MLST And PFGE Of Oenococcus Oeni From Different Wine Producing Regions In China And The Research On Their Stress Related Genes’ Expression

Oenococcus oeni is a major starter used for the malolactic fermentation of wine. It is massively used for the manufacture of wine products. In this study, O. oeni were isolated from dry red wine samples from different wine producing areas around China. Those isolates were identified by 16 S rRNA gene sequence comparison. Meanwhile, multilocus sequence typing(MLST) and pulsed-filed gel electrophoresis(PFGE) were used to study the genetic polymorphism and evolutionary relationship of the isolates. Through the two typing methods, different genotypes in those isolates were confirmed, and the strains with different genotypes were sent for the wine-making condition adaptive test, from which the highly wine-making condition adaptive strains were confirmed, then those highly adaptive strains with different genotypes were had their stress related genes’ expressions during MLF studied. The results were showed as follow:First, Through comparing the 16 S rRNA gene sequences, 55 strains were identified as O. oeni. Sixteen O. oeni were isolated from Shandong, 4 strains were isolated from Hebei, 2 strains were isolated from Shaanxi, 11 strains were isolated from Xinjiang, 12 strains were isolated from Ningxia, and 10 strains were isolated from Inner Mongolia.Second, fifty-five representative isolates in previous study with two model strains O. oeni PSU-1(CP000411) and ATCC BAA-1163(AAUV00000000) were analyzed by MLST. Seven stress related genes, cfa、clpL、clp P、ctsR、mle A、mleP and omr A, were chose and used for MLST analysis. The result showed that the method separated the strains into two clusters which are correlated with the wine-producing regions. Furthermore, positive selective pressure was detected for these genes. In addition, the population structure was a mixture of evolutionary pathways, and the strains exhibited both clonal and panmictic characteristics.Third, the fifty-five representative isolates in previous study with the two model strains were also analyzed by PFGE. The result showed that the method also separated the strains into two clusters which are correlated with the wine-producing regions, but they were different from the two clusters shaped by MLST. Meanwhile, Not I was selected as the major restriction enzyme of O. oeni for PFGE. Beside, this has been the first time that PFGE and MLST results have been combined to shape a unique dendrogram. Thus, the combination of results from both typing methods allowed a better discrimination of genotypes.Fourth, the wine condition adaptive test was carried out for the strains with different genotypes which typed by the combined method. The result shows that the genotypes of strain SD-2a, NX-3b, SD-lb, SD-2h and SD-2ji correlated with better wine condition adaptability. In addition, the level of pH and the content of alcohol are important factors that affecting the malolactic enzyme activity of O. oeni strains during wine making process. The results of the changes of free amino acid after MLF in the selected strains showed that after MLF, total free amino acids content increased, among which, Ser, Glu, Lys and Arg content increased more than others.Last, we evaluated the different behaviors of five highly adaptive strains with different genotypes by means of measuring the evolution of MLF. The initial activities of malate decarboxylase and citrate lyase would determine a faster MLF and earlier citrate consumption, respectively. Moreover, the strains that performed best in wine-like conditions presented a much higher relative expression of several stress responsive genes, particularly hsp18, clp P, cts R, cfa and rmlB.