Complete Genome Sequence And Annotation Of Bacteriophage PaP2, With Biological Characterization Of This Phage

Bacteriophages are viruses of bacteria. They are ubiquitous in nature, and are nowrecognized to be the most numerous entities in the biosphere. Lysogenic phages areimportant vectors for the gene lateral transfer between bacterial strains, and cause a genestream among different organisms, then bring the biological diversity. Lytic phages can lyseand kill the host bacteria, and lysogenic phages also have a lytic period. So that phages arein the hope to be developed as anti-bacteria pharmaceutics. Deep-looking insight into thegenetic background of phage is very important for no matter investigating the interaction ofphage and its host bacteria, and revealing the biological diversity mechanism resulting fromgene horizontal transfer caused by phages, or engineering remodeling of phage andphage-therapy. Now the genomic era has re-focussed researchers' attention onto theimportance of bacteriophages. To date, there are totally 214 completed bacteriophagegenomes available in public databases, and 63 of them were sequenced just last year. In thisdissertation, we are focused on the whole genome sequence and annotation of Pseudomonasaeruginosa phage 2 ( PaP2 ). The results are as follows: 1. The biological characterization of PaP2. The phage PaP2 is a temperate phage ofPseudomonas aeruginosa. Electron microscopy revealed that the phage has an isometrichead (about 52 nm in diameter) and a short tail (about 16 nm in length and 5 nm in width),and it belongs to the Podoviridae family. The optimal multiplicity of infection (MOI) ofPaP2 is about 10, and hosts infected by the phages with an MOI of 10 will produce highestphage titer at 3.5h after infection. One-step growth kinetics of the phage showed that thelatent period is about 90 min, the rise period is about 75 min, and the average burst size isabout 34 phage particles per an infected cell. 2. The general characterization of PaP2 genome. The complete nucleotide sequence of*This work is supported by the Grant from Chongqing City (No.6458) 4第三军医大学博士学位论文phage PaP2 is 43,783 bp. The G+C content of the PaP2 genome averages 45.36%, which issignificantly lower than that of P. aeruginosa. There are six regions with significantlyhigher G+C content than the average, among which two genes, gene 1 and gene 13, havingthe highest G+C content, may be acquired genes by horizontal transfer. The codon usagefrequency analysis showed that the phage PaP2 and its host bacteria have obvious codonusage bias and there is no accordance between them. So it is probably not long revolutiontime for PaP2 to reside in P. aeruginosa. Restriction analysis showed that the genome is alinear dsDNA molecule. The 5'-terminal fragment produced by the digestion of restrictionenzyme AatⅡwas recovered by agarose gel electrophoresis, and then was blunted by S1nuclease digestion. The 5'-terminal fragment was sequenced by primer walking. Thesequencing results showed that the unblunted 5'-terminal fragment has 16 bases more thanthat of the blunted fragment. So the conclusion can be drawn that the phage genome has a5'-protruded cohesive terminal, and the sequence of the cohesive end at the 5'-terminal ofthe genome is: 5'-TCAGGCTATAGTGGTT-3'. 3. The bioinformatics analysis of phage genome. 156 ORFs longer than 100bp arefound out by ORF finder software at NCBI website. Predictions using the GeneMark andSoftberry programs revealed that the phage genome has 58 predicted genes. No potentialtRNA coding regions were found in the phage genome. The predicted genes are tightlyorganized with little space between them, and there are 19 overlapping gene regions withtotal 437 bp, ranging from 1 to 170 bp. Despite absence of homology at the DNA level, 4 ofthe phage-encoded putative proteins show homology to known proteins in the databaseusing blastp program. The function of these 4 genes are respectively: gene 31 codes DNApolymeraseⅠ, gene 54 codes exonuclease ?...