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Applied and Environmental Microbiology, May 2001, p. 2029-2036, Vol. 67, No. 5
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.5.2029-2036.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Changes in Protein Synthesis and Morphology during
Acid Adaptation of Propionibacterium
freudenreichii
Gwénaël
Jan,1,*
Pauline
Leverrier,1
Vianney
Pichereau,2,3 and
Patrick
Boyaval1
Laboratoire de Recherches de Technologie
Laitière, Institut National de la Recherche
Agronomique,1 and UMR CNRS 6026,
Département osmoadaptation chez les bactéries,
Université de Rennes I, Campus de
Beaulieu,2 35042 Rennes, and Laboratoire
de Microbiologie de l'Environnement, USC INRA EA956,
Université de Caen, 14032 Caen,3 France
Received 2 October 2000/Accepted 4 February 2001
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ABSTRACT |
Survival of bacteria in changing environments depends on their
ability to adapt to abiotic stresses. Microorganisms used in food
technology face acid stress during fermentation processes. Similarly,
probiotic bacteria have to survive acid stress imposed within the
stomach in order to reach the intestine and play a beneficial role.
Propionibacteria are used both as cheese starters and as probiotics in
human alimentation. Adaptation to low pH thus constitutes a limit to
their efficacy. Acid stress adaptation in the probiotic SI41 strain of
Propionibacterium freudenreichii was therefore
investigated. The acid tolerance response (ATR) was evidenced in a
chemically defined medium. Transient exposure to pH 5 afforded
protection toward acid challenge at pH 2. Protein neosynthesis
was shown to be required for optimal ATR, since chloramphenicol reduced
the acquired acid tolerance. Important changes in genetic expression were observed with two-dimensional electrophoresis during adaptation. Among the up-regulated polypeptides, a
biotin carboxyl carrier protein and enzymes involved in DNA synthesis and repair were identified during the early stress response, while the
universal chaperonins GroEL and GroES corresponded to a later response.
The beneficial effect of ATR was evident at both the physiological and morphological levels. This study constitutes a
first step toward understanding the very efficient ATR described in
P. freudenreichii.
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INTRODUCTION |
Bacteria are periodically exposed to
abiotic stresses in a variety of environments. In this context,
survival involves sensing changes in environmental parameters such as
temperature, pH, or the presence of toxic compounds and adapting
quickly in order to exhibit a greater tolerance. Bacteria have evolved
a set of inducible responses, including stress proteins, leading to
tolerance, which implies the complex regulation of gene expression
(41).
Acid stress is of particular importance for bacteria used in food
technology. Indeed, a variety of food products are acidified during
fermentation by lactic acid bacteria. Probiotic microorganisms, in
particular, are usually provided in the form of fermented milk and
suffer lactic acid stress. Consequently, probiotics, including Bifidobacterium and Lactobacillus strains,
undergo severe mortality during the processing and storage of such
products. For this reason, less acidified products such as cheeses were
proposed as carriers of these bacteria (8). Probiotics are
further challenged by extreme acid stress when reaching the stomach
lumen where hydrochloric acid is present. It is thus clear that the
ability to efficiently adapt to acid stress is a sine qua non condition
for a probiotic microorganism in order to reach the intestine and exert
the expected beneficial effects (10).
As for other stresses, a sublethal acidic environment can trigger an
adaptive response in bacteria and offer protection toward a subsequent
exposure to a lethal acidic pH, a mechanism known as acid tolerance
response (ATR). ATR has been well documented for a number of
gastrointestinal or food-borne pathogenic bacteria, such as
Escherichia coli (36), Salmonella
enterica serovar Typhimurium (7), Aeromonas
hydrophila (18), Vibrio
parahaemolyticus (45), Helicobacter pylori
(27), Listeria monocytogenes (4),
and Enterococcus faecalis (6), as well as the
oral cariogenic organism Streptococcus mutans
(12). Indeed adaptation and survival at low pH might be
important factors in the pathogenicity of gastrointestinal bacteria and
are of great concern in food safety and health. In contrast, less is
known about the mechanisms of acid tolerance in beneficial
microorganisms used in the dairy industry, except Lactococcus
lactis (13) and Lactobacillus acidophilus
(21).
