| Enterococcus faecium are common commensal bacteria in the gastrointestinal tract of mammals.However,due to the abuse of antibiotics,drug-resistant Enterococcus faecium has gradually evolved into an important pathogen on a global scale.Bacitracin is a broad-spectrum polypeptide antibiotic synthesized by non-ribosomal synthases of Bacillus licheniformis and Bacillus subtilis.It is widely used as a growth promoter and preventive drug in animal husbandry industry of China,and plays an important role in promoting animal growth and inhibiting the growth of pathogenic bacteria in the gastrointestinal tract of animals.The large-scale application of bacitracin in aquaculture makes E.faecium in the gastrointestinal tract of animals highly resistant to bacitracin and proliferates in large quantities,which causes contamination of the food chain of agricultural and livestock products and threaten food and public health safety.The discovery and utilization of bacitracin has a history of more than 70 years,but there is still a lack of comprehensive and systematic research on its mechanism of action and tolerance.Therefore,this study took E.faecium as the research object,mainly based on transposon insertion sequencing(Tn-seq)and transcriptome sequencing(RNA-Seq),to explore its bacitracin tolerance mechanism from a genome-wide scale.The main research contents and results are as follows:(1)The complete genome sequence of E.faecium E980 was obtained by whole genome sequencing.The results revealed that E980 consists of a circular chromosome with the size of 2,653,961 bp and two circular plasmids with the size of 215,852 bp and10,977 bp,respectively.According to the coding gene prediction,the E980 contains a total of 2969 genes with an average gene length of 837 bp,of which 2701 genes are on chromosome and 268 genes are on plasmids.In addition,the functions of coding genes are also annotated through different databases to lay the foundation for subsequent functional genomic analysis and mechanism research.(2)The transposon mutant libraries of E980 were constructed based on the thermosensitive plasmid p GPA-2 carrying Mariner transposon,and the quality were preliminarily evaluated by colony counting,inverse PCR(IPCR)and DNA footprinting.The results showed that the transposon insertions of the mutant library are random and high-density.The mutant pools were propagated in BHI medium with and without bacitracin,followed by extraction of genomic DNA,and then the DNA libraries required for sequencing on the Illumina platform were constructed by the Tn-seq method for high-throughput sequencing.(3)Data processing and analysis of Tn-seq were performed to mine and identify essential genes for E980 in BHI and conditionally essential genes under bacitracin conditions.The results exhibited that the E980 transposon mutant libraries include at least 33,916 unique mutants,with an average of one transposon insertion mutation per84 bp in the genome.In BHI,a total of 631 E980 essential genes were identified,and their functions were significantly concentrated in 48 metabolic pathways that maintain the basic physiological needs of cells,such as ribosomes,substance synthesis and metabolism.Under the stress of bacitracin,a total of 20 conditionally essential genes were identified,and their functions were mainly focused on six physiological functions of transporter,regulator,biosynthesis,metabolism,DNA repair and phage.Subsequently,a series of studies were carried out on two newly discovered bacitracin resistance genes,E980＿1899(encoding glycosyltransferase)and E980＿1049(encoding the major tail protein of Phi13 family phage).Targeted gene deletion mutants(Δ1899::gm and Δ1049::gm)were constructed by homologous recombination technology and their characteristics were studied.The results showed that E980＿1899is a bacitracin essential gene and inactivated E980＿1049 can enhance the tolerance of E.faecium to bacitracin.Further analysis found that cell wall synthesis and phage activation play an important role in the tolerance of E.faecium to bacitracin,which also provides a theoretical basis for in-depth exploration of the mechanism of bacitracin.(4)The differentially expressed genes of E980 between under the stress of bacitracin and in the BHI were investigated by transcriptome sequencing(RNA-Seq).The results showed that there are 601 significantly differentially expressed genes under bacitracin stress,of which 235 were significantly up-regulated,and their functions were mainly concentrated in the ribosomal pathway and biological processes such as gene expression,translation and peptide metabolism.There are 366 significantly downregulated genes,mainly related to physiological functions such as redox process,carbohydrate transport and metabolism.This study systematically and comprehensively identified the key genes of E.faecium resistance to bacitracin,and explored its molecular mechanism.It provides a potential drug target for the prevention and treatment of drug-resistant E.faecium,which will help reduce the pollution of drug-resistant Enterococcus to the food chain of agricultural and livestock products and improve the level of health protection. |
