Investigation Of Comparative Genomics And Functional Analysis Of Bacteriocin Biosynthetic Genes In Two Lactobacillus Spp.

Lactobacillus spp. are probiotics that have been recognized to have great benefits in promoting human health, such as the improvement of the intestinal flora and the enhancement of the immune function. Most of researches on Lactobacillus so far are their physiological and biochemical characteristics and their probiotic functions. With the rapid development of DNA sequencing technology and genomics, investigations on the genomics and the mining of their important fuctional genes are becoming more and more important in the researches and applications of Lactobacillus. As the broad and heavy utilization even abuse of antibiotics, more and more antibiotics super-resistant bacteria occur. Therefore, it is necessary to develop new antimicrobial substances to partly substitute antibiotics. Bacteriocins are the one of best alternatives to antibiotics. Although bacteriocins of Lactobacillus spp. have been relatively broad screened and isolated, there is still a very few bacteriocins which have been well characterized from Lactobacillus spp. because traditional even current methods for isolation and purification of bacteriocins are time-consuming and laborious. Thus, investigation of comparative genomics and functional analysis of their bacteriocin biosynthetic genes is not only an effective but also a pivotal and crucial method for the discovery and development of novel bacteriocins from Lactobacillus spp.In this study, L. plantarum FMNP01, isolated from fresh mango fruits with excellent probiotic property, was sequenced at whole genome level using the secondgeneration sequencing technology. Comparative genomics between L. plantarum FMNP01 and L. sp FMNP02 which whole genome was previously sequenced by our laboratory was performed and analyzed in order to elucidate the genomic mechanism of the molecular evolution, bacteriocin biosynthesis, proteolytic system, carbohydrate metabolism between these two Lactobacillus strains. At the same time, the functions of potential bacteriocin biosynthetic genes in these two Lactobacillus strain genomes were analyzed by experimental biology method. The main results are as follows:(1) L. plantarum FMNP01 was sequenced using the second-generation sequencing technology. The genome comprises a single circular chromosome(3,313,644 bp), with an overall G+C content of 44.48%. Coding sequence(CDS) prediction was carried out with Glimmer to provide 3,147 CDSs, covering 83.70% of the genome. 64 t RNAs were predicted by t RNAscan and 16 r RNAs were predicted by r RNAmmer1.2.(2) Comparative genomic analysis between L. plantarum FMNP01 and L. sp FMNP02 was conducted. Phylogenetic tree based on 16 s r RNA sequences was constructed. The results reveal that the evolutionary relationships between these two Lactobacillus strains are relatively far inside the lactobacillus genus. L. plantarum FMNP01 is closed to L. kimchi, and L. sp FMNP02 is closed to L. casei.(3) The analyses based on the gene sequence homology and collinearity reveal that there are only 1459 core genes in these two Lactobacillus strain genomes. L. plantarum FMNP01 has 1688 specific genes and L. sp FMNP02 has 1632 specific genes. The results show that there are large number of gene differences between these two Lactobacillus strain genomes, almost half of the genes are specific genes.(4) The comparative analysis of the proteolytic system between L. plantarum FMNP01 and L. sp FMNP02 showed that the number of peptide transport system and peptidase of L. sp FMNP02 was much more than that of L. plantarum FMNP01, suggesting that the proteolytic activity and ability of L. sp FMNP02 is stronger than that of L. plantarum FMNP01.(5) The comparative analysis of carbohydrate metabolism between L. plantarum FMNP01 and L. sp FMNP02 showed that lager numbers of PTS system proteins existed in both genomes. L. plantarum FMNP01 genome has 58 PTS system proteins and L. sp FMNP02 genome has 69. However, there were a lot of differences between the two Lactobacillus strains in terms of the classification of the PTS system proteins.(6) A bacteriocin biosynthetic gene cluster was found in L. plantarum FMNP01 genome. When compared with L. plantarum WCFS1 genome, this bacteriocin biosynthetic gene cluster in L. plantarum FMNP01 missed four genes which were pln Q?pln I?pln F and pln S. Pln JK gene in L. plantarum FMNP01 genome was heterologously expressed in E. coli BL21(DE3)-p ET-32a(+) expression system, but the expression product did not show antimicrobial activity.(7) 28 genes were predicted to be bacteriocin biosynthetic genes in L. sp FMNP02 genome, including two genes F1 and F2 which might be class ? b bacteriocin biosynthetic genes. F1 and F2 genes were heterologously expressed in E. coli BL21(DE3)-p ET-28(+) expression system, and the expression product F1-28 a showed antimicrobial activities against Vibrio parahaemolyticus, and F2-28 a can inhibit the growth of E. coli.