Genomic Polymorphism Of The Genus Lactobacillus And Microevolution Of Lactobacillus Delbrueckii Subsp.Bulgaricus

Lactobacillus is the largest genus of lactic acid bacteria that plays important roles in balancing the microbiota of human and animal gastrointestinal tracts. They are the major dairy starter strains and probiotic products. Lactobacillus delbrueckii subsp. bulgaricus is one of the essential dairy starters used for manufacturing of fermented dairy products. Phylogenetic and species differentiation analysis is an extremely important for the development and utilization of Lactobacillus strains. In this study, the whole genomes of146Lactobacillus type strains were deciphered using whole-genome re-sequencing technology. Phylogenomic and evolutionary analysis of149Lactobacillus type strains was performed based on the whole genome level, which combined with the complete genome sequence of three Lactobacillus type strains. Simultaneously, the microevolution of305L. delbrueckii subsp. bulgaricus was determined using multi-locus sequence typing (MLST) technology. The results are as follow:(1) The whole genomes of146Lactobacillus type strains were deciphered. Only72homologous genes were found to be shared by all lactobacilli, and they were assigned to the core-genomes. More than70,000gene families were identified to pan-genomes, with its size growing continuously with the number of genomes. The results integrated with genome size, GC content, the number of protein-coding, ANI and TNI values, indicated an extremely high genetic diversity of the genomes of Lactobacillus. The genetic diversity is larger than typical families.(2) A phylogenetic tree with representative genomes of421genera from26phyla was reconstructed with Lactobacillus strains. The results showed that the Lactobacillus clade actually included strains from five genera, Oenococcus, Pediococcus, Weissella, Leuconostoc and Lactobacillus, thus, it was proposes that these five genera should be grouped under the Lactobacillus complex. In contrast, Lactococcus, Streptococcus, and Enterococcus, were located immediately before the Lactobacillus complex.(3) The phylogenetic tree of the Lactobacillus type strains was reconstructed based on the72core genes. All type strains of the Lactobacillus genus were divided into2big branches. Furthermore, the ancestral lineages in the phylogenetic tree were facultatively heterofermentative (FHE), which were at the beginning of the genus of Lactobacillus formation. In addition, the type strains, isolated from humans and animals, appeared to go through niche shift events on branch1but evolved parallelly with strains in other niches on branch2.(4) A total of305strains of L. delbrueckii subsp. bulgaricus were identified, belonging to121STs and14clonal complexes, using multilocus sequence typing (MLST) technology based on8housekeeping genes. Five ancestral lineages possibly existed among all isolates of L. delbrueckii subsp. bulgaricus by the analysis of STRUCTURE. Our results also revealed that the isolates from different regions in Mongolia were closer to ancestral lineages of L. delbrueckii subsp. bulgaricus.(5) The results further demonstrated that the isolates from different regions in Mongolia were the closest to the ancestral lineages of L. delbrueckii subsp. bulgaricus, which spreaded from Mongolia to Buryatia, Tuva, Kalmyki of Russia, and Xinjiang, Qinghai, Tibet of China. It was also demonstrated that there was a direct relationship between isolates of L. delbrueckii subsp. bulgaricus and their isolation location and origin.