Isolation And Identification Of Pseudomonas Aeruginosa Phage Genomics Research

Pseudomonas aeruginosa (PA) is of multi-drug resistance ability and high adaptive to environment. It's an opportunistic pathogen to human being, and frequently isolated from patients suffering from bacteremia in trauma, burn and immunity-compromised hosts, so it is seriously threaten to human health. Bacteriophage is bacterial virus, which created evolutionally intimate relationship with its host bacterium. First, the lysogenic bacteriophages, can alter the biologic features of host bacterium by its inheritance material, meanwhile, they can change host's inheritance material constituent in the process of the DNA recombination, integration, transposition and incision in host genome, as a result, bringing the microbes gene diversity. This kind of gene horizontal transfer causes a "gene stream" besides plasmids, in bacteria and phages. Secondary, based on the facts of both lytic and lysogenic phages (the later could be induced into lytic growth cycle) having ability to lyses host bacteria, bacteriophage is in the hope to be developed as anti-bacteria pharmaceutics. Deep-looking insight into the genetic background of phage and its host is very important for no matter investigating the interaction of bacteria and phage, and revealing the biological diversity mechanism resulting from gene horizontal transfer caused by phages, or engineering remodeling of bacteriophage and phage-therapy. Recently, the whole genome sequencing of P. aeruginosa PAO 1 had been completed. But the genomics work of P. aeruginosa phages was quite limited. Here we isolated 3 new bacteriophages of P. aeruginosa and conducted whole genome sequencing of 2 phage strains, one being lytic, another being lysogenic, both having a same host. The genome sequencing of one of which, PaP3, has been completed, and the gene functions of this genome has been initiated. Our works covered: I. Three new P. aeruginosa phages were been isolated from sewage by using clinically isolated P. aeruginosa stains as host bacteria. They were named respectively as PaPI (Pseudomonas aeruginosa phage 1) PaP2 and PaP3. PaP1 is lytic phage; PaP2 and PaP3 are lysogenic ones. Electronic microscope showed that PaP1 is tail phage, and PaP2 and PaP3 are of short tail. PaP1 belongs taxonomically to Myoviridae, both PaP2 and PaP3 belong to Pedoviridae. All of them are dsDNA lv * This work is supported by national natural science fundation (30070037) phages. Both PaP2 and PaP3 have a same host PA6. 2. "Flora substitution phenomena? phage-sensitive host bacteria being substituted for phage-resistant ones under the phage's selective pressure, had been observed. The mutation frequency of P. aeruginosa resistant to phage is about 1.4-7.9 × 10-7 determined by end-point-titer method. 3. In order to observe whether nitrogen ion tractus could induce lysogenic bacteriophage to lytic conversion, PaP3 and its lysogenic bacteria were treated with N ion tractus, and then observed if there were clear phage plaque coming out on the plate. But unfortunately there was no change on the form and number of plaques. The experiment got a negative result. 4. Genomic DNA was extracted respectively from phage PaPI and PaP3, then shot-gun library constructed, and picked up randomly clone for genome sequencing. Up to now, the whole genome sequencing of PaP3 has been completed. Restrict enzyme spectrum and sequencing results showed that genome of PaP3 is a dsDNA molecule with blunt end, and consisting of 44,139 bp.