Characterization Of Lactobacillus Prophage And Its Effect On Intestinal Microbiota

The human gut microbiome is a dense consortium of microorganisms.In recent years,considerable attention has been received by the bacterial which has been known as one of important factors to determine the health of host.Moreover,the physiological significance and function of human intestinal phage have gradually been studied for the past few years.Lactobacillus is a dominant species in the human gut and has benefits for human health.Prophages are ubiquitous in Lactobacillus and have the potential to alter presence forms.Thus,the characterization of the prophage carried by Lactobacillus,and its effect on intestinal microbiota and health are worthy to be explored.In this study,we predicted prophages carried by 639 Lactobacillus strains to analyze their distribution characteristics,functional genome and evolutionary direction in Lactobacillus.Then we used mitomycin C to induce lysogenic phage in 207 Lactobacillus strains to evaluate simultaneous the host infestation range,morphological characteristics,and functional genes through double-layer plate method,transmission electron microscope observation,and genome extraction sequencing experiments,respectively.Finally,Lactobacillus lysogenic phages are intragastrically administered to C57BL/6J mice to study its effect on intestinal microbiota structure,and explore the influence on host intestinal health.This study provides data and theoretical basis for the research and application of Lactobacillus phage diversity and the influence of phage on intestinal health.The main findings are as follows:Firstly,based on the prediction analysis,489 of 639 Lactobacillus strains carried 832 intact prophages.The results showed that there was difference in the number of prophages between different species of Lactobacillus,and the proportions of Lactobacillus plantarum and Lactobacillus paracasei strains carried two or more prophages were 81.1% and 64.0%,respectively.However,the proportions in Lactobacillus fermentum,Lactobacillus gasseri,Lactobacillus rhamnosus and Lactobacillus salivarius were all in the range of 20.0%~31.0%.Strains isolated from different sources also had difference in the number of prophages,71.7% of strains isolated from feces carried only one or less prophage,while 74.6% of strains isolated from fermented food carried two or more prophages.It is indicated that Lactobacillus strains isolated from fermented food carried more prophages.Secondly,the genomic analysis of 832 Lactobacillus prophages showed that the average genome size of them was 39.90±14.93 Kb.The GC content was 30.78%~49.77%,it was consistent with the host.Some prophages would confer host adaptation to habitat through gene exchange and horizontal transfer functional genes.Average nucleotide identity was used to study the evolution of Lactobacillus prophages,and found that the main evolutionary direction of Lactobacillus prophage was closely related to the species of host.Thirdly,29 strains of 207 Lactobacillus were induced to release phage particles.Proportions of inducible strains in Lactobacillus fermentum,Lactobacillus gasseri,Lactobacillus salivarius and Lactobacillus brevis were higher than 20.0%,while Lactobacillus paracasei was 5.8%.Lactobacillus rhamnosus and Lactobacillus plantarum had not inducible strain.It was found that the host range of Lactobacillus lysogenic phage was highly specific.Observation by transmission electron microscopy revealed that the lysogenic phage consisted with a polyhedron heads of 68 nm~85 nm in diameter and a contractile or non-contractile tail of 136 nm~200 nm in length.Functional gene analysis indicated that most functional genes in Lactobacillus lysogenic phage belonged to five core modules: lysogenic,packaging,replication,morphogenesis,and lysis.Finally,the inhibitory effect on the body weight gain of mice was found by Lactobacillus lysogenic phage.? diversity of the intestinal microbiota in mice increased in the early stage of Lactobacillus lysogenic phage intervention,but decreased with continuous intervention.? diversity was significantly affected in Day 42.Lactobacillus lysogenic phage decreased the relative abundance of Bacteroidetes,while Actinobacteria and Firmicutes were increased.At the genus level,the relative abundance of Bacteroides was decreased,while Clostridiales genus and Bifidobacterium were increased.It was indicated that Lactobacillus lysogenic phage affected intestinal microbiota diversity and structural composition.