Propionibacteria are gram-positive, nonmotile, anaerobic to
aerotolerant bacteria producing propionic acid, acetic acid, and carbon
dioxide as products of the fermentation of sugars and lactic acid. They
are thus used for the industrial fermentative production of propionic
acid (30). Dairy propionibacteria, such as
Propionibacterium freudenreichii, are traditionally used as
starter cultures in cheese technology. They are also considered
probiotics due to their ability to inhibit the growth of undesirable
flora (23), to stimulate the growth of
bifidobacteria (3), and to beneficially modify the
enzymatic activities within the gut (25). It is likely that the acid tolerance of dairy propionibacteria contributes to their
potentiality as both starters and probiotics.
Acid susceptibility was shown to be highly dependent on the species and
strain of propionibacteria studied (34). In a previous report, we described the ability of a P. freudenreichii
strain isolated from Swiss-type cheese to develop ATR in a complex
medium (17). Here, we investigated ATR mechanisms in a
probiotic strain of the same species. Although a chemically defined
medium is radically different from conditions encountered in situ, it
allows physiological and molecular investigations under reproducible
and controllable conditions. We thus developed such a medium for dairy
propionibacteria and demonstrated that growth and ATR (this study) were
similar to what was observed in rich medium (17). This
allowed us to study changes in protein synthesis in order to elucidate
the mechanism involved in ATR. Acid-induced polypeptides were
determined, their relative rate of synthesis was monitored in a
time-dependent manner, and five proteins were identified by N-terminal
sequencing. In addition, changes in cell morphology were observed by
scanning electron microscopy during acid adaptation and extreme acid challenge.
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MATERIALS AND METHODS |
Microorganism and growth conditions.
The SI41 strain of
P. freudenreichii subsp. shermanii used in this
study was kindly provided by Standa Industrie (Caen, France) and is
part of the probiotic preparation Propiofidus. This strain was
routinely cultured in yeast extract lactate (YEL) medium
(24) and stored long-term at 
80°C in the same medium
complemented with 10% glycerol. However, for this study, we developed
a chemically defined medium, MDP, to favor the incorporation of
radiolabeled amino acids. MDP medium contained (per liter of distilled
water) 12.8 g of sodium lactate, 0.6 g of
KH2PO4, 0.4 g of
potassium acetate, 50 mg of
MgSO4O · 7H2O, 5 mg of
MnSO4 · 4H2O, 2.5 mg of
FeSO4 · 7H2O, 2.5 mg of
CuSO4, 2.5 mg of NaCl, 0.25 mg of cobalt acetate,
15 µg of ZnSO4, 1 µg of
H3BO3, 1 µg of
Na2MoO4, 50 µg of
thiamine, 100 µg of pyridoxal, 50 µg of calcium pantothenate, 50 µg of riboflavin, 100 µg of nicotinamide, 10 µg of
p-aminobenzoic acid, 4 µg of biotin, 20 µg of folic
acid, and 2 µg of cyanocobalamin. Amino acids were supplied at the
following concentrations (per liter): 50 mg of
L-Ala, 160 mg of L-Arg, 150 mg of L-Asn, 250 mg of
L-Asp, 140 mg of L-Cys, 80 mg of Gly, 190 mg of L-Glu, 150 mg of
L-Gln, 100 mg of L-His, 180 mg of L-Ile, 300 mg of
L-Leu, 220 mg of L-Lys, 60 mg of DL-Met, 460 mg of
L-Pro, 170 mg of L-Phe, 180 mg of L-Ser, 150 mg of
L-Thr, 50 mg of L-Trp, 60 mg of L-Tyr, and 480 mg of
DL-Val. Five milligrams each of the bases
adenine, guanine, uracil, and xanthine were also added. The pH of the
medium was adjusted to 7.0 with NaOH prior to filter sterilization
(0.45-µm pore diameter; Millipore, Bedford, Mass.). Growth was
carried out at 30°C without shaking and monitored
spectrophotometrically at 650 nm as well as by CFU counting.
Acid adaptation and extreme acid challenge.
Log-phase cells
were obtained as follows. A starter culture (10 ml in MDP medium) was
diluted 1,000-fold in fresh MDP medium. During exponential growth, this
preculture was again diluted 1,000-fold in 100 ml of fresh medium. When
the culture reached a cell density of 5 × 108 cells per ml (optical density at 650 nm
[OD650] = 0.5), bacteria were harvested by
centrifugation (6,000 × g 30°C, 5 min). For acid
adaptation, cells were recovered in an equal volume of MDP medium
adjusted to pH 5.0 and incubated at 30°C for 60 min.
