FEMS Microbiology Letters 160 (1998) 43^47
Increase of sul¢te tolerance in Oenococcus oeni by means
of acidic adaptation
Jean Guzzo *, Michel-Philippe Jobin, Charles Divieés
Laboratoire de Microbiologie, ENS.BANA, Universiteè de Bourgogne, 1 Esplanade Erasme, F-21000 Dijon, France
Received 14 December 1997 ; accepted 23 December 1997
Abstract
Sulfite is an antimicrobial agent used at the beginning of winemaking to avoid development of undesirable microorganisms.
However, Oenococcus oeni, which is mainly responsible for the malolactic fermentation, has to grow in wine and therefore has
to be resistant to sulfite. This study showed that acid-adapted cells of O. oeni survived better than non-adapted cells in the
presence of a high sulfite concentration (30 mg l31 ). Addition of a sub-lethal concentration of sulfite (15 mg l31 ) during the
adaptation step in acidic medium increases the sulfite tolerance. Moreover, sulfite appeared to be able to induce a heat shocklike response. Our results suggest that pH homeostasis mechanisms and stress protein synthesis could be involved in the
induction of sulfite tolerance in O. oeni. z 1998 Federation of European Microbiological Societies. Published by Elsevier
Science B.V.
Keywords : Oenococcus oeni; Malolactic fermentation; Sul¢te tolerance ; Heat shock protein
1. Introduction
Oenococcus oeni is a lactic acid bacterium (LAB)
known to carry out the malolactic fermentation
(MLF) in wine [1,2]. MLF corresponds to the decarboxylation of L-malate to L-lactate and CO2 . The
consequences of this reaction are the deacidi¢cation
of wine and the increase in microbial stability of the
product. The growth of O. oeni in wine requires its
capacity to resist acidity, high ethanol and sul¢te
concentrations. Recently, O. oeni was chosen as a
model to study the stress response in LAB. The major heat shock proteins (Hsp) have been identi¢ed [3]
and a gene hsp18 encoding an 18-kDa small Hsp
* Corresponding author. Tel.: +33 3 80 39 66 73;
Fax: +33 3 80 39 66 40; E-mail: [email protected]
(smHsp) called Lo18 has been characterised [4]. To
avoid the growth of undesirable microorganisms, it
is a common practice to add sul¢te to grape must at
the beginning of the vini¢cation process (50^100 mg
l31 ). Sul¢te is recognised as a powerful antimicrobial
agent especially at low pH like that of wine, where
sul¢te predominates as free SO2 , the more active
form of sul¢te [5]. The cell death caused by sul¢te
could be a consequence of ATP depletion in Saccharomyces cerevisiae [6,7]. In vitro experiments have
shown that sul¢te reacts with proteins, nucleic acids
and with some cofactors [5].
Nevertheless, MLF occurs in wine even in the
presence of added SO2 . Consequently, LAB and
more particularly O. oeni appear to be able to develop a tolerance to sul¢te. How bacteria resist SO2 is
still unknown, but it appears that the pH of the
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 0 0 7 - X
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J. Guzzo et al. / FEMS Microbiology Letters 160 (1998) 43^47
medium has a great in£uence on the capacity of cells
to develop resistance to sul¢te. Thus, at pH 4, the
resistance mechanisms take longer to develop than at
pH 3.5 [8].
This study aims to investigate the enhancement of
sul¢te tolerance in O. oeni by prior growth at acidic
pH in the presence or absence of a sub-lethal concentration of sul¢te. The e¡ect of sul¢te on O. oeni
protein synthesis, especially heat shock proteins, will
be considered.
2. Materials and methods
2.1. Strain, medium and reagents
O. oeni Lo84.13 was grown at 30³C in FT80 medium [9]. Concentrated sul¢te solution (1 g l31 ) was
prepared by weighing 0.148 g of Na2 S2 O5 in 100 ml
of distilled, boiled and cooled water. The acidic form
of SO2 is in equilibrium with the alkaline form
HSO3
3 with a pKa of 1.78. Sul¢te concentrations of
15, 30 and 60 mg l31 were added in the medium at
pH 3.5 or 5.3. The lower pH value was chosen to
increase the quantities of free SO2 .
