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Previous Article | Next Article 
Journal of Bacteriology, April 2001, p. 2677-2681, Vol. 183, No. 8
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.8.2677-2681.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Characterization of Brucella suis clpB
and clpAB Mutants and Participation of the Genes in
Stress Responses
Euloge
Ekaza,
Jacques
Teyssier,
Safia
Ouahrani-Bettache,
Jean-Pierre
Liautard, and
Stephan
Köhler*
Institut National de la Santé et de la
Recherche Médicale U-431, Université Montpellier II,
F-34095 Montpellier, France
Received 23 October 2000/Accepted 26 January 2001
 |
ABSTRACT |
Pathogens often encounter stressful conditions inside their hosts.
In the attempt to characterize the stress response in Brucella suis, a gene highly homologous to Escherichia coli
clpB was isolated from Brucella suis, and the deduced
amino acid sequence showed features typical of the ClpB ATPase family
of stress response proteins. Under high-temperature stress conditions,
ClpB of B. suis was induced, and an isogenic B. suis
clpB mutant showed increased sensitivity to high temperature, but
also to ethanol stress and acid pH. The effects were reversible by
complementation. Simultaneous inactivation of clpA and
clpB resulted in a mutant that was sensitive to oxidative
stress. In B. suis expressing gfp, ClpA but not
ClpB participated in degradation of the green fluorescent protein at 42°C. We concluded that ClpB was responsible for tolerance to several
stresses and that the lethality caused by harsh environmental conditions may have similar molecular origins.
| |
TEXT |
Brucella suis is a
gram-negative, facultative intracellular bacterium and one of the
causative agents of brucellosis in humans and animals (2).
Oral infection, which is considered a natural route of infection by
brucellae, and localization of the bacteria inside the host cell
phagosome most likely expose the pathogen to various stresses. In
response to environmental stress conditions such as elevated
temperatures, variation in pH, and starvation, a certain number of
proteins are induced or repressed in Brucella spp., among
which are the GroE and DnaK heat shock proteins (12, 16,
24). In intracellular brucellae, which encounter stressful conditions such as acidic pH (23) and possibly oxidative
stress due to the presence of oxygen radicals, the known stress
proteins GroEL and DnaK are induced during infection, and DnaK is
essential for multiplication of B. suis in macrophage-like
cells (12, 17). The HtrA (high-temperature requirement)
stress protein is involved in high-level spleen colonization with
Brucella abortus in mice (5). Several other
stress response proteins including ClpA, ClpB, ClpC, and ClpX, are
members of a family of proteins called the Clp ATPases (HSP100
proteins), represented in prokaryotes and eukaryotes with a high degree
of conservation (28). Clp stress proteins can be induced
by high temperature, oxidative stress, high salt or ethanol
concentration, and iron limitation (14, 26, 28). In
addition to being sensitive to various stresses, Listeria
monocytogenes clpC mutants are attenuated for virulence in mice
(26).
Our article describes the isolation and characterization of a B. suis gene encoding a homolog of the ClpB stress response proteins.
An isogenic clpB mutant and the complemented strain were
constructed and compared with the wild type and a clpA
mutant (4) for survival at high temperature, at different
ethanol concentrations, at acid pH, and in the presence of hydrogen
peroxide. In addition, the effect of hydrogen peroxide on a
clpAB double mutant was studied, and the possible
participation of ClpA and ClpB in protein degradation was analyzed.
B. suis 1330 (biovar 1) was grown in tryptic soy (TS) broth
for 24 h or on TS agar for 72 h at 37°C. When needed,
antibiotics were added (25 µg of chloramphenicol per ml and 50 µg
of kanamycin per ml). Escherichia coli strains were grown in
Luria-Bertani (LB) broth or on LB agar. Antibiotics used were
ampicillin (50 µg/ml), kanamycin (50 µg/ml), and chloramphenicol
(25 µg/ml).
ClpB is highly conserved in B. suis.