Adapted and nonadapted cells were harvested by centrifugation and
recovered in an equal volume of MDP medium at pH 2.0. Viable-cell counts were determined after 30 min of acid challenge. Samples were
diluted in peptone water (0.1% peptic digest of meat; Biokar Diagnostics, Beauvais, France), pH 7.0, containing 0.9% NaCl and poured into YEL medium containing 1.5% agar for maximal recovery. The
number of CFU was determined after 6 days of anaerobic incubation at
30°C.
Chloramphenicol treatment.
The MIC of chloramphenicol was 4 µg/ml, and chloramphenicol was used at a concentration of 40 µg/ml
where bacteriostasis occurred and where no lethal effect was observed
in P. freudenreichii. It was added to the culture 60 min
before adaptation and during adaptation at pH 5.0 and acid challenge at
pH 2.0.
Radioactive labeling and whole-cell protein extraction.
Log-phase cells grown in MDP medium were labeled essentially as
described by Flahaut et al. (6). Bacteria were harvested by centrifugation (6,000 × g, 30°C, 5 min) and
recovered in an equal volume of MDP medium devoid of cysteine and
methionine, at either pH 7.0 (control cells) or pH 5.0 (adapted cells).
A 1-ml subsample of bacterial suspension was labeled with 500 µCi of
[35S]methionine/cysteine protein labeling mix
(ICN Pharmaceuticals, Orsay, France) for 30 min at 30°C.
Incorporation was stopped by rapidly washing the bacteria in 1 ml of
stop solution (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM methionine,
1 mM cysteine, 1 mg of chloramphenicol per ml, 0.4 mM
phenylmethylsulfonyl fluoride [PMSF]). Washed cells were then
recovered in protoplastization solution (25 mM Tris-HCl [pH 7.5], 0.5 M sucrose, 5 mg of lysozyme per ml, 1 mg of chloramphenicol per ml, 0.4 mM PMSF) and incubated for 5 min at 37°C prior to centrifugation
(6,000 × g, 30°C, 5 min). The cell pellet was
recovered in 200 µl of lysis solution consisting of 50 mM Tris-HCl
[pH 7.5], 0.3% sodium dodecyl sulfate (SDS), 200 mM dithiothreitol
(DTT), and 0.4 mM PMSF and immediately sonicated on ice with a Vibra
Cell sonicator (Bioblock Scientific, Illkirch, France) equipped with a
tapered microtip (three bursts of 1 min at 1-min intervals, output of
2.5). The lysate was brought to 95°C for 10 min to improve protein
solubilization, and insoluble materials were removed by
centrifugation (10 000 × g, 10 min, room temperature).
Four volumes of ice-cold acetone was added, and proteins were
precipitated for 30 min on ice prior to centrifugation (12 000 × g, 10 min, 4°C) and then air dried.
Two-dimensional electrophoresis.
Preliminary experiments
performed with pH 3 to 10 gradients have shown that the majority of
P. freudenreichii polypeptides were focused in the pH 4 to 7 range. For this reason, pH 4 to 7 Dry Strips (Amersham Pharmacia
Biotech, Uppsala, Sweden) were used in this study. Air-dried protein
pellets were solubilized in sample solution containing 7 M urea, 2 M
thiourea, 25 mM DTT, 4%
3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS),
and 2% IPG-Buffer (Amersham Pharmacia Biotech). The surfactant CHAPS
and the chaotropic agent thiourea were used throughout the isoeletric
focusing to improve protein solubility and transfer to the second
dimension (33). Equal amounts of radioactivity (106 dpm) were loaded onto the gel in the first
dimension. Isoelectric focusing was carried out as described by
Görg et al. (11) with Immobiline Dry Strips on a
Multiphor II electrophoresis system (Amersham Pharmacia Biotech). The
strips were equilibrated in the presence of DTT and then iodoacetamide
before being placed on the top of the second-dimension gel.
SDS-polyacrylamide gel electrophoresis (PAGE) (12.5% polyacrylamide)
was performed according to the method of Laemmli (19) in
slab (16 by 16 by 0.1 cm) gels (Protean II; Bio-Rad, Hercules, Calif.).
For analysis of small proteins, the second dimension of some
two-dimensional gels was performed by discontinuous SDS-PAGE in
Tris-Tricine buffer according to the method of Schägger and von
Jagow (38). This electrophoretic system consisted of a
separating gel, a spacer gel, and a stacking gel at concentrations of
16, 10, and 4%, respectively. The radioactivity in the dried gels was
detected with a Storm Phosphorimager (Amersham Pharmacia Biotech).
