320
Anthrax (Bacillus anthracis), Molecular Biology of
5% CO
2
and 37
C.
AtxA
strains show
signifcantly
reduced
production
oF
all
three toxin proteins in culture and are
avirulent in a mouse model.
AtxA is an activator oF toxin synthesis
that
acts
at
the
transcriptional
level.
Although
pagA
,
lef
,and
cya
are transcribed
as part oF an AtxA-dependent regulon,
there are no obvious sequence similarities
upstream oF the virulence genes that would
suggest an AtxA binding site, nor does
the AtxA protein sequence suggest any
characteristics oF DNA-binding proteins,
so the mechanism it uses to
regulate
transcription oF these genes is not clear.
The temperature requirement For toxin
synthesis
is
at
the
level
oF
the
atxA
transcript, as steady state levels oF
atxA
mRNA are signifcantly increased in cells
grown at 37
C compared to cells grown
at 28
C. Higher levels oF AtxA do not
directly
translate
into
increased
levels
oF
toxin
production,
however.
Strains
that carry
atxA
on a multicopy plasmid
have high levels oF AtxA, but they do
not
produce
more,
and
some
strains
even produce signifcantly less, protective
antigen than do matched strains carrying
atxA
in a single copy on pXO1. ±urther,
although CO
2
-enhanced toxin expression
is AtxA-dependent, CO
2
does not aFFect
AtxA levels. Although altering AtxA levels
does not directly lead to an alteration
oF toxin levels, it can have a regulatory
eFFect. It has been reported that some
B. anthracis
strains carrying a multicopy
plasmid encoding
atxA
produce toxin in
aCO
2
-independent Fashion in contrast
to
the
CO
2
-dependence
seen
in
the
parental strain.
The
toxin
genes
are
not
the
only
B. anthracis
genes that are AtxA-regulated.
T
h
ep
r
e
s
e
n
c
eo
Fp
XO
1a
l
s
oi
n
c
r
e
a
s
e
s
capsule
synthesis.
AtxA
appears
to
be
necessary
and
suFfcient
For
this
stimulation,
as
indicated
by
enhanced
transcription
oF
a
capB-lacZ
Fusion
in
an
AtxA-dependent
manner.
To
identiFy additional AtxA-dependent gene
expression, HoFFmaster and Koehler used
a
lacZ
-containing transposon as a marker
For
genes
that
were
induced
by
CO
2
in an AtxA-dependent manner. In this
scheme,
lacZ,
which is a gene whose
expression can be easily monitored, will
be placed randomly under the control
oF the regulatory sequences oF the gene
into which the tranposon inserts.
lacZ
expression will thereFore mark genes that
are expressed under the
conditions oF
interest – in this case, the presence oF CO
2
and AtxA. In this experiment, there were
10
independent
transposon
insertions
with AtxA-dependent expression that did
not
map
to
the
toxin
genes.
Two-
dimensional
electrophoresis
oF
protein
extracts From
atxA
+
versus
atxA
–cells
also indicates that synthesis oF nontoxin
proteins is influenced by AtxA. Some oF
these AtxA-regulated genes may play a
role in the growth oF
B. anthracis
,because
atxA
-null mutations cause
B. anthracis
to
grow poorly on certain minimal media,
and in sporulation, because an
atxA
-null
mutant sporulates more eFfciently than
the parent strain when grown in rich
media. ±inally,
atxA
,bu
tn
o
tth
et
o
x
in
genes, is required For release oF
B. anthracis
From macrophages, so an
atxA
-regulated
gene is likely to be involved in this process.
4.2
PagR
One oF the CO
2
-enhanced,
atxA
-dependent
loci on pXO1 is a repressor oF the
pagA
operon that serves to limit
pagA
expression
under conditions optimal For PA synthesis.
It was named
pagR
For p
rotective a
ntig
en
r
epressor.
pagR
is located downstream oF
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