A 380-bp DNA
fragment was amplified by PCR from the second ATP-binding domain of the
Escherichia coli clpB gene with the oligonucleotides 5'-TGGCGGATCCAAATCGCCCG-3' and
5'-CGACCGTTCTCCCTTGCCCG-3' (11, 32),
radiolabeled, and hybridized to a genomic Southern blot of B. suis DNA under the previously described conditions
(4). A 2.7-kb HindIII fragment containing
the 3' end of B. suis clpB recognized by the probe was
cloned into pUC18, and a 4-kb EcoRV fragment with the 5' end
was isolated in a second cloning step and fused in the proper
orientation to generate the intact clpB gene, yielding
plasmid pUC-CLPB. The fusion site was verified by sequencing.
Nucleotide sequence analysis performed with the automated Applied
Biosystems 373A DNA sequencer revealed an open reading frame of 2,625 nucleotides coding for 874 amino acids, flanked by a putative
ribosome-binding site and a potential rho-independent transcription
terminator. The theoretical molecular mass of this protein was 97 kDa,
which was in agreement with the mass of the protein recognized by
anti-E. coli ClpB antiserum in lysates of B. suis
(Fig. 1). Using the FASTA algorithm
(22), B. suis ClpB showed 59% amino acid
identity over its entire length with ClpB of E. coli
(9) and of Haemophilus influenzae
(6). A multiple sequence alignment of the three protein
sequences performed with ClustalW 1.60 (34) revealed the
typical features of the Clp ATPases, with two nucleotide-binding
regions, each containing the A and B box nucleotide-binding motifs
separated by a long spacer of 123 amino acids, typical of the ClpB
subfamily (31). The two characteristic internal signature
sequences DASNLLKPALARG and RIDMSEFMEKHSVSRLIGA
were found at positions 295 to 307 and 632 to 650, respectively,
of the protein sequence, and the sensor and substrate discrimination
domain, involved in substrate recognition by Clp ATPases
(30), was identified as the motif GARPLKRVI at positions 814 to 822.
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FIG. 1.
Expression of b-clpB in B. suis at
37°C (lanes 1 to 3) and 46°C (lanes 4 to 6). Lysates equivalent to
109 bacteria were loaded per lane. Western blot analysis
was performed with polyclonal anti-E. coli ClpB antibodies.
Lanes 1 and 4, wild-type B. suis with control plasmid
pBBR1MCS; lanes 2 and 5, B. suis clpB null mutant with
pBBR1MCS; lanes 3 and 6, B. suis clpB mutant complemented
with pBBR1-CLPB. The arrow indicates the position of ClpB.
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ClpB is involved in survival of B. suis at high
temperature, in the presence of ethanol, and at acid pH.
In order
to evaluate a possible role of the ClpB homolog of B. suis
in stress response, we constructed a clpB deletion mutant. In the construct containing the 3' half of clpB, a 400-bp
EcoRV-Bsu15I fragment located within the open
reading frame encoding ClpB was replaced by the kanamycin resistance
gene from pUC4K. The whole insert was blunted and recloned into the
Brucella suicide vector pCVD442 (3) prior to
electroporation of B. suis (4). clpB inactivation by double recombination was confirmed by Southern blot
analysis (not shown) and by Western blot using polyclonal anti-E.
coli ClpB cross-reacting with the homologous protein of B. suis (Fig. 1). For complementation, cloned clpB of
B. suis was inserted as a 5-kb PvuII fragment
into the broad-host-range cloning vector pBBR1MCS (13),
introduced as plasmid pBBR1-CLPB into the clpB null mutant
of B. suis, and expression of clpB was visualized
by immunoblot (Fig. 1). Growth rates at 22, 37, and 42°C in TS medium
were not significantly different for wild-type bacteria and the mutant.
For Western blot analysis with anti-E. coli ClpB antibodies,
bacteria were grown to an optical density at 600 nm (OD600)
of 0.5 in TS broth at 37°C, centrifuged, washed once in
phosphate-buffered saline (PBS), and resuspended in 1 ml of TS broth at
the appropriate temperature, 37 or 46°C. The bacteria were incubated
for 2 h, followed by heat killing at 65°C for 45 min and one
wash in PBS. For sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), total protein equivalent to 109
bacteria was loaded onto SDS-12.5% polyacrylamide gels and separated according to standard protocols. Following transfer of proteins, ClpB
from B. suis was detected by chemiluminescence using a
1,000-fold-diluted rabbit anti-E. coli ClpB antiserum
(35) and anti-rabbit immunoglobulin horseradish
peroxidase-conjugated secondary antibody. B. suis ClpB was
induced approximately threefold under heat stress conditions at 46°C
(Fig. 1, lanes 1 and 4), as measured using SigmaGel software (SPSS
Science, Chicago, Ill.), confirming this characteristic property
previously described for ClpB of E. coli
(11). In the strain overexpressing clpB, the
induction was not readily visible, possibly due to a multicopy effect
of plasmid pBBR1-CLPB and high levels of expression at 37°C (Fig. 1,
lanes 3 and 6).