Image analysis, gel matching, and quantification of the radioactivity
in individual spots were performed with the Melanie II software
(Bio-Rad). Molecular masses were calibrated by comigration of
low-molecular-mass markers (94.0, 67.0, 43.0, 30.0, 20.1, and
14.4 kDa) and peptide markers (16,949, 14,404, 10,700, 8,159, 6,214, and 2,512 Da) with bacterial proteins. Similarly, isoelectric points
were calibrated with the broad pI kit (isoelectric points 4.55, 5.20, 5.85, 6.55, and 6.85 were resolved on such gels). All markers were
obtained from Amersham Pharmacia Biotech. Relative rates of synthesis
were determined by calculating the ratio of radioactivity in a spot to
radioactivity in the entire gel. Results are the means of
determinations from at least three independent experiments, with a
standard deviation of less than 15%.
N-terminal amino acid sequence determination.
Cells from a
200-ml culture in MDP medium were lysed and proteins were extracted
with SDS as described above. Protein content in the extract was
determined according to the Lowry method (22) by using
bovine serum albumin as a standard. Aliquots of 200 mg of total
proteins were acetone precipitated and separated by two-dimensional electrophoresis. The resulting two-dimensional gels were transferred to
polyvinylidene difluoride (PVDF) membranes (Hyperbond; Beckman Instruments, Inc., Fullerton, Calif.) by immersed electroblotting in 10 mM 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) according to the
method of Matsudaira (26). Proteins were visualized by staining with Coomassie blue R-250 by the method of Pryor et al. (32). Spots were cut from the membrane and applied to an
automatic Beckman/Porton LF3000 protein sequencer (Beckman
Instruments) as described by the manufacturer. Sequence homologies were
searched with the FASTA program (31).
Electron microscopy.
P. freudenreichii cells were
laid by gentle filtration onto 0.2-µm-pore-diameter membranes
(Isopore membrane filters; Millipore). Membranes were fixed for 48 h with 2% (wt/vol) glutaraldehyde in 0.1 M sodium cacodylate buffer
[pH 6.8] and rinsed in the same buffer. Samples were dehydrated with
ethanol (10, 25, 50, 75, 95, and finally 100%), critical point dried
by the CO2 method, and coated with gold. Cells
were examined and photographed with a Philips XL 20 scanning electron
microscope operating at 10 kV.
| |
RESULTS |
ATR of P. freudenreichii SI 41 in MDP medium.
Propionibacteria are polyauxotrophic bacteria routinely cultivated
on complex medium supplemented with yeast extract, such as YEL medium.
For a better understanding of stress adaptation, we developed the MDP
defined medium and compared it with YEL medium. In this medium, the
SI41 strain displayed growth characteristics slightly different from
those observed in the complex one. The generation time was 6 h,
and the final OD was 2, corresponding to a cell density of
5.108 CFU/ml, while the generation time was
5 h, and the final OD was 2.5, corresponding to
109 CFU/ml in YEL medium. Therefore, MDP medium
fulfilled the nutrient requirements of P. freudenreichii and
was used in these experiments.
ATR was then explored for the probiotic SI41 strain grown in this
medium. Cells were challenged during exponential growth
in MDP medium.
(The pH of such cultures was typically between
6.5 and 7.0.) They
underwent severe mortality when shifted to
pH 2.0 (Fig.
1). Indeed, only 4.3% of viable
propionibacteria
were detected after 30 min of this challenge. In
contrast, when
identical cultures were exposed to a sublethal pH of 5.0 for 60
min prior to challenge, they survived at pH 2.0 without
significant
loss of viability. Furthermore, the same protective effect
of
acid pretreatment was observed when SI41 was grown in rich YEL
medium (data not shown). This is clearly evidence of an ATR for
P. freudenreichii SI 41 grown in both media.
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FIG. 1.
ATR in P. freudenreichii SI41 and effect
of protein synthesis inhibition. Log-phase cells were harvested and
submitted to acid adaptation at pH 5 with (B) or without (A)
chloramphenicol treatment. Viability was then determined after 30 min
of a subsequent extreme acid challenge at pH 2. As a control,
nonadapted cells were subjected to the same challenge (C). Results are
expressed as the percentage of viable cells at the end of the
challenge. Error bars represent the standard deviation for triplicate
experiments.
