Experimental evolution forcing Oenococcus oeni acid tolerance highlights critical role of the citrate locus

Oenococcus oeni is the main lactic acid bacterium associated with malolactic fermentation (MLF) of wines. MLF plays an important role in determining the final quality of wines. Nevertheless, due to the stressful conditions inherent to wine and especially acidity, MLF may be delayed. This study aimed to explore by adaptive evolution improvements in the acid tolerance of starters but also to gain a better understanding of the mechanisms involved in adaptation toward acidity. Four independent populations of the O. oeni ATCC BAA-1163 strain were propagated (approximately 560 generations) in a temporally varying environment, consisting in a gradual pH decrease from pH 5.3 to pH 2.9. Whole genome sequence comparison of these populations revealed that more than 45% of the substituted mutations occurred in only five loci for the evolved populations. One of these five fixed mutations affects mae, the first gene of the citrate operon. When grown in an acidic medium supplemented with citrate, a significantly higher bacterial biomass was produced with the evolved populations compared to the parental strain. Furthermore, the evolved populations slowed down their citrate consumption at low pH without impacting malolactic performance.