Study On DNA Methylation Diversity Of Lacticaseibacillus Paracasei

As one of the important bacterial resources,Lacticaseibacillus paracasei(L.paracasei)not only has a wide range of sources,but also has various potential probiotic functions such as regulating the balance of human intestinal flora and enhancing immunity.As a form of epigenetic modification,DNA methylation not only plays an important role in bacterial restriction modification systems and phage exclusion systems,but also enriches bacterial genetic diversity by changing genetic phenotypes.Studies on DNA methylation are rarely reported.Therefore,this study used Illumina and Pac Bio SMRT sequencing technology to analyze the genome and methylome of 28 L.paracasei strains,and used gene knockout technology to construct methyltransferase mutant strains to explore the relationship between DNA methylation and strains.The main findings of the relationship between phenotypes are as follows:(1)A genome dataset of 28 L.paracasei strains was obtained,and it was found that the average nucleotide identity of all strains was greater than 98.72%,the genetic distance was relatively close,and the functional genes between different strains were relatively similar,mainly carbohydrate metabolism-related genes.The proportion is18.12±2.15%.(2)The methylome map of 28 L.paracasei isolates was drawn,and a total of 41m6 A methylation motifs and 1 m4 C methylation motif were identified.Overall methylation levels were high in L.paracasei,and m6 A methylation was highly variable between strains and was enriched in carbohydrate metabolism-related genes.(3)The restriction modification system of L.paracasei showed high diversity.A total of 30 genes related to restriction modification systems were identified,and the type I restriction modification system genes were more widely distributed.(4)Based on the Cre-lox P gene knockout system,the L.paracasei IMAU60143 methyltransferase LPP＿2007 gene mutant strain was successfully constructed,and combined with the third-generation sequencing results,it was proved that the LPP＿2007gene knockout reduced the methylation sites in the genome 41.58%,other methylation information did not change significantly.It indicated that LPP＿2007 gene plays an important role in the epigenetic modification of L.paracasei IMAU60143.(5)Using the Biolog phenotype microarray technology,it was found that there were differences in the utilization of eight carbon sources between mutant strains and wild strains.It is speculated that m6 A methylation may regulate the expression of carbohydrate-encoding genes to some extent.In conclusion,this study revealed the distribution pattern of DNA methylation and the composition of restriction modification system in L.paracasei from the perspectives of genomics and epigenetics;By constructing the L.paracasei IMAU60143 methyltransferase mutant strain,it was found that the methyltransferase LPP＿2007 gene was involved in the regulation of the methylation phenotype of the strain to a certain extent;There are certain differences in the utilization capacity of different carbon sources.It is speculated that m6 A methylation may be a mechanism regulating carbohydrate metabolism in L.paracasei,but further research is needed.This study opens up new ideas for the study of bacterial methylation,and also provides an objective and detailed theoretical basis for the subsequent development and utilization of L.paracasei.