Complete Genome Sequence And Annotation Of Klebsiella Phage P13with The Properties Of Exopolysaccharide Depolymerase

Bacteriophage is virus of bacteria and ubiquitous in the nature where bacteriaexists. However, the quantity of known and researched phage is still small so far, theEBI data base records1178phage genome sequences up to the beginning ofFebruary2013. Genome sequencing and functional genomics analysis of phage arerelatively easy, because the size of its genome is small. Therefore, the researching ofphage genomics makes significance sense for figuring out biodiversity, generegulation, growth metabolism and other biological phenomena of phage.Klebsiella phage P13was studied in this dissertation. One step growth curve ofphage P13shows that the latent period is about20min and the outbreak period isabout40min. It certifys that phage P13is ascribed to virulent phage with the rapidlytic cycle. Transmission electron microscope revealed that phage P13consists of asmall spherical head with a diameter of50nm and a short stumpy tail spikes. Thesequence of phage P13genome has been obtained by high throughput sequencingand its size is45976bp. The restriction enzyme test indicates that phage P13geneticmaterial is liner double–stranded DNA.The bioinformatics analysis of phage P13genome shows that there are282ORFs longer than100bp and50predicted genes are found out by GeneMark,Softberry and Glimmer programs. The total gene length of phage P13is41639bp,and the average gene length is833bp. Forty of the phage-encoded putative proteinsshow homology to known proteins in database in GenBank. The functions of26putative proteins have been clear by BLASTp program and13in these putativeproteins have highly conserved sequence mosaic structure. The homology of the50putative genes and the whole genome indicates that phage P13is similar toEnterobacteria phage SP6, Pectobacterium phage PP1, Enterobacteria phageUAB_Phi78, Enterobacteria phage K1-5, Enterobacteria phagevB_EcoP_ACG-C91and Enterobacteria phage K1E, and these sixty kinds of phage all belongs to SP6-like genus phage. Meanwhile, in view of the morphology, it isconfirmed that Kleblsiella phage P13is belonged to SP6-like virus genus,Autographivirinae subfamily, Podoviridae family.Four different kinds of repeat sequences are found in phage P13genome andthese repeat sequences are related to the transcription, recombination of DNA andgenomic evolution. The promoter and terminator structure are found in the upstreamand downstream sequence of RNA polymerase gene and major capsid protein gene,respectively which makes proteins transcription and expression fast. Overlappingregion structures by one base are existed between gene30(bacteriophage head to tailconnecting protein) and gene31(putative scaffolding protein), gene33(ail tubularprotein A) and gene34(putative tail tubular protein), gene40(putative smallterminase subunit) and gene41(large terminase subunit protein), which is similar tothe trp operon. The main function of this structure is to make the adjacent genesexpression efficient and equivalent, which is beneficial to facilitate collaborative roleof biological. The G+C content of phage P13genome is slightly lower to the contentof Klebsiella pneumoniae KTCT2242and their codon bias have many similarities.The similar base composition structure between phage P13and its host illustrate thatthe infection history between them is long.The bacteriophage depolymerase which possess good heat-stability could beproduced by phage P13when it infect the host. The main function of thedepolymerase is to hydrolyze the exopolysaccharide (EPS) which is around the hostcell, and then allowing the phage to get access to receptor of the bacterial cell tofinish the lytic cycle. The hydrolysis characterization of EPS depolymerase isbeneficial for further application, such as biofilm degradation, preparation ofbioactive oligosaccharide, structure analysis of polysaccharide and medical aspects.Both the enzyme activity and phage titer are detected during the process ofdepolymerase purification. About27.5U enzyme activity is recovered from220mLphage suspension after acetone precipitation, dialysis and ultrafiltration. Inconsideration of the function of the50putative genes and the common genomestructure of SP6-like phage, the putative gene49and gene50are possible depolymerae protein. Based on the molecular biology experiment method, theengineering bacteria which has recombinant plasmid that contain the depolymerasegene has been prepared successfully.