Whole Genome Sequencing And Comparative Genomics Analysis Of Pasteurella Multocida

Pasteurella multocida can infect a wide range of animal species. The bacterial pathogen is the causative agent of many economically important diseases that include swine atrophic rhinitis, swine pneumonia, avian fowl cholera, bovine and buffalo, sheep, and rabbit haemorrhagic septicaemia, and so on. It is also one of the etiologic agents of porcine respiratory disease complex. Pasteurella multocida is widespread around the world and has been caused great economic losses in livestock breeding industry. Many key virulence factors have been identified in Pasteurella multocida, including capsule, lipopolysaccharide, adhesins, toxins and outer member proteins. However, there are still some crucial virulence factors are worth to be exploited in this pathogen. We have finished the following works to interpret the relationships between the genotypes and pathogenic phenotypes of Pasteurella multocida at the genome level.1. General feactures of7newly seqeuced Pasteurella multocida genomesThe draft genomes of7Pasteurella multocida strains were sequenced by using Illumina Solexa platform and de novo assembled. The sizes of genomes are ranged from2.22Mb to2.36Mb. Physical gaps in the genomes of HB03and HN06were filled. HB03contains a single circular chromosome of2,307,684bp （GenBank accession number: CP003313）, the strain contains2,118CDSs,53tRNA genes and six16s-23s-5s rRNA operons. The average GC content is40.4%. HN06contains a circular chromosome with2,402,218bp and the average GC content is40.2%. The HN06genome encodes2,258CDSs,56tRNA genes and six16s-23s-5s rRNA operons. Moreover, HN06contains a plasmid pHN06（GenBank accession number: CP003314） with5,360bp in length and encoding7CDSs. Additionally, some specific genetic elements were identified, including pseudogenes, phage-associated genes and two-component systems related genes.2. Comparative genomic analysis of4Pasteurella multocida complete genomesWhole genomic nucleotide sequence alignments were genetated by using complete genome sequences of strains HB03, HN06, Pm70（GenBank accession number: NC₀02663）, and36950（GenBank accession number: CP003022）. Some insertions and deletions, as well as inversions regions were identified. Compared to the other three strains, Pm70genome contains five large inversion regions. Meanwhile, insertions and deletions large than90bp were determined. The average total length of strain specific sequences between the3P. multocida strains and HB03was226kb, approximately9.8%of HB03genome. The average deletions and insertions length of HB03were6,704bp and7,117bp, respectively. Compared to the other three strains, HB03habors two large specific regions with37,342bp and18,757bp in length, respectively. The37,342bp region contains phage related genes and the18,757bp region encoding tetracycline resistance related proteins. We also investigated3insertion regions larger than36kb exited in the HN06genome but absented in the HB03genome. The largest insertion region was a prophage region, in which includes an important toxin gene toxA. The other two regions are bacteriophages liked sequence, which may horizontal transferred from other bacteria. These3regions were absent in Pm70and36950genomes, they were strain-specific regions in HN06genome.3. The comparative analysis of protein-coding gene compositions among4Pasteurella multocida genomes Two methods were used to determine the distribution of protein-coding genes of4Pasteurella multocida genomes. Based on the BSR method,1,812genes of HB03were highly conserved among the4genomes, and79proteins were showed great genetic diversity between HB03and other three strains, respectively. The total number of orthologous of4Pasteurella multocida was2,687. Based on the blastclust method,1,789orthologous were identified as core gene clusters that shared by all strains, accounts for66.6%of the all orthogolous.261genes （9.7%） were dispensable gene clusters that were found to be shared by at least two strains but not all, and the remaining23.7%were strain-specifc genes. HB03and36950shared the largest clusters. Strain HN06contained297strain-specific genes, which was the most one of the4Pasteurella multocida strains. Furthermore, the genomic regions involved in biogenesis of capsular polysaccharide, lipopolysaccharide, and Flp fimbriae were identified compared to show the linkages of gentic diversity to the phenotypic diversification.4. The phylogenetic relationship of4Pasteurella multocida strainsThe phylogenetic relationship between the4P. multocida strains was predicted by comparison of45,871single nucleotide polymorphism sites （SNPs）. The phylogenetic tree shows that the4strain distributed in3branches and strain HB03is not close to strain HN06, but rather much closer to strain36950. Interestingly, this result is consistent with the result of comparative genomics analysis.