To study sensitivity at high temperature, log-phase cultures at 37°C
were resuspended in an equal volume of heated TS medium and incubated
for 2 and 4 h at 50°C with shaking. Serial dilutions of samples
were plated on TS agar to determine the concentration of viable
bacteria. The decrease in viability of the clpB mutant was
significantly greater at 2 and 4 h than that of the wild type (Fig. 2; P < 0.001).
Complementation of the mutant in trans with the intact
clpB gene restored a lower sensitivity to 50°C, comparable to the results obtained with the wild type (Fig. 2). In contrast, a
clpA mutant of B. suis (4) behaved
like the wild type, and the decrease in viability of the
clpAB mutant was comparable to that of the clpB
mutant (not shown). The clpAB double mutant was obtained
from a clpB null mutant in which clpA was
inactivated in addition as described recently (4), but
using a chloramphenicol resistance gene as the selection marker
(25). This second inactivation was verified using
polyclonal anti-ClpA antiserum (4).
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FIG. 2.
High-temperature survival at 50°C of wild-type
B. suis 1330 with plasmid pBBR1MCS ( ), the
clpB mutant containing pBBR1MCS ( ), and the mutant
complemented in trans by clpB on plasmid pBBR1MCS
( ) in TS broth at 0, 2, and 4 h postinoculation. Experiments were
performed three times in duplicate, and results represent means ± standard deviations (SD). Asterisks indicate significant differences
between the clpB mutant and the other two strains
(P < 0.001).
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Members of the HSP100 proteins have also been reported to be involved
in the ethanol-induced stress response in Bacillus subtilis and Saccharomyces cerevisiae (14, 27). To
address this point for ClpB, B. suis from a stationary-phase
culture was diluted 100-fold in TS supplemented with ethanol and
incubated at 37°C for 6 h prior to plating of serial dilutions
onto TS agar. In B. suis, ClpB participated in resistance to
ethanol stress, as the clpB mutant showed a 1,000-fold
decrease over the incubation period in 10% (vol/vol) ethanol, compared
to the wild-type strain (Fig. 3). The
minimal effective ethanol concentration was determined to be 8%. In
the complemented mutant, in contrast, resistance to ethanol was
restored and not significantly different from that measured for the
wild-type strain. The clpAB mutant behaved like the
clpB mutant, and no role could be assigned to ClpA during ethanol stress (not shown).
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FIG. 3.
Effect of ethanol at a final concentration of 10%
(vol/vol) on survival of the B. suis wild type (WT), the
clpB null mutant (ClpB ) containing pBBR1MCS, and the null
mutant complemented with clpB cloned into pBBR1MCS (ClpB-
compl.) in TS broth at 0 h (hatched bars) and 6 h (solid bars)
postinoculation. Experiments were performed three times in duplicate.
Error bars indicate SD. The value marked by an asterisk was
significantly different from the others (P < 0.001).
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|
Acid pH as a stress factor may be encountered by brucellae upon their
entry into the host organism via the oral route. To study the effect of
acid pH stress, a stationary-phase culture of B. suis was
diluted 100-fold in TS broth adjusted to pH 4.0 with citric acid (2 M)
and incubated at 37°C for up to 48 h. Samples were plated on TS
agar to determine the concentration of viable bacteria at the time
points indicated. We noticed a 100-fold initial decrease in viability
for all strains within 8 h of inoculation. Between 8 and 48 h, the clpB mutant showed an additional 105-fold
decrease, compared to a less than 103-fold decrease for the
wild type and the complemented strain (Fig. 4).
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FIG. 4.
