alone does not appear to be
suf±cient to distinguish anthrax from en-
vironmental samples. The relationship of
the Ba813-carrying, nonanthrax
Bacillus
strains to veri±able
B. anthracis
is not yet
clear. Genetically, these strains are closely
related to each other but distinct from
B. anthracis
.
Other assays for speci±c chromosomal
regions have been developed, including
one for variability in
rpoB
, which is almost
exclusively found in
B. anthracis
strains.
Interestingly, the one exception to this
was a nonanthrax
Bacillus
strain that also
carried Ba813.
A variable region in the
B. anthracis
chromosome has been identi±ed using
arbitrarily primed PCR. Only one complete
open reading frame was present in this
region,
and
it
was
dubbed
vrrA
,f
o
r
variable region with repetitive sequence.
PCR of this region ampli±ed a fragment
of distinct size from
B. cereus
,
Bacillus
mycoides
,and
B. anthracis
, allowing these
closely related species to be distinguished
from each other. Although most of this
variation was located in
vrrA
,m
an
yo
f
the single nucleotide substitutions that
differed between
B. anthracis
and the other
bacilli were third position synonymous
mutations
that
would
not
change
the
encoded amino acid sequence. The role
of the
vrrA
-encoded protein is unknown,
but the gene most closely resembles
shp2
,
a
gene
that
encodes
a
sheath
protein
in
micro±larial
parasites.
A
repetitive
sequence, QYPQ, in the parasite protein is
also predicted in the
vrrA
-encoded protein,
and this sequence has been implicated
in the covalent cross-linking of multiple
protein subunits.
Rather than looking at one locus at
a
time,
a
more
effective
method
of
distinguishing
B. anthracis
from related
species is to use techniques that look
at multiple loci simultaneously. One of
the
methods
that
has
been
used
is
ampli±ed fragment length polymorphism,
or
AFLP.
In
this
method,
restriction
fragments of DNA from the test sample are
ligated to linker fragments that are then
used to prime an ampli±cation reaction
(Fig. 3).
The
labeled
primers
used
in
this reaction can be made more or less
speci±c, so that a manageable number of
fragments are produced in the ±nal output.
These ampli±ed fragments can then be
screened for length polymorphisms using
gel
electrophoresis,
with
the
idea
that
there will be a species-speci±c pattern of
restriction polymorphisms.
AFLP has the advantage that a large
number of loci
are
examined at
once
and
that
no
prior
knowledge
of
the
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