Increase of sul te tolerance in Oenococcus oeni by means of acidic adaptation

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 l 31 ). Addition of a sub-lethal concentration of sulfite (15 mg l 31 ) 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 CO 2. 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: jguzzo@u-bourgogne.fr (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 l 31 ). Sul te is recognised as a powerful antimicrobial agent especially at low ph like that of wine, where sul te predominates as free SO 2, 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 SO 2. Consequently, LAB and more particularly O. oeni appear to be able to develop a tolerance to sul te. How bacteria resist SO 2 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 S0378-1097(98)00007-X

44 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 l 31 ) was prepared by weighing 0.148 g of Na 2 S 2 O 5 in 100 ml of distilled, boiled and cooled water. The acidic form of SO 2 is in equilibrium with the alkaline form HSO 3 3 with a pk a of 1.78. Sul te concentrations of 15, 30 and 60 mg l 31 were added in the medium at ph 3.5 or 5.3. The lower ph value was chosen to increase the quantities of free SO 2. 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 l 31 of SO 2 ) at an initial cell density of 10 7 colony-forming units (CFU) ml 31. 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 l 31 ) in FT80 ph 3.5 and re-incubated in the same medium with 30 mg l 31 of sul te. Cells were inoculated in FT80 medium adjusted to ph 5.3 and grown at 30³C up to an OD 600nm of 0.6. After incubation with various concentrations of sul- te (15, 30 or 60 mg l 31 ) for 30 min, cells were harvested by centrifugation. The isolation of total RNA and the Northern blot experiments were performed 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 l 31 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 l 31 of sul te. Within 24 h, almost all the cell died and no CFU was counted after this period of time. With 30 mg l 31 of sul te, a rapid decrease in cell population was observed 2.3. Northern and Western blot analyses Fig. 1. Survival of O. oeni cells in the absence (b) or in the presence of various concentrations of sul te: 15 mg l 31 (F); 30 mg l 31 (R); 60 mg l 31 (8). The data presented are mean values from two separate experiments.

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) of15mgl 31 sul te and re-inoculated in a medium containing 30 mg l 31 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 l 31 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 l 31 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 l 31 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 l 31 ) (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 l 31 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 l 31 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.

46 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 l 31 ). 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 l 31 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 l 31 ; 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 l 31 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 l 31 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 SO 2 concentration in the cell and consequently attenuate the lethal e ect. In fact, the free SO 2 entering the bacterial cell by di usion would be converted into HSO 3 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 l 31 ). 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. References [1] Kunkee, R.E. (1991) Some roles of malic acid in the malolactic fermentation in wine making. FEMS Microbiol. Rev. 88, 55^72. [2] Davis, C.R., Wibowo, D.J., Lee, T.H. and Fleet, G.H. (1986) Growth of lactic acid bacteria during and after malolactic fermentation of wines at di erent ph. Appl. Environ. Microbiol. 51, 539^545. [3] Guzzo, J., Delmas, F., Pierre, F., Jobin, M.-P., Samyn, B., Van Beeumen, J., Cavin, J.-F. and Divieés, C. (1997) A small heat shock protein from Leuconostoc oenos induced by multiple stresses and during stationary growth phase. Lett. Appl. Microbiol. 24, 393^396. [4] Jobin, M.-P., Delmas, F., Garmyn, D., Divieés, C. and Guzzo, J. (1997) Molecular characterization of the gene encoding an 18-kDa small heat shock protein associated with the membrane of Leuconostoc oenos. Appl. Environ. Microbiol. 63, 609^614. [5] Schimz, K.L. (1980) The e ect of sulphite on the yeast Saccharomyces cerevisiae. Arch. Microbiol. 125, 89^95. [6] Hinze, H. and Holzer, H. (1986) Analysis of energy metabolism after incubation of Saccharomyces cerevisiae with sulphite or nitrite. Arch. Microbiol. 145, 27^31. [7] Schimz, K.L. and Holzer, H. (1979) Rapid decrease of ATP content in intact cells of Saccharomyces cerevisiae after incubation with low concentrations of sulphite. Arch. Microbiol. 145, 225^229. [8] Del ni, C. and Morsiani, M.G. (1992) Resistance to sulphur dioxide of malolactic strains of Leuconostoc oenos and Lactobacillus sp. isolated from wines. Sci. Aliments 12, 493^511. [9] Cavin, J.F., Prevost, H., Lin, J., Schmitt, P. and Divieés, C.

J. Guzzo et al. / FEMS Microbiology Letters 160 (1998) 43^47 47 (1989) Medium for screening Leuconostoc oenos strains defective in malolactic fermentation. Appl. Environ. Microbiol. 55, 751^753. [10] Guzzo, J., Cavin, J.F. and Divieés, C. (1994) Induction of stress proteins in Leuconostoc oenos to perform direct inoculation of wine. Biotechnol. Lett. 16, 1189^1194. [11] Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680^ 685. [12] Guzzo, J., Murgier, M., Filloux, A. and Lazdunski, A. (1990) Cloning of the Pseudomonas aeruginosa alkaline protease gene and secretion of the protease into the medium by Escherichia coli. J. Bacteriol. 172, 942^948. [13] Salema, M., Poolman, B., Lolkema, J.S., Loureiro Dias, M.C. and Konings, W.N. (1994) Uniport of monoanionic L-malate in membrane vesicles from Leuconostoc oenos. FEBS Lett. 124, 1^7.