2.2. Evaluation of survival
Bacteria were grown in FT80 medium at pH 5.3,
30³C and re-inoculated in survival medium (FT80
medium at pH 3.5 with 0, 15, 30 or 60 mg l31 of
SO2 ) at an initial cell density of 107 colony-forming
units (CFU) ml31 . Vials were incubated at 30³C for
6 days and periodically analysed for survival. Viability was measured as previously described [10]. For
the experiments on adaptation to sul¢te, bacteria
were grown in the absence or presence of sul¢te
(15 mg l31 ) in FT80 pH 3.5 and re-incubated in
the same medium with 30 mg l31 of sul¢te.
formed as described by Jobin et al. [4] using an internal fragment of the hsp18 gene as a probe.
A fraction of the same cell pellet was solubilised in
Laemmli sample bu¡er [11] and mechanically disrupted as previously described [10]. Equal amounts
of proteins (10 Wg) were loaded on a 15% SDS-polyacrylamide gel (SDS-PAGE). The proteins were
transferred to nitrocellulose (Schleicher and Schuell,
0.2 Wm) for 30 min. Immunodetection was performed
as previously described [12] but slight modi¢cations
were carried out. For the revelation step, the ECL
kit (Amersham) was used to enhance the detection
sensitivity.
3. Results
3.1. Sul¢te tolerance
Cells grown in FT80 medium at pH 5.3 were used
as inoculum to study the survival in FT80 medium at
pH 3.5 in the presence of 0, 15, 30 or 60 mg l31 of
sul¢te (Fig. 1). Cell viability was periodically analysed as described in Section 2. As shown in Fig. 1,
cell survival was markedly decreased after 24 h of
incubation in the presence of sul¢te. Total death
was observed with 60 mg l31 of sul¢te. Within 24 h,
almost all the cell died and no CFU was counted
after this period of time. With 30 mg l31 of sul¢te,
a rapid decrease in cell population was observed
2.3. Northern and Western blot analyses
Cells were inoculated in FT80 medium adjusted to
pH 5.3 and grown at 30³C up to an OD600nm of 0.6.
After incubation with various concentrations of sul¢te (15, 30 or 60 mg l31 ) for 30 min, cells were
harvested by centrifugation. The isolation of total
RNA and the Northern blot experiments were per-
Fig. 1. Survival of O. oeni cells in the absence (b) or in the presence of various concentrations of sul¢te: 15 mg l31 (F); 30 mg
l31 (R) ; 60 mg l31 (8). The data presented are mean values
from two separate experiments.
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J. Guzzo et al. / FEMS Microbiology Letters 160 (1998) 43^47
45
Fig. 3. Northern blot analysis of hsp18 mRNA induction. Total
RNA was prepared either from cells incubated for 30 min in the
presence of various concentrations of sul¢te, from heat-shocked
cells at 42³C (HS) or from unshocked cells (C).
Fig. 2. E¡ect of di¡erent adaptation conditions on sul¢te tolerance. Cells were adapted at pH 3.5 in the absence (F) or presence (R) of 15 mg l31 sul¢te and re-inoculated in a medium containing 30 mg l31 of sul¢te at pH 3.5. As a control, cells were
grown at pH 5.3 and re-inoculated at pH 3.5 in a medium without sul¢te (b) or containing 30 mg l31 of sul¢te (8). The data
presented are mean values from two separate experiments.
after 24 h. However, viable cells were present even
after 144 h. As reported by Del¢ni et al. [8], in the
presence of inhibiting concentrations of sul¢te, the
resistant cells can remain latent without necessarily
losing their reproductive capacity. In the presence of
15 mg l31 of sul¢te, most cells die within 3 h. However, certain cells then appeared to develop tolerance
and were able to start regrowth after 24 h. This
growth in the presence of sul¢te suggested an induction of sul¢te tolerance. To verify this hypothesis,
the sul¢te tolerance of cells subjected to acidic conditions (pH 3.5) was examined. Growth in an acidic
medium (pH 3.5) without sul¢te led to a signi¢cant
degree of protection to a challenge medium containing 30 mg l31 of sul¢te (Fig. 2). For the acid-adapted
cells, the number of viable counts after 24 h of incubation was 2 log higher when compared to the
non-adapted cells. It is worth noting that in this
case, a signi¢cant bacterial growth was observed
over the remainder of the incubation. This result
indicates that sul¢te tolerance can be developed in
response to acid treatment. Furthermore, the sul¢te
tolerance of O. oeni was enhanced by pre-incubating
the cells at pH 3.5 in the presence of a sub-lethal
concentration of sul¢te (15 mg l31 ) (Fig. 2). In
this case, a decrease of less than 1 log in the cell
population was immediately followed by bacterial
growth extending to about four generations, over
6 days.
