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Journal of Bacteriology, December 1999, p. 7629-7633, Vol. 181, No. 24
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Conditional Sigma Factor Expression, Using the
Inducible Acetamidase Promoter, Reveals that the Mycobacterium
tuberculosis sigF Gene Modulates Expression of the
16-Kilodalton Alpha-Crystallin Homologue
Yukari C.
Manabe,1,*
Jong Min
Chen,2
Chiew G.
Ko,3
Ping
Chen,3 and
William R.
Bishai1,2,3
Division of Infectious Diseases, Department
of Medicine, Johns Hopkins University School of
Medicine,1 and Department of
Microbiology and Molecular Immunology,2 and
Center for Tuberculosis Research, Department of International
Health,3 Johns Hopkins University School of
Hygiene and Public Health, Baltimore, Maryland
Received 1 July 1999/Accepted 4 October 1999
 |
ABSTRACT |
A chemically inducible acetamidase promoter-sigF fusion
gene was integrated into the chromosome of Mycobacterium
bovis BCG. Two-dimensional protein gel analysis permitted the
identification of a number of protein spots whose expression was SigF
related. One spot upregulated by inappropriate induction of
sigF expression corresponded to the 16-kDa antigen
alpha-crystallin.
| |
TEXT |
Despite effective antimicrobial
therapy, tuberculosis remains a leading cause of death worldwide.
Identifying important regulatory genes which enable Mycobacterium
tuberculosis to survive environmental stress could have important
therapeutic implications for a disease for which treatment is prolonged
and multidrug resistance a daunting problem worldwide. Alternate sigma
factors are activated under specific stress conditions and control the
expression of specific regulons (6). Several M. tuberculosis sigma factors, including SigB, SigC, SigE, SigF, and
SigH, appear to be responsive to stress conditions (11, 12,
20).
The sigF gene in M. tuberculosis was discovered
by using degenerate PCR and appears to be upregulated in stationary
phase (4). It shows significant homology with stress
response sigma factors in Streptomyces coelicolor and
Bacillus subtilis (3). Since stress responses in
vitro are likely to reflect the array of mycobacterial genes expressed
upon exposure to the host's immune system, the identification of genes
controlled by these sigma factors may provide insight into the
pathogenesis of M. tuberculosis. Using a chromosomally
integrated, chemically inducible Mycobacterium smegmatis
acetamidase promoter fused to the sigF gene, we were able to
express SigF inappropriately during exponential phase when it is absent
in wild-type cultures of both Mycobacterium bovis BCG and
M. tuberculosis (10, 13, 14, 16). By comparing two-dimensional gel protein patterns of SigF-expressing strains with
noninduced cultures and wild-type M. bovis BCG, we sought to
identify proteins that were SigF dependent. This conditionally expressing construct has been particularly useful as we have previously attempted to overproduce SigF by using other promoters and showed that
high-level, constitutive production of SigF is lethal in M. bovis BCG (3).
Induction of sigF expression with the inducible
acetamidase promoter.
A 0.7-kb sigF-containing
NdeI-SpeI segment from pYZ99 (Table
1) (4) and a 3-kb acetamidase
promoter locus-containing BamHI-NdeI fragment
from pAMI1 (10) were ligated to one another and cloned into
the BamHI-SpeI-digested pNBV1 to produce pCK0218.
This fusion contained the four open reading frames upstream of the
M. smegmatis acetamidase gene up to the ATG of the
acetamidase gene. The acetamidase ATG was followed by sigF
codon 2 (ACG) in the correct translational reading frame. The
BamHI-SpeI acetamidase promoter-sigF
fusion gene fragment was excised, ligated to PacI linkers,
and cloned into PacI-digested pMH94, a mycobacterial
integrative vector, to yield pCK0275 (9). Using M. bovis BCG (Pasteur strain) and M. tuberculosis CDC1551
(18) transformed with pCK0275, which results in the
single-copy integration of the acetamidase promoter-sigF fusion gene (Pace::sigF), we tested
whether acetamide induction produced inappropriate SigF expression
during logarithmic phase.
Whole-cell lysates of log-phase
M. bovis BCG (optical
density [OD], 0.5) containing an integrated copy of the fusion gene
with and without acetamide induction were separated on a sodium
dodecyl
sulfate - 10% polyacrylamide gel electrophoresis minigel.
Figure
1 shows a Western blot of lysates from
these bacteria developed
with anti-SigF antibodies. A 30-kDa band
consistent with expression
of SigF in exponential phase was seen in the
acetamide-induced
strain (Fig.
1, right lane) but not in the uninduced
cells (middle
lane). Purified SigF is shown in the left lane. A similar
level
of induction of SigF expression was seen with
M. tuberculosis containing the fusion gene (data not shown). Purified
SigF and
polyclonal rabbit antibodies against SigF were prepared as
described
previously (
3).
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FIG. 1.
