Whole Genome Sequencing And Comparative Genomics Analysis Of Avian Pathogenic Escheerichia Coli

Avian pathogenic Escherichia coli （APEC） is the pathogen of avian colibacillosis which causse sepsis, pericarditis and airbag inflammation, etc.. APEC, a widespread disease which are estimated to multimillion dollar annual losses for the world’s poulty industries. However, the mechanism of APEC virulence remain largely unknown. The genomic diversity among in different sources and different virulence strains and frequently horizontal gene transfer, increasing the difficulty of the study on APEC pathogenesis.With the popularity of the next generation high-throughput sequencing technology and the development of the next-next generation sequencing technology, the costs of sequencing is lower and lower and more and more pathogenic microorganisms get their sequences information, to reveal the pathogenic mechanism and principles of pathogenic microorganism provides a new effective method. At present, the NCBI genome database has only a few whole genome sequence information of the APEC strains. In order to study genetic rules and pathogenic mechanism of APEC, we chosed the two strains of078serotype of APEC strains, having finished the whole genome sequencing and comparative genome analysis research. The main research was described as follows:1. Genome sequencing of APEC strainsIn this study, the whole genomes of APEC strains ACN001and ACN002were sequenced by using Roche company454GS-FLX technology platform. The whole genome of ACN001strain, which consists of a circular chromosome sized4,936,576bp and. The genome contains4794protein-coding genes and86tRNA genes and seven16s-23s-5s ribosomal rRNA operon. The genome GC content is50.67%on average; The whole genome of ACN002strain, which consists of a circular chromosome sized4,879,931bp. The genome contains4,677protein-coding genes and85tRNA genes and seven16s-23s-5s ribosomal rRNA operon. The genome GC content is50.69%on average. The strains ACN001and ACN002contain6plasmids and7plasmids respectively, which include virulence plasmid and multiple-drug resistance plasmids. At the same time, the basic characteristics of inserted sequences and phage related genes of the two strains are forecasted.39and37IS elements and8and6prophage regions are predicted in the chromosome genomes of ACN001and ACN002respectively. These prophage regions contain binding sites, integrase, enzymes, tRNA and phage assembly related genes.2. Comparative genomic analysis of APEC strainsWhole genomic nucleotide sequence collinearity comparative analysis were generated by using the complete genome sequences of the strains ACN001and ACN002. The results indicated that strain ACN001has a good linear with the strain ACN002genome, seven insertions or deletions regions and two translocations regions have been found and located. According to the result of functional annotation speculate that these areas are bacteriaphages liked sequences, which may be horizontal transfer genes. Compared to the strain ACN001, strain ACN002genome contains one inversion regions with685kb in length. Biology function analysis in the inversions, insertions and deletions regions, we found that encoding adhesion gene aidA and encoding transmembrane protein gene tonB in ACN001genome is the complete, whereas in ACN002genome is incomplete. Meanwhile, we compared and analyzed the similarities and differences between ACN001and ACN002genomes in the virulence plasmid, multiple-drug resistance plasmids, iron uptake systems, flagella gene clusters and type I fimbriae gene clusters, founding that there are some differences in ACN001and ACN002genomes in type I fimbriae fim gene cluster, iron uptake related Ybt system, flagella flg gene cluster, virulence plasmid and multiple-drug resistance plasmids. Through these analysis, achieving the molecular basis of pathogenicity differences in gene level, providing the basis to further explore in the pathogenic mechanism of the APEC strains.Orthologous analysis were completed by using genome sequences of the strains ACN001, ACN002, APEC01（GenBank accession number: NC₀08563） and APEC078（GenBank accession number: CP004009）. The total number of orthologous of4APEC strains was5739, in which there are3246orthologous were determined as the core gene clusters that shared by all4strains, accounting for56.56%of the total orthologous. Significantly, strains ACN001, ACN002and APEC O78share the largest gene clusters （689） more than other combinations, however, strain APEC01shared gene clusters with other strains is always less than other combinations. In the analysis of various strains of the specific gene cluster, found that strain APEC O1contains723strain-specific genes, which is the most one of the4APEC strains. Results indicated that strains ACN001and ACN002are close to strain APEC078in evolutionary relationships, but far away with strain APEC O1.3. The phylogenetic relationship analysis of APEC strainsIn our study, the phylogenetic relationship was predicted by series connection of the E. coli seven housekeeping genes （adk, fumC, gyrB, icd, mdh, pur A, and recA）. Strains in the phylogenetic tree including48strains of E. coli,5strains of shigella and1strains of Escherichia fergusonii genome sequences. The phylogenetic tree shows that strain ACN001is close to strain ACN002, and they two are much closer to strain APEC078rather than strain APEC O1. Interestingly, The above results are also consistent with the COG results in the genomics analysis and orthologous analysis results in the comparative genomics.