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To determine the requirement for neosynthesis of specific protective
proteins in
P. freudenreichii SI 41 ATR, cells were treated
with the protein synthesis inhibitor chloramphenicol before and
during
adaptation at pH 5.0. In this case, a notable mortality
was observed
during subsequent acid challenge at pH 2.0. Indeed,
43% of the adapted
cells survived the extreme acid challenge,
while 100% survived after
adaptation in the absence of antibiotic.
However, these cells displayed
a reduced tolerance toward acid
stress, and survival was still higher
than that of nonadapted
bacteria. Thus, inhibition of protein
neosynthesis prevented ATR,
at least partly, suggesting a role of
acid-induced stress proteins
in acid
adaptation.
Morphological changes of P. freudenreichii upon
exposure to acid stress.
Scanning electron micrographs revealed
the characteristic morphology of dairy propionibacteria for cells grown
at pH 7.0: pleomorphic rods arranged in "Chinese characters" were
observed, and the average length of individual cells (calculated from
at least 100 measurements for each observation) was 1.22 µm (Fig. 2A). In contrast, cells incubated at pH
2.0 showed dramatic changes in morphology. The rod shape was lost, and
bacteria showed a segmented aspect in which individual segments were
shorter (0.48 µm on average). In addition, bleb-like structures on
the surface of a significant number of bacterial cells were observed
(Fig. 2B). Less morphological disturbances were observed upon exposure
to pH 5.0. The rod shape was generally preserved, and deformations of
the cell surface were almost absent (Fig. 2C). In addition, an
elongation of certain cells was observed, and the average size of
bacteria was then 1.57 µm. Furthermore, when challenged at pH 2.0, these bacteria adapted at pH 5.0 did not present modifications
comparable to those undergone by nonadapted bacteria (Fig. 2D). This
illustrates, at the morphological level, the protective effect of ATR.
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FIG. 2.
Analysis of morphological changes of P.
freudenreichii SI41 during acid stress. Nontreated cells (A),
cells undergoing extreme acid challenge at pH 2 (B), and pH 5 acid-adapted cells (C) were analyzed by scanning electron microscopy.
For comparison, cells subjected to acid adaptation (pH 5) and then to
extreme acid challenge (pH 2) were also analyzed (D).
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Changes in protein synthesis during ATR.
The requirements of
ATR-specific polypeptides for maximal protection having been shown, we
studied the rate of protein synthesis during acid adaptation. After
pulse-labeling, we analyzed whole-cell protein extracts of the SI41
strain by two-dimensional electrophoresis. Electrophoregrams of control
cells growing at pH 7.0 and cells undergoing acid adaptation were
compared (Fig. 3).
Pulse-labeling was performed during four successive 15-min periods
during 60 min of acid adaptation.
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FIG. 3.
Two-dimensional analysis of protein expression during
acid adaptation in P. freudenreichii SI41. Protein
synthesis was monitored during 60 min of pH 5 adaptation. Cells were
pulse-labeled between 0 and 15 min (B), 15 and 30 min (C), 30 and 45 min (D), or 45 and 60 min (E) of adaptation. As a control, nontreated
cells were labeled at pH 7 for 15 min (A). Whole-cell protein extracts
were analyzed by two-dimensional electrophoresis followed by
autoradiography. The arrows indicate polypeptides displaying an
increased relative rate of synthesis during acid adaptation compared
with that of nonadapted cells. Two polypeptides displaying,
respectively, an unchanged ( ) and a reduced ( ) relative rate of
synthesis are also shown.
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On autoradiograms of control gels, the synthesis of 433 discrete
cellular proteins was detected, in a molecular mass range
of 14 to 130 kDa and a pI range of 4 to 7 (Fig.
3A). During the
first 15 min of acid
adaptation, 17 protein synthesis inductions
were detected (out of the
46 induced polypeptides observed on
these gels). This is the case with
the acid stress proteins (ASPs)
7, 14, 34, and 45 (Fig.
3B). Among
these early inductions, some
were maintained throughout the experiment
(see ASP 45 in Fig.
3 and ASP 47 in Fig.
4 and 5), while others (ASPs 14 and 22)
returned
to their initial level (Fig.
3C,
3D and
5). Certain stress
proteins,
such as ASP 8, were induced only at the end of the adaptation
(Fig.