Western blot of M. bovis BCG culture lysates
incubated with anti-SigF antibody showing expression of SigF by use of
the inducible acetamidase promoter from M. smegmatis. Lanes:
left, purified M. tuberculosis SigF; middle,
exponential-phase (OD, 0.5) uninduced BCG containing the
Pace::sigF fusion gene construct;
right, exponential-phase BCG containing the
Pace::sigF fusion gene construct
induced with acetamide.
|
|
Comparison of protein expression in BCG
Pace::sigF with and without acetamide
induction using 2-D gel electrophoresis.
Recently, 2-D gels have
been used extensively to examine culture filtrates as well as
cell-associated proteins expressed by M. tuberculosis
(15, 17, 19). In addition, 2-D gels have been used to
compare bacterial proteins expressed during macrophage infection and
under certain stress conditions (1, 8). We sought to
identify SigF-dependent proteins by using 2-D gel electrophoresis. We
compared protein patterns in whole-cell lysates of exponential-phase M. bovis BCG harboring the
Pace::sigF fusion gene with or without acetamide induction. Only small differences in spot intensity were
noted on Coomassie blue staining of gels. Hence, we opted for a
pulse-labeling approach to visualize recently synthesized proteins. For
metabolic labeling, 250 µCi of [35S]methionine and
cysteine was added per 10 OD units of culture. Immediately thereafter,
half of each labeled culture was exposed to 0.2% acetamide. After a
4-h incubation, bacteria were pelleted by centrifugation, washed twice
with sterile phosphate-buffered saline (PBS), and then resuspended with
lysis solution (3 M urea, 0.5% Triton X-100, 3.25 µM dithiothreitol,
2% Pharmalyte, phenylmethylsulfonyl fluoride [100 µg/ml],
leupeptin [2 µg/ml]). By using 0.1-mm-diameter glass beads, the
samples were homogenized twice. The samples were centrifuged, and 250 µg of soluble protein was rehydrated overnight into freeze-dried
isoelectric focusing strips (Pharmacia Biotech). First-dimension
electrophoresis and second-dimension electrophoresis with 8 to 18%
gradient gels were carried out in accordance with the manufacturer's instructions.
Proteins initially absent in exponential phase but inducible with the
addition of acetamide were readily apparent by this
approach. Figure
2 shows 2-D gels of exponential-phase
(OD, 0.6)
P
ace::
sigF M. bovis BCG
lysates induced with acetamide (Fig.
2A)
and uninduced lysates (Fig.
2B). Several proteins appeared to
be upregulated in the induced
culture. The appearance of these
spots was dependent upon the
P
ace::
sigF fusion as they were not
present in acetamide-treated wild-type cell lysates (data not
shown).
In addition, proteins which were absent in the induced
culture were
noted in the uninduced cell lysates.
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FIG. 2.
2-D polyacrylamide gel electrophoresis of
Pace::sigF M. bovis BCG whole-cell
culture lysates without acetamide (B) and after 4 h of induction
with acetamide (A). All cultures were treated for 4 h with
[35S]cysteine-methionine. Arrows denote protein pattern
differences between gels.
|
|
In another experiment, stationary-phase cultures were diluted only 1:50
and grown to ODs at 600 nm of 0.2 and >1.0. Figure
3 shows 2-D electrophoresis of these
whole-cell lysates. A 16-kDa
protein with an estimated isoelectric
point of 5 was visible during
early exponential phase with the addition
of acetamide (Fig.
3A)
although absent under the same conditions in
uninduced lysates
(Fig.
3B) and in wild-type lysates (Fig.
3C). In
stationary-phase
cultures where the native
sigF gene is
known to be expressed,
the 16-kDa protein spot is uniformly present in
all cell lysates.
It appears to be relatively upregulated in
acetamide-induced cell
lysates of
M. bovis BCG containing
the fusion gene (Fig.
3D).
This inducible spot correlates with the
predicted molecular weight
and isoelectric point of the
alpha-crystallin homologue, Acr (
21).
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FIG. 3.
A comparison of wild-type M. bovis BCG and
the Pace::sigF strain during early
exponential phase (OD, 0.2) and stationary phase (OD, >1.0) with and
without acetamide. Whole-cell lysates labeled with
[35S]methionine-cysteine of both strains were compared by
2-D gel electrophoresis. (A) Exponential-phase
Pace::sigF BCG with acetamide; (B)
exponential-phase Pace::sigF BCG
without acetamide; (C) exponential-phase wild-type BCG with acetamide;
(D) stationary-phase Pace::sigF BCG
with acetamide.
|
|
Analysis of 16-kDa protein.
We then sought to determine if
SigF was necessary for the elaboration of the 16-kDa protein in
aerobically grown cultures. Using polyclonal rabbit antibodies directed
against Acr, we compared 2-D Western blots of wild-type M. tuberculosis stationary-phase cultures (Fig.
4A) and stationary-phase cultures of an
M. tuberculosis sigF knockout strain (M. tuberculosis 
sigF) (Fig. 4B) (P. Chen, R. E. Ruiz, and W. R. Bishai, Abstr. 99th Gen. Meet. Am. Soc. Microbiol.
