Diversity Of Methylation-Omics And Restriction-Modification Systems Of Lactococcus Lactis Subsp.lactis Isolated From Natural Fermented Milk

Lactococcus lactis subsp.lactis(L.lactis subsp.lactis)is one of the most common starter strains in the production of fermented dairy products,which has high economic value.Its fermentation properties and application value are closely related.Restriction-modification systems and DNA methylation can affect the physiological activities and the phenotypic characteristics of bacteria.However,few studies have been reported on the correlation between DNA methylation-omics,restriction-modification systems and important production characteristics,such as acid production,proteolysis ability and viscosity production.It has a great significance for the development and utilization of L.lactis subsp.lactis to analyze the diversity of DNA methylation-omics and restriction-modification systems,and to explore the influence of DNA methylation-omics on the phenotype of strains.In this study,23 strains of L.lactis subsp.lactis with different fermentation characteristics(acid-producing ability,protein hydrolysis ability and viscosity producing ability)were selected as research objects.The diversity of DNA methylation-omics and restriction-modification systems of L.lactis subsp.lactis was analyzed by PacBio SMRT sequencing technology,and the effect of DNA methylation-omics on the phenotype of the strains was explored.The main results were as follows:(1)In this study,the whole genomes of 23 L.lactis subsp.lactis isolated from natural fermented milk were sequenced by PacBio SMRT sequencing technology.It was found that there was a high Average nucleotide consistency(ANI>98.06%)between the strains.Moreover,their functional genes were similar,mainly carbohydrate and protein metabolism related genes,which were 18.48±0.67%and 9.94±1.20%,respectively.(2)The methylation-omics of 23 strains of L.lactis subsp.lactis were mapped.And nine m4C motifs and forty-five m6A motifs were identified in this study.There were 22 complementary motifs in m6A,which existed in 70%strains of L.lactis subsp.lactis,and each motif was detected in no more than 2 strains,which had a high strain specificity and could be used as a"biological label" for strain identification and protection.L.lactis subsp.lactis showed rich methylation patterns.According to the main motif types,the strains could be divided into two groups of A and B,in which m6A and m4C were the main motif types in groups A and B with TNNNDNNH and GNNNNNNH in sequence respectively.(3)Main restriction-modification systems of distribution of L.lactis subsp.lactis was type ? restriction-modification systems,followed by type ?restriction-modification systems.14 kinds of restriction-modification enzyme genes of L.lactis subsp.lactis(76.92%)were mostly detected in the plasmids.Compared with group B,group A strains had more abundant restriction-modification enzyme genes.This study speculated that the diversity of restriction-modification systems might be the reason for diversity of their motifs(4)The number of m6A sites in the CoiA gene showed a significant positive correlation with the ratio of average viscosity rising(R=0.67,p=0.003).There was a significant difference in the number of m6A modification sites in the CoiA genes between the group with high average viscosity rising rate and the group with low average viscosity rising rate(p=0.01),and the former generally contained more m6A sites.It was suggesting that the number of m6A sites in CoiA genes might affect the ratio of average viscosity rising of the strains during after-ripening.In summary,this study revealed the diversity of L.lactis subsp.lactis methylation patterns,type ? and type ? restriction-modification systems,from the perspective of whole genomics and epigenetics,and explored the reasons for the phenotypic differences of L.lactis subsp.lactis.It was found that the phenotypic differences of strains with close genetic relationship were significant,indicating that epigenetic effects had a significant impact on their phenotypes.The difference in number of m6A sites in CoiA genes might be the reason for the difference in viscosity production property of L.lactis subsp.lactis.In this study,the epigenetic characteristics affecting the phenotypic differences of the strains of L.lactis subsp.lactis were studied,which laid a theoretical foundation for the further development and utilization of excellent starter cultures in China and the screening of strains with excellent fermentation characteristics.