3D and
5), while others were only transiently overexpressed
(ASPs
11, 35, and 39). The most important changes concerned the
ASPs 1, 2, and 25, which showed maximal induction factors of 4.8,
3.9, and 3.8, respectively. The induction factors of the other
acid-induced
polypeptides were weaker: between 1.5 and 3.5. Finally,
keeping the
bacteria at pH 5.0 for up to 60 min led to a shutdown
of synthesis of
the majority of the polypeptides expressed in
growing cells. Indeed,
only 303 polypeptides were labeled between
45 and 60 min of treatment,
and most of them were almost undetectable
on the autoradiograms (Fig.
3E). In contrast, some ASPs were still
expressed with a consequently
high relative rate of synthesis
(ASPs 5, 8, and 15).
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FIG. 4.
Two-dimensional analysis of expression of small
polypeptides during acid adaptation in P. freudenreichii
SI41. Protein extracts were prepared as described in the legend to Fig.
3, and two-dimensional electrophoresis was performed with discontinuous
Tris-Tricine gels in the second dimension. The arrows indicate
polypeptides displaying an increased rate of synthesis during acid
adaptation (B) compared with that of nonadapted cells (A).
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Stress-induced polypeptides with molecular masses below 14 kDa have
been described previously. We thus looked for acid-induced
proteins by
using discontinuous SDS-PAGE as a second dimension.
As seen in Fig.
4,
these gels were able to efficiently separate
polypeptides in the
2.5- to 14.4-kDa range. Five additional ASPs
were detected on these
gels (ASPs 47 to 51). ASPs 50 and 51, in
particular, are not detected
on control gels and correspond to
neosynthesized proteins in response
to moderate acid
stress.
The induction factors varied in a time-dependent manner, as shown in
Fig.
5. This provides further evidence of
the early,
transient, late, or permanent nature of acid-induced
proteins.
As shown on the electrophoregrams, most of the cellular
proteins
were repressed, while some remained unchanged (two examples
are
illustrated in Fig.
3 and
5).
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FIG. 5.
Variations in induction factors during acid adaptation.
For each acid-induced polypeptide, the induction factor (defined as the
ratio between relative rates of synthesis in adapted and control cells)
was determined at 15, 30, 45, and 60 min of adaptation. Results are
shown for ASPs 5 ( ), 8 ( ), 11 ( ), and 47 ( ) as well as for
the unchanged () and repressed () polypeptides indicated in Fig.
3.
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Analysis of ASPs.
From the observed ASPs, five belonged to the
different classes of induction rate and were obviously detectable on
Coomassie blue-stained gels. They were then subjected to N-terminal
sequencing. Databases were screened and revealed homologies to known
proteins. Results of five identifications are shown in Table
1.
ASP 8 was shown to be homologous to the protein chaperone GroEL from
Bacillus subtilis (
42), but also displayed
homologies
to 60-kDa chaperonins from various bacterial species.
Similarly,
ASP 49 showed homologies with the GroES chaperonin from
Mycobacterium bovis (
46) and with a series of
10-kDa chaperonins of both bacterial
and eucaryotic origins. Moreover,
ASPs 8 and 49 displayed molecular
masses (67 and 11 kDa, respectively)
and isolelectric points (4.5
and 4.6, respectively) in agreement with
those reported for the
GroE system of
B. subtilis
(
39) and
E. coli (
43).
ASP5 revealed homologies to the RecR protein involved in DNA repair and
genetic recombination in
B. subtilis (
1), and
ASP
11 was homologous to an
L. lactis plasmid-encoded
replication
protein, RepB (
40).
ASP 47 showed 100% identity with biotin carboxyl carrier protein
(BCCP), a biotin containing a 1.3S carboxyl carrier subunit
contained
in
P. freudenreichii transcarboxylase (
28).
| |
DISCUSSION |
In P. freudenreichii, we demonstrated a very efficient
ATR, in both rich and chemically defined media. The ATR affords
protection toward a pH as low as 2.0 without mortality, in
contrast to nonadapted cells. To better understand the mechanisms
underlying this phenomenon, in this work, we evaluated changes in
morphology and protein synthesis during development of ATR.
