Genome Sequencing And Identification Of Genes Essential For In Vivo Infection In Fish Pathogen Vibrio Anguillarum MVM425

Vibrio anguillarum belongs to Vibrionaceae and Vibrio genus,and is a Gram-negative bacterium.V.anguillarum is one of leading pathogens infecting various marine fish.Vibrio anguillarum MVM425 was isolated from infected seabass with the endogenous plasmid pEIB202 sequenced and the draft genome sequence finished.This project applied Pacbio sequencing platform,the third-generation sequencing technology,to complete the assembly of Vibrio anguillarum MVM425 whole genome.The genome size is 4.2 Mbp.The chromosome I(ChrI)includes 3,164,924 bp and the chromosome ?(Chr?)contained 1,141,928 bp,and the plasmid pEIBl contained 66,521 bp.On Chrl,there are 2771 CDSs,28 rRNAs and 98 tRNAs,and the Chrll encodes 972 CDSs and 4 tRNAs.We constructed a mariner-transposon-based mutant library of MVM425 and set up Tn-seq platform for the functional genomics analysis to identify essential genes related to growth in LB containing 2%NaCl,and the conditional essential genes related to infection in vivo in fish.We developed effective bioinformatics tools to analyze our Tn-seq data with relatitively low TA coverage.Tn-seq identified 473 and 359 putative genes essential for its growth in rich medium and during the in vivo infection processes,respectively.In addition,our analysis revealed genes displayed various fitness levels in liver,spleen and kidney,suggesting the dilferent genetic requirements for colonization in these organs.Furthermore,we validated our Tn-seq data by using the gene knock out mutants and in vivo infection experiments.Comprehensive genomics and functional genomics analysis highlighted conditional essential genes for important pathways involving energy metabolism,nitrogen metabolism,nucleotide synthesis,amino acid synthesis,cofactors,siderophore synthesis,secretion(T2SS and T6SS),quorum sensing,and flagellum biosynthesis.This study provides wealth of information about V anguillarum genes related to the infection in fish,and will facilitate the understanding of its pathogenesis as well as development of diagnostics and vaccines against the pathogen.