1999, abstr. U-101, p. 653, 1999). After gel electrophoresis, proteins
were transferred to nitrocellulose membranes by gravity diffusion
overnight at 4°C and then blocked with 5% nonfat milk in PBS with
0.1% Tween 20 (PBS-T) for 1 h. Membranes were then incubated
overnight in PBS-T with rabbit-specific polyclonal antibody at the
appropriate concentration at 4°C. After washing the membranes, they
were incubated with anti-rabbit horseradish peroxidase-conjugated antibody for 2 h and then with a chemiluminescent substrate for autoradiograph exposure. Polyclonal rabbit antibodies against Acr were
the gift of C. E. Barry (Bethesda, Md.).
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FIG. 4.
2-D gel Western blot of M. tuberculosis
culture lysates incubated with anti-alpha-crystallin antibody. (A)
Western blot of the stationary-phase M. tuberculosis sigF
knockout strain lysate; (B) Western blot of stationary-phase wild-type
M. tuberculosis culture lysate. The arrows show the location
of the alpha-crystallin protein.
|
|
While the anti-alpha-crystallin antibody nonspecifically binds to other
heat shock protein complexes, it shows a discrete
spot at the predicted
molecular weight and isoelectric point of
5 for Acr. This 16-kDa
protein uniformly appears only in wild-type
cultures and is completely
absent in
M. tuberculosis sigF culture
lysates.
To further establish the identity of the 16-kDa protein, we excised the
protein and performed sequence analysis on the gel
piece through the
Harvard Microchemistry Facility where samples
are proteolytically
digested, separated by microcapillary high-pressure
liquid
chromatography, and analyzed with tandem mass spectrometry.
Correlating
these results with known sequences by using the algorithm
Sequest,
three unique polypeptide protein fragments, including
a hypothetical
protein (Rv3686c), lumazine synthase (RibH, Rv1416)
involved in the
riboflavin synthesis pathway, and Acr (HspX, Rv2031c),
were identified
in the excised gel piece (
5). The molecular
weights and
isoelectric points of all three of these proteins
are similar. In
summary, the 16-kDa SigF-dependent spot was both
immunoreactive with
anti-Acr antibodies and found by mass spectrometry
to contain peptide
sequences matching
Acr.
The 16-kDa alpha-crystallin homologue, Acr, is a member of the small
heat shock protein superfamily. Proteins in this family
act as
molecular chaperones preventing the thermal aggregation
of proteins and
suppressing denaturation (
2). The
M. tuberculosis Acr protein has been shown to accumulate in the transition to
stationary phase similar to SigF. However, as noted by Yuan et
al.,
SigF may not be the sole regulator of alpha-crystallin expression
as
many of the conditions shown to upregulate SigF expression
have no
effect on the expression of the 16-kDa protein (
12,
21).
Moreover, the expression of the
acr gene has been shown
to
be complex, with evidence for a strong promoter and a repressor
binding
site modulating expression (Y. Yuan, D. D. Crane, D. R.
Sherman, M. Hickey, and C. E. Barry, Proceedings of the
Thirty-Second
U.S.-Japan Cooperative Medical Science Program,
Tuberculosis-Leprosy
Research Conference, Cleveland, Ohio). In other
recent data from
Hu and Coates, Acr appears to be posttranscriptionally
modified
to account for its accumulation in stationary phase rather
than
solely transcriptionally regulated (
7).
In agreement with Yuan et al., our data show that in ordinary
exponential-phase culture, very little alpha-crystallin can
be detected
even with SigF induction using the acetamidase promoter
(
21). In contrast, in cultures recently diluted from
stationary
phase, SigF induction leads to alpha-crystallin expression.
Furthermore,
in
M. tuberculosis sigF knockout strains,
alpha-crystallin is
absent in stationary-phase cultures. Overall,
alpha-crystallin
expression appears to be controlled by transcriptional
and translational
mechanisms of expression and accumulation which may
be modulated
by different stress conditions such as anaerobiosis. Our
data
suggest that alpha-crystallin requires SigF in conjunction with
other stationary-phase elements in order to be produced at detectable
levels in stationary phase under aerobic culture
conditions.
We have used the
M. smegmatis-inducible acetamidase promoter
fused to
sigF to produce the transcription factor at a
nonphysiologic
time. 2-D gel electrophoresis comparisons reveal several
candidate
proteins that may be regulated by SigF either as a direct
transcriptional
effect or indirectly through other regulatory proteins
controlled
by SigF. In addition, Western blots of 2-D gels offer an
alternative
method for separating and distinguishing individual
proteins.
Using these techniques, we have shown a putative link between
two genes which are upregulated in the transition to stationary
phase:
sigF, encoding an alternative sigma factor, and
acr, encoding
the alpha-crystallin
homologue.
| |
ACKNOWLEDGMENTS |
Y.M. is supported by a Heiser Foundation Research Grant. This work
is also supported by NIH grants AI36973 and AI37856.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center for
Tuberculosis Research, Department of Medicine, Division of Infectious
Diseases, Johns Hopkins University, 1830 E. Monument St., Room
455, Baltimore, MD 21205.
| |
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Journal of Bacteriology, December 1999, p. 7629-7633, Vol. 181, No. 24
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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