Adaptive evolution to improve acid tolerance in Oenococcus oeni

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Adaptive evolution to improve acid tolerance in Oenococcus oeni

Frédérique Julliat, N. Tourti, Stéphane Guyot, Herve Alexandre, Cosette Grandvalet

To cite this version:

Frédérique Julliat, N. Tourti, Stéphane Guyot, Herve Alexandre, Cosette Grandvalet. Adaptive evo- lution to improve acid tolerance in Oenococcus oeni. 11th international synposium of oenology of Bordeaux, Jun 2019, Bordeaux, France. �hal-03150729�

-1 -0,9 -0,8 -0,7 -0,6 -0,5 -0,4 -0,3 -0,2 -0,1 0

Log(N/No)

Parental B2 C2 D2

Adaptive evolution to improve acid tolerance in

Oenococcus oeni

1 F. Julliat,, ¹ N. Tourti ^1, S. Guyot, ¹ H. Alexandre, ¹ C. Grandvalet

1

Univ. Bourgogne Franche-Comté, AgroSup Dijon, PAM UMR A 02.102, F-21000 Dijon, France

Strategy involved

Parental versus evolved strains to extreme acidity challenge Background

Conclusions and perspectives

1. Intracellular pH in growth conditions or during acid stress at pH 1.9

Oenococcus oeni

Saccharomyces cerevisiae

Alcoholic fermentation

Malolactic fermentation

Winemaking

Oenococcus oeni is a lactic acid bacterium (LAB) mainly responsible for the malolactic fermentation (MLF) in wine. MLF plays an important role in determining the final quality of wines ^[1] . Even though this LAB is naturally present in musts, wines and oenological environment, spontaneous MLF are usually unpredictable because of the stressful conditions and especially due to acidity ^[2] . The consequence of the mismanagement of this step might lead to the depreciation of wine quality.

To obtain a clone more tolerant to acidity, we undertook a replication of O. oeni until 450 generations in a temporally varying environment (pH 5.3 to 2.9) to improve acid tolerance. To discriminate stress tolerance of evolved populations versus parental strain an acid stress was performed to both population.

2. Plasma membrane permeability after acid stress at pH 1.9

3. Lost of cultivability of evolved strains after acid stress at pH 1.9

Oenococcus oeni

ATCC BAA-1163

6 Independent clones

Propagation until they reached the stationary phase

PARENTAL STRAIN

x6

FT80m ^[3]

pH 5.3

PARENTAL STRAIN

450

generations

From pH 5.3 to 2.9

3 EVOLVED

STRAINS (E2, E3,E4)

FT80m x6

pH 4.5 FT80m

pH 5.3

FT80m pH 4

FT80m pH 3.7

FT80m pH 3.5

FT80m pH 3.2

vs

Physiological properties :

• Membrane permeability

• Intracellular pH

• Survival tests

FT80m pH 3

FT80m pH 2.9

Cultivability following stress treatment. Parental strain or evolved strains were gown respectively at pH 5.3 or 2.9 until mid-exponential growth phase. Cultures were transferred into acidified FT80m (pH 1.9). Cultivability was estimated by plating on agar medium after 60min treatment.

0 1 2 3 4 5 6 7

T0 T30

In tr acel ular pH

Time (minute) Parental E2 E3 E4

0 10 20 30 40 50 60 70 80 90 100

T30 T90

Per cen tag e of PI pos iti ve cell s

Time (minute)

Parental E2 E3 E4

Intracellular pH. Parental strain or evolved strains were gown respectively at pH 5.3 or 2.9 until mid-exponential growth phase. Intracellular pH was measured using CFDA-SE as probe. A first measurement has been made and then cultures were transferred into acidified FT80m (pH 1.9) and another measurement has been made after 90 min.

Percentages of permeable parental and evolved strains. Parental strain or evolved strains were gown respectively at pH 5.3 or 2.9 until mid-exponential growth phase. Cultures were transferred into acidified FT80m (pH 1.9). Plasma membrane permeability was performed using propidium iodide as a probe at 30min or 90min after acid stress at pH 1,9.

Cultivability following stress treatment. Parental strain and evolved strains were gown respectively at pH 5.3 until mid-exponential growth phase. Cultures were transferred into acidified FT80m (pH 1.9). Cultivability was estimated by plating on agar medium after 60 min treatment.

➔ Evolved strains maintain their intracellular pH even when they grow at pH 2.9

➔ Either parental nor evolved strain are able to regulate their intracellular pH during an acid stress at pH 1.9

➔ Compared to parental strain, evolved strains hold their membrane integrity for at least 30min during an acid stress

➔This state seems to be temporary : all strains become permeable after 90 min

➔ Evolved strains resist better than parental strain at an acid stress at pH 1.9

➔ This resistance may be due to adaptation or acclimatization of cells

➔ Return to intial pH 5.3 disrupt avantage of evolved strains

➔ The resistance of evolved strains at pH of 1.9 does not seem to be due to adaptation but rather to a transitional acclimatization of cells

3. Lost of cultivability of evolved strains after a recovery to initial pH 5.3

a

b b b

a a

b b

• Evolved strains maintain the same intracellular pH in acidic conditions (pH 2.9) than the parental strain in optimal conditions (pH 5.3)

• Tolerance to acidity of evolved strains is a transitional state which could optimize MLF performance in oenological conditions

• Further works will focus on genome sequencing and transcriptome (RNAseq).

-6 -5 -4 -3 -2 -1 0

Log(N/No)

Parental E2 E3 E4

***

2% (v/v)

[1] Bartowsky, E.J. (2005). Oenococcus oeni and malolactic fermentation – moving into the molecular arena.

Australian Journal of Grape and Wine Research 11, 174–187.

[2] Bauer, R., and Dicks, L.M.T. (2004). Control of malolactic fermentation in wine. A review. S. Afr. J. Enol.

Vitic 25, 74–88.

[3] Cavin, J.F., Prevost, H., Lin, J., Schmitt, P., and Divies, C. (1989). Medium for Screening Leuconostoc oenos Strains Defective in Malolactic Fermentation. Appl Environ Microbiol 55, 751–753.

References