Whole Genome Analysis Of Two Klebsiella Pneumoniae Strains Isolated From Moschus Berezovskii And Prokaryotic Expression Of Novel Gene Kp05372

Musk deer?Moschus berezovskii?as a national-level protected animal,the artificial breeding of musk deer has been restricted by pneumonia.Pneumonia was erupted in a Musk deer farm in a certain area of Sichuan Province,with a high mortality.Our research group paid close attention to the emergency infections,and isolated one pathogenicity K.pneumoniae strain?LPKP?,which might responsible for the prevalence of disease.To investigate the difference of pathogenicity between LPKP and gut pathogenic bacteria GPKP,the pathogenicity phenotypic and genotypic of the two strains were compared by animal experiment and genomic sequence analysis.And construction of prokaryotic expression vector of novel gene kp05372 was carried out.In this dissertation,the following experiments were conducted:1.Isolation,identification,and pathogenicity analysis of lung pathogenic K.pneumoniae?LPKP?In this study,lung pathogenic LPKP was isolated and identified from suppurative pneumoniae in musk deer by conventional methods and by 16S rRNA sequence analysis.The isolate was 99%identical to K.pneumoniae,and named K.pneumoniae LPKP.Animal experiment showed that the LD₅₀0 of LPKP and GPKP are 4.1×10⁹ CFU/mL and 6.3×10⁷CFU/mL,respectively.Pathological change was observed in the lung,liver and kidney of two bacterial infected groups by paraffin sections.Moreover,the jejunum of GPKP infected group,histopathological change had been found.In addition,a clear capsule band was observed around the two strains by Indian ink stain.2.Genome analysis of the two K.pneumoniae strainsThe genome of LPKP was assembled de novo into 50 contigs,which together comprised4,736,259 bp with 57.32%GC content,the genome contains 5,516 predicted ORFs.The genome of GPKP was assembled de novo into 45 contigs,which together comprised4,879,707 bp with 57.33%GC content,the genome contains 5,490 predicted ORFs.Phylogenetic analysis found that LPKP and KP-1 belong to one branch,and GPKP and YH43belong to one branch.MLST analysis revealed that the LPKP sequence type was ST29 and the GPKP sequence type was unknown.At the same time,wzi and wzc genotyping methods could not classify GPKP capsular membrane,while LPKP capsule type was K54.The K54cps gene clusters were encoded by 20 CPS-related genes and share highly conserved?99.96%similarity in nucleotide sequences?gene contents among four strains.Comparative analysis of the structures of the cps gene clusters showed that a manCB cluster was found at the 3'end of GPKP cps gene cluster,which had been verified by PCR amplification,and was different from the previously reported 79 capsular types.Furthermore,comparative genomics analysis identified 840 genes in the GPKP genome that are absent in other reference strains.By functional annotation of these specific genes,5 specific virulence-related genes were found,which were coded RfbU,FbpC,FbpB,FbpA and IrgA.3.Construction of a novel gene kp05372 prokaryotic expression vectorA novel gene kp05372 had been found in GPKP genome.The encoding product of kp05372 gene consists of 302 amino acids which has no signal peptide.Additionally,it is a kind of fat soluble and unstable protein,and plays a physiological role in the cell.The protein N-terminal contains a conserved HTH DNA binding motif,while C-terminal contains a LysR coinducer recognition.The promoter analysis found two non-tyical-35 box and-10 box with multiple locations in the promoter region.Bioinformatics analysis indicatrd that kp05372 is a LTTR.The kp05372 gene was cloned into prokaryotic expression vector pET-32?a?and constructed the recombinant expression plasmid pET-32?a?/kp05372,transformed into BL21?expression strain?.Based on the PFGE and western-blotting,the optimal expression conditions?37?,0.6 mmol/L IPTG induced 6 h?had been determined,and the recombinant protein of about 53.2 KDa was detected.In conclusion,a pathogenic K.pneumoniae LPKP succeed isolated from Moschus berezovskii lung.LD₅₀ of LPKP was 65-fold higher than GPKP.The specific virulence related genes and novel capsular type of GPKP might be responsible for the unique phenotype of GPKP.The bioinformatics analysis and construction of prokaryotic expression vector were laid a foundation for function analysis of novel LTTR gene kp05372.