312
Anthrax (Bacillus anthracis), Molecular Biology of
pXO1 or the
B. anthracis
genome, nor is
there a difference in codon usage between
them, so the pathogenicity island must
have evolved in a species very similar to
B. anthracis
.
In fact, other members of the
B. cereus
group possess many sequences related
to
the
pXO1
coding
sequences.
A
re-
cent hybridization study found that DNA
from three members of this group (two
B. cereus
strains and a
Bacillus thuringien-
sis
strain) each hybridized with over half
of
the
probes
that
were
used
to
rep-
resent
the
pXO1
open
reading
frame
complement. This conservation of pXO1
genes is independent of evolutionary re-
latedness in terms of the chromosomal
sequences, further supporting the idea that
these sequences may shuffle between re-
lated species.
Lest the
reader
fear that
the
deadly
anthrax toxin genes are jumping around
between bacteria, I will point out that
approximately 20% of the open reading
frames
tested
in
this
study,
including
those encoding the anthrax toxin, were
not found in any of the related species
that were tested, and are, as far as we
know, unique to anthrax. Further, the
results of a phylogenetic analysis of
pag
sequences from several
B. anthracis
strains
largely agreed with those from a similar
analysis of chromosomal sequences. This
suggests that the
pag
gene did not evolve
separately from the chromosome, so this
gene probably did not undergo horizontal
transfer during the recent evolution of
B. anthracis
.
3
Molecular Identifcation oF
B. anthracis
Standard microbiological techniques can
be used to distinguish
B. anthracis
from
other bacilli. Distinguishing characteris-
tics include lack of motility and hemolysis,
a typical colony morphology (including
a flat, tacky appearance and gray-white
color), and susceptibility to lysis by gamma
phage. Although these techniques can be
used to identify
B. anthracis
in most cases,
the threat of bioterrorism makes the de-
velopment of rapid molecular surveillance
methods, for use in environmental sam-
ples, a priority. Microbiological techniques
rely upon isolation and culture of bacteria,
and they generally take at least 24 h to
complete. This fact can limit their suc-
cessful use in certain situations. It would
be desirable to develop faster diagnostic
methods so that anthrax outbreaks can
be identi±ed and managed as rapidly as
possible. A second limitation to standard
microbiological techniques is the fact that
anthrax victims who are treated with an-
tibiotics before blood cultures are taken
often have blood cultures that are negative
for
B. anthracis
. For accurate diagnosis of
these patients, molecular diagnostic tech-
niques must be perfected.
As mentioned above,
B. anthracis
is ge-
netically closely related to
B. cereus
and
B. thuringiensis
and,
in
fact,
some
re-
searchers have proposed on the basis of
multilocus enzyme electrophoresis as well
as 16S and 23S rRNA sequence analy-
sis, that these organisms be classi±ed as
subtypes of the same species. The relat-
edness of these bacteria has complicated
efforts to develop
B. anthracis
-speci±c tests
because the detection of related species as
false-positives can be a problem.
3.1
Molecular Methods to Distinguish
B. anthracis
From Other Bacteria
The presence of virulence plasmids, in-
cluding pXO1 and pXO2 in
B. anthracis
,
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