P. freudenreichii cells subjected to extreme acid
environments were shown to undergo dramatic changes in morphology,
while viability decreased. In contrast, cell integrity was preserved in
adapted bacteria, while viability was not affected. This indicates that
bacteria preexposed to a sublethal environment can adapt and retain a
normal shape during the challenge. Stress-induced morphological changes
in nonsporulating bacteria were previously described. Shrinkage of
E. coli cells, resulting from the induction of the
bolA morphogene belonging to the rpoS
regulon, is triggered by entry into stationary phase and a variety of
stresses, including low pH (37). Similarly, modifications
of the cell morphology in the gram-positive organism E. faecalis are caused by stress-induced modifications of the
transcription of many genes (9).
ATR has been reported to be linked to a concomitant modification of
protein synthesis. However, inhibition of ASP synthesis did not abolish
ATR in all the studies. Protein neo-synthesis inhibition by
chloramphenicol reversed ATR in L. monocytogenes (4), in A. hydrophila (18), and in
the enterobacteria S. enterica serovar Typhimurium and
E. coli (7, 36). In contrast, it showed no
effect on acid adaptation in L. lactis subsp.
lactis (13) and in E. faecalis
(5). In L. acidophilus, blocking of
protein synthesis reduced ATR during adaptation at pH 5, but had no
effect on ATR developed at pH 4.2 (21). In
P. freudenreichii, we showed that chloramphenicol
reduced ATR, suggesting an important role of ASPs. However,
this reduction was not complete, suggesting that both an inducible
preexisting system and a mechanism dependent on de novo protein
synthesis coexist in this Propionibacterium and that they
together afford maximal protection against extreme acid stress, as
suggested for L. acidophilus (21).
Analysis of ASPs revealed that general stress response proteins,
indicative of damage to macromolecules, were induced during ATR in
P. freudenreichii. Indeed, ASPs 8 and 49 showed homologies with the highly conserved chaperonins GroEL and GroES. These heat shock
proteins are also induced by acid stress in E. coli
(16), L. lactis (13), and
Lactobacillus delbrueckii subsp. bulgaricus (20). The similar levels and kinetics of induction for
these chaperonins are consistent with the previous report that they belong to the same operon, under the control of a regulatory agent, CIRCE (controlling inverted repeat for chaperone expression), in
B. subtilis (14). ASPs 5 and 11 showed
homologies with proteins involved in DNA synthesis or repair
(RecR and RepB). Such proteins, which belong to the SOS regulon, have
been shown to be induced by mutagenic agents, starvation, and entry
into stationary phase (44). Indeed, most stresses that
cause growth arrest may be responsible for oxidative stress, at least
in E. coli (29).
ASP 47 was unambiguously identified as P. freudenreichii
BCCP (28). This 12-kDa polypeptide is part of a multimeric
(30 subunits) transcarboxylase responsible for decarboxylation of methylmalonyl-coenzyme A and carboxylation of pyruvate, a metabolic reaction specific to propionibacteria (15). It is
noteworthy that this carboxyl carrier protein is labile under acidic
conditions (28). Glutamate, lysine, and arginine
decarboxylases are key actors of the pH homeostasis in enterobacteria
(2). Because glutamate is by far the most abundant
intracellular free amino acid in P. freudenreichii
(35) and BCCP is a very abundant polypeptide, such a
crucial role of a biotin-containing carboxyl carrier protein in the
P. freudenreichii adaptative response to acidic environments must be investigated.
In conclusion, a chemically defined medium was developed and the
two-dimensional electrophoresis method was adapted to monitor protein
synthesis in P. freudenreichii. This allowed a kinetic study
of both general and specific stress proteins during acid adaptation in
this bacterium to be carried out. The present work constitutes a first
insight into the mechanisms leading to efficient acid tolerance in
dairy propionibacteria. Moreover, it will allow a proteomic study of
other stresses, which should lead to the characterization of stress
regulons in P. freudenreichii.
| |
ACKNOWLEDGMENTS |
We thank A. Rouault for expert technical assistance, M. Gautier
for interest in this work, V. Rioux for help with protein blotting, and
Y. Auffray and J. C. Giard for critical reading of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Recherches de Technologie Laitière, Institut National de la
Recherche Agronomique, 65 rue de St. Brieuc, 35042 Rennes
Cedex, France. Phone: (33) 2 23 48 57 41. Fax: (33) 2 23 48 53 50. E-mail: gjan{at}labtechno.roazhon.inra.fr.
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Applied and Environmental Microbiology, May 2001, p. 2029-2036, Vol. 67, No. 5
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.5.2029-2036.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