Effect of acid pH on long-term survival of wild-type
B. suis 1330 with plasmid pBBR1MCS (), the
clpB mutant containing pBBR1MCS (), and the mutant
complemented in trans by clpB on plasmid pBBR1MCS
() in TS broth adjusted to pH 4.0. Experiments were performed four
times, and results represent means ± SD. Asterisks indicate
significant differences between the clpB mutant and the
other two strains at 32 and 48 h (P < 0.00001).
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|
clpAB double mutant of B. suis is sensitive
to oxidative stress.
Because of reports that Clp proteins play a
role in oxidative stress response (26), we investigated
the role of ClpB in resistance to increasing concentrations of
H2O2. Disk assays were performed as follows: 75 µl of B. suis cultures adjusted to an optical density of
0.2 were plated on TS agar, and sterile 6-mm paper disks saturated with
10 µl of H2O2 at concentrations ranging between 0.9 and 8.8 M were layered on top prior to incubation at 37°C
for 2 days and measurement of inhibition zone diameters. The
single-gene null mutants clpB and clpA were not
more sensitive to H2O2 than the wild type, but
a clpAB double mutant showed increased sensitivity (Fig.
5). Both Clp ATPases probably
participated in the oxidative stress response and could substitute for
each other.
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FIG. 5.
Sensitivity of wild-type B. suis 1330 (open
bars), the clpA mutant (hatched bars), the clpB
mutant (striped bars), and the clpAB double mutant (solid
bars) to various concentrations of H2O2. Disk
assays were performed three times in triplicate; error bars indicate
SD. At each concentration, the values represented by the solid bars
were significantly different from the others (*, P < 0.05; **, P < 0.01).
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ClpA but not ClpB participates in stress-dependent GFP degradation
in vivo.
It has been shown recently that the protease ClpP of
L. monocytogenes contributes to stress-dependent degradation
of the model foreign protein green fluorescent protein (GFP)
(7). In E. coli, the proteolytic activity of
ClpP is dependent on the formation of a complex with ClpA
(18). Following our observation that ClpA of B. suis can functionally replace ClpA of E. coli
(4), we investigated whether ClpA or ClpB of B. suis participates in protein degradation in vivo. The B. suis wild type and 
clpA, clpB, and
clpAB mutants were transformed with plasmid
pBBR1-GFP-SOG, carrying a strong constitutive B. suis
promoter fused to gfp, resulting in fluorescent bacteria
(21). The fluorescence intensity of each strain was
determined by flow cytometry at 37 and 42°C (the maximum temperature
for growth of the clpA and clpAB mutants), and unlabeled strains were used as controls. Bacteria were grown to an
OD600 of approximately 1.0 in TS broth at 37 or 42°C. GFP expression was monitored after excitation at 488 nm using a FACSCalibur flow cytometer (Becton Dickinson). Bacteria were electronically gated,
and a total of 50,000 bacteria per sample were analyzed using CellQuest
software. The Kolmogorov-Smirnov two-sample test included in the
software was used for statistical analysis. The experiments were
performed three times for all strains. Fluorescence intensity was
similar for all strains grown at 37°C, but at 42°C wild-type
B. suis was fourfold less fluorescent than the
clpA mutant (Fig. 6), and
hence significantly different (P < 0.001). A similar
difference was observed between the wild type and the clpAB mutant (P < 0.001), whereas the
clpB mutant behaved like the wild-type strain. These
results suggested that ClpA participated in the degradation of a
foreign protein under high-temperature stress conditions in B. suis and that ClpB was not involved in this activity.
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FIG. 6.
Flow cytometry analysis of GFP expression in wild-type
B. suis 1330 (WT; black histograms) and in the
clpB (dark grey), clpA (light grey), and
clpAB (white) null mutants transformed with plasmid
pBBR1-GFP-SOG. The X values indicate mean relative fluorescence
intensities for the strains at 37°C (left) and at 42°C (right).
Experiments were performed three times, and one representative
experiment is shown.
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Conclusions.