3.2. E¡ect of sul¢te on hsp18 expression
Because Lo18 synthesis is faintly induced by acid
shock at pH 3.5 (data not shown), it was di¤cult to
distinguish between the e¡ects of acidity and sul¢te
on Hsp synthesis in O. oeni. To circumvent this
problem, we studied the e¡ect of sul¢te at a pH
value of 5.3, which does not induce a stress response
[3]. The in£uence of increasing concentrations of sul¢te on the extent to which the lag phase persisted
before bacterial growth at pH 5.3 was investigated.
O. oeni cells were always sensitive to the inhibitory
e¡ect of sul¢te. With 15 mg l31 of sul¢te, the growth
started after a delay of 6 h when compared to the
corresponding control. Longer lag phases of 23 and
28 h were observed with 30 and 60 mg l31 of sul¢te,
Fig. 4. Western blot analysis of Lo18. Total proteins were extracted either from cells incubated for 30 min without sul¢te (C)
or with various concentrations of sul¢te, or from heat-shocked
cells (42³C; HS). Proteins were separated by 15% SDS-PAGE
and immunodetection was carried out using an antiserum raised
against Lo18 smHsp from O. oeni.
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J. Guzzo et al. / FEMS Microbiology Letters 160 (1998) 43^47
respectively. Thus, a clear growth inhibition by sul¢te appears, even at pH 5.3. Under these experimental conditions, we investigated the expression of
hsp18 after sul¢te shock at various concentrations
(15, 30 and 60 mg l31 ). After 30 min of incubation
with sul¢te, total RNA was isolated from O. oeni
cells and subjected to Northern blot analysis (see
Section 2). The hsp18 transcript was detected only
in cells incubated with 60 mg l31 of sul¢te (Fig. 3).
No detectable signal was observed with lower concentrations of sul¢te. These results were con¢rmed
by Western blot experiments using an antiserum
raised against Lo18. This smHsp was detected in
the presence of the highest concentration of sul¢te
(60 mg l31 ; Fig. 4) but only faintly detected for the
other lower concentrations.
4. Discussion
In this study, we attempted to characterise the
sul¢te shock response of O. oeni. One of the objectives of this study was to demonstrate an adaptation
phenomenon to sul¢te and to elucidate the cellular
mechanisms involved in this tolerance. Under our
culture conditions (30³C, pH 3.5), exposure of O.
oeni to 30 mg l31 of sul¢te dramatically decreased
the viability. The remaining viable cells did not start
to grow even after 6 days. Since increased cell survival and growth were observed in the presence of 30
mg l31 of sul¢te only after growth of the cells in an
acidic medium (pH 3.5), one must conclude that the
growth in such acidic conditions induces molecular
and/or physiological changes in cells allowing sul¢te
tolerance. To explain this result, we propose that
sul¢te tolerance of adapted cells at pH 3.5 could be
due to a maintenance of intracellular pH. It has been
reported that O. oeni is able to maintain a rather
constant intracellular pH (5.8^6.3) in the pH range
3.0^5.5 [13]. This pH homeostasis phenomenon
could induce a decrease of the free SO2 concentration in the cell and consequently attenuate the lethal
e¡ect. In fact, the free SO2 entering the bacterial cell
by di¡usion would be converted into HSO3
3 , which is
less toxic. In wine, the acidic pH appears favourable
for the development of sul¢te tolerance.
Moreover, our results demonstrated that the combined action of a low sul¢te concentration and acidic
pH enhanced tolerance to a highly inhibitory concentration of sul¢te suggesting the involvement of
several adaptation mechanisms.
In O. oeni, the smHsp Lo18 is induced by multiple
stresses and during stationary growth phase [3].
Moreover, the hsp18 gene encoding Lo18 is expressed only under stress conditions and the regulation occurs at the transcriptional level [4]. Thus, we
think that this gene can be used as a general stress
marker in O. oeni. Northern and Western blot experiments showed a weak induction of hsp18 expression in the presence of a high concentration of sul¢te
(60 mg l31 ). These results suggest that sul¢te is able
to induce a heat shock-like response. Our work
shows also that sul¢te tolerance can be induced in
response to acidic conditions that require a maintenance of intracellular pH. Thus, we propose that
sul¢te tolerance in wine could involve cellular pH
homeostasis mechanisms and stress protein synthesis.
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