Genomic Analysis Of The Pseudomonas Aeruginosa Phage PaP1: Evidence Of A Novel Immunity Mechanism Of Bacteria Mediated By EndonucleaseⅤ

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen, frequently foundin clinical treatment and resistant to many kinds of antibiotics. Pseudomonas aeruginosa isone of the main pathogens in hospital infection. Phages are viruses specifically infectingbacteria and ubiquitous in the biosphere. Estimations of phage numbers are approximatelytenfold higher than those of bacteria. Phages are structurally simple, gnomically small andeasily cultured, making them constantly become important objects and materials of manybasic vital phenomena. Identification and classification of phages are groundwork in phagestudies. Currently, most of the newly identified phages have been categorized into differentgenera; whereas there are still some phages remain unclassified.Whole genome sequencing and annotation are important work in phage studies.Shot-gun sequencing method had been the most popular genome sequencing method for along time. Some phages could not be successfully sequenced by shot-gun strategy or thesequencing process had to stop halfway. Early in the year2004, our laboratory encounteredPseudomonas aeruginosa phage PaP1with its genome hard to sequence. At that time,research students of our laboratory sent the PaP1genome DNA to a commercial sequencingcompany to have it sequenced by shot-gun strategy, but it failed. Then they sent it toanother commercial sequencing company to have it sequenced by shot-gun strategy, itfailed again. So the sequencing of PaP1genome has to be postponed.In the year2008, the second generation sequencing method had spread worldwide. Wesent the PaP1genome DNA to a commercial sequencing company to have it sequenced byRoche/454sequencer. It gives us a whole PaP1genome sequence with a length of91,715bp. In this thesis, we will fully annotate the PaP1genome, perform comparative genomicanalysis of this genome, and extensively analyze the reason behind the PaP1genome hard to sequence by shot-gun sequencing method. The results are as follows:1. Additional analysis of the common bionomics of Pseudomonas aeruginosa PA1andphage PaP1. We observed the growth characteristics, gram-negative morphology,extracellular matrix and flagella of Pseudomonas aeruginosa PA1. The PaP1particles wereconcentrated by using PEG8000, and purified by ultrafiltration. The morphology of PaP1was observed by transmission electron microscope. The result shows that PaP1have anicoschedra head⁶9nm in diameter and a contractile tail¹39nm in length. This is animportant evidence for that PaP1is a member of myovirus.2. Identification of the terminal redundancy of phage PaP1. Roche/454sequencingmethod reveals a whole PaP1genome sequence91,715bp in length. The linear dsDNAfeature and the location of the PaP1genome ends were characterized by restriction enzymedigestion analysis. We recovered the3’ and5’ end fragments of the PaP1genome, and thenterminal run-off sequencing of the two terminal fragments were carried out to identify theterminal sequence of the PaP1genome. We designed a new strategy to explore the terminalsequence of the PaP1DNA, and the result shows that the PaP1genome has1190bpterminally redundant regions.3. Bioinformatic and comparative genomic analyses of the PaP1genome. GC skewanalysis reveals that the replication origin of the PaP1genome locates within the terminallyredundant region. The PaP1genome has169putative genes （including12tRNA genes）,based on the analysis of open reading frames and coding sequences. BlastP analysis revealsputative functions of these genes. Both the genome sequence and the annotationinformations were submitted to GenBank under the accession number: NC₀19913.Comparative genomic analyses of four Pseudomonas aeruginosa myovirus （PaP1, JG004,PAK_P1and vB_PaeM_C2-10_Ab1） were performed. We constructed the phylogenetic treeof these four phages and proposed a new genus: PaP1-like phages.4. Identification of phage PaP1structural proteins. SDS-PAGE was used to visualizeeach structural protein of phage PaP1in the gel. At least17proteins with molecular weightsranging from6kDa to80kDa were resolved. Each protein band was then excised forHPLC-MS, permitting the allocation of15protein bands to12corresponding PaP1genes.5. Analysis of the puzzle of the PaP1genome hard to sequence by shot-gun strategy.Shot-gun sequencing of the PaP1genome only gives us43contigs. We located these contigs to the PaP1genome sequence revealed by Roche/454sequencer and found thatapproximately half of PaP1genome can not be sequenced by shot-gun strategy. Then wefound that EndonucleaseⅤ（EndoⅤ）, produced by E. coli DH5α （the host bacteria ofshot-gun sequencing）, can digest the PaP1genome DNA. But with the same condition, thePaP3genome DNA （successfully sequenced by shot-gun strategy） can not be digested byEndo Ⅴ. This result provides a hint for the puzzle of the shot-gun sequencing of the PaP1genome.6. Analysis of EndoⅤ mediated immunity of bacteria. We knocked out the EndoⅤcoding gene （nfi） of E. coli DH5α using λ-Red recombination system and the recombinantstrain was named E. coli DH5α Δnfi. Then shot-gun sequencing of the PaP1genome DNAwas successfully completed using E. coli DH5α Δnfi as the host bacterium. SMRTsequencing revealed7557modified bases （including152m4C and51m6A） in the PaP1genome. These results indicate that the well-known DNA repair protein EndoⅤ ofE. colican mediate the immunity of modified bases containing invasive phage DNA.In conclusion, this work fully annotated the PaP1genome, constructed a new genusnamed “PaP1-like phages” within the Myoviridae family, identified12PaP1structuralprotein coding genes, and reveal a new immunity mechanism for bacteria to resist phageDNA.