The participation of ClpB proteins in response to
stress has been investigated (1, 27, 31, 32), and the
demonstration that ClpB interacts with DnaK, DnaJ, and GrpE in
suppressing and reversing protein aggregation by the formation of a
bichaperone system in vitro (8, 19, 20, 36) represents an
important contribution to the understanding of the unique role of ClpB
in thermotolerance and stress recovery. The clpB B. suis
cells were rapidly killed at 50°C, as has been shown previously for
E. coli (33) and Helicobacter pylori
(1), hence establishing for B. suis the vital
role of ClpB in stress recovery. Nevertheless, the genetic elements
participating in this stress response have not been identified, and
from the recent publication of a 
32 consensus sequence
for B. abortus (25), we could not detect a 35
or a 10 region typical of heat shock promoters preceding the
clpB coding sequence. Acid pH is a second stress factor, and our results showed the induction of an adaptive acid tolerance response
during the first 8 h, as previously reported (15). Survival of the clpB null mutant continued to drop over the
next 24 h, and ClpB therefore appeared to be involved in this
tolerance response. In brucellae, ethanol and acid pH may be signals
sensed and transduced by two-component systems, but very little is
known about this subject yet. In contrast to our earlier results with ClpA (4), overexpression of clpB restored the
wild-type phenotype for the various stress conditions studied. An
excess of ClpB obviously did not affect the postulated interaction of
this chaperone with the DnaK-DnaJ-GrpE system.
During oxidative stress, only simultaneous inactivation of both
clpA and clpB resulted in increased sensitivity
of B. suis to various concentrations of hydrogen peroxide.
ClpA and ClpB have in common that their functions contribute to the
elimination of damaged or aggregated proteins. Studies performed in
vitro with these Clp ATPases from E. coli revealed that the
mechanisms involved are nevertheless different. ClpA is part of the
two-component protease ClpAP, and it binds protein substrates and
presents them to the protease ClpP for degradation. ClpB participates
in suppression and reversion of protein aggregation. It is conceivable
that the accumulation of damaged proteins is the event leading to
increased sensitivity of B. suis clpAB null mutants to
H2O2 and that the reduction in the
concentration of altered proteins by degradation or refolding increases
resistance to this specific stress.
Flow cytometry experiments presented here led to the conclusion that
the Clp ATPase ClpB cannot replace ClpA in its role in protein
degradation in B. suis. The higher fluorescence intensity measured in the isogenic clpA and clpAB mutants
was obviously due to the absence of ClpA. Three recent publications
were of great interest for the interpretation of our results. (i) A
similar shift in fluorescence was described following inactivation of the protease ClpP in L. monocytogenes (7). (ii)
In vitro experiments showed that E. coli ClpA can bind
unfolded GFP and that the complex ClpAP can degrade unfolded GFP but
not native GFP (10). (iii) ClpX, in contrast, which also
presents substrates for degradation to ClpP, cannot trap unfolded
substrates unless they have a specific binding motif (29).
We concluded that at 42°C, GFP molecules that were sufficiently
unfolded in B. suis probably allowed specific binding by
ClpA and presentation to ClpP in vivo, resulting in degradation of the
substrate in the wild type and in the clpB null mutant.
We have characterized the important role of the ClpB homolog of
B. suis in response to several environmental and in vitro stresses. The inactivation of clpB of B. suis
demonstrated that, in this organism, a single protein was responsible
for tolerance to heat, ethanol, and acid pH, adding proof to the
hypothesis that the molecular causes of lethality are similar in very
different environmental circumstances. The possible participation of
ClpB in resistance of the pathogen B. suis to host defense
mechanisms remains to be determined.
Nucleotide sequence accession number.
The nucleotide sequence
of B. suis clpB has been submitted to the GenBank database
(accession number AJ251205).
| |
ACKNOWLEDGMENTS |
We thank C. H. Chung for his generous gift of anti-E.
coli ClpB antiserum and M. T. Alvarez-Martinez for help with
the computer software.
This work was supported in part by grant QLK2-CT-1999-00014 from the
European Union.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: INSERM U-431,
Université Montpellier II, C.P. 100, Pl. E. Bataillon, 34095 Montpellier, France. Phone: (33) 4 67 14 42 38. Fax: (33) 4 67 14 33 38. E-mail: kohler{at}crit.univ-montp2.fr.
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Journal of Bacteriology, April 2001, p. 2677-2681, Vol. 183, No. 8
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.8.2677-2681.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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