P. Aeruginosa is an opportunistic pathogen which usually lives in the soil and surfaces of a watery environment[2]. P. aeruginosa infection could cause a strong neutrophil response inducing significant damage to host tissues even death due to the antibiotic resistance [8]. Phage therapy is considered to be an effective approach for the treatment of P. aeruginosa infections. Here, in this study, we isolated and analyzed a new bacteriophage PaYy-2 that may specially infect Pseudomonas aeruginosa.
Bacteriophage PaYy-2 was isolated from a lake of south China in Yueyang city, Hunan province as described in previous research[9].And a large-scale preparation of bacteriophages was performed from 1 L of liquid culture whcih was reported by Boulanger[1]. Then the phage genome was extracted following the kit instructions of the Lambda Bacteriophage Genomic DNA Rapid Extraction Kit (DN22, Aidlab, China). Genome sequencing was performed by Illumina HiSeq1500 technology (USA). The clean reads were then assembled into a complete genome sequence with the software SOAP denovo [10]. ORFs were predicted by the software GeneMark.hmm[5]. The functions of the ORFs were predicted using BlastP[3] against the non-redundant protein database of the National Center for Biotechnology Information (NCBI). Phylogenetic analysis based on the terminase large subunit was performed using MEGA 6 with the neighbor-joining method[11]. The prediction of tRNA coding regions was performed by the tRNAScan-SE server v.1.21[12] program. Genomic comparisons were performed with the software Easyfig2.2.3[7].
Transmission electron microscopy shown as Fig 3, implies that PaYy-2 had a capsid of approximately 47.9 nm (n=3) in width and a tail length of 144 nm (n=3). Morphologically, the PaYy-2 phage belongs to a genus of the Myoviridae family. Phage PaYy-2 has a 92,348 bp double-stranded DNA genome encoding 168 ORFs, all of the encoding proteins with various sequence similarities to those of the Pseudomonas phages of the Pakpunavirus genus (Min 34%, Max 100%), with a G+C content of 49.33%. We predicted 15 tRNAs on the genome (Fig.1), three more than the Pseudomonas phage YS35, suggesting that the new phage may probably encode tRNAs to circumvent codon usage bias for better adaptability [6]. Based on the BLASTP homology scan[4], 20 structure-related proteins were found in the PaYy2 phage genome including the tail sheath protein, head-tail joining protein unique to the new phage except the two tail fiber proteins, two baseplate proteins, one tape measure protein, one major capsid protein in the reference genome . There are also other specific structural proteins in the new phage consisting of one putative protease subunit, one macro domain protein, one toxic anion resistance protein, one SprT-like family protein, one constituent protein, one portal protein and three more putative unknown structural proteins. In addition, four lysis related proteins were discovered in the new phage, including one putative endolysin, likely playing a predominant role in potentially hydrolysable, and three other common proteinsone CMP/dCMP deaminase with zinc-binding domain, one cell wall hydrolase, one putative phosphoesterase also in the reference phage YS35. The new phage also carried 22 genes coding replication and metabolism proteins. The proteins were only predicted in the genome including one RNA ligase/tail attachment protein, one phosphoribosylpyrophosphate synthetase, one nictotinate phosphoribosyltransferase, two DNA recombination-mediator protein A, one ribonucleoside-diphosphate reductase alpha chain, one ribonucleoside-diphosphate reductase beta subunit, one 3'-phosphatase, 5'-polynucleotide kinase, one nucleotide pyrophosphohydrolase, one RNA ligase, one pyrophosphatase and one putative RNA polymerase except the other nine common proteins in the two phages (Supplement tableS1,S2). The DNA replication and metabolisms predicted proteins suggest that phage has probably its own completely replication and metabolisms ways regulated by the related genes. Annotation of the genome also found two packaging and assembly associated proteins including peptidoglycan binding protein and tail fiber assembly protein that are only present in PaYy-2 phage.
PaYy-2 phage genome sequence is partiely similar to that of Pseudomonas phage YS35 with 68% similarity as shown in the Fig1, total of 23 predicted functional proteins are shared between phage Payy2 and Pseudomonas phage YS35 (85%-99%). To classify the phage species, phylogenetic analysis based on the RNA polymerase protein (Fig.2 a-18.25kD) and the Terminase large subunit protein sequence (Fig.2 b-57.0kD) which are considered conservative in the phage genome were carried out. The RNA polymerase protein of PaYy-2 is homologous to those of Pseudomonas phages SRT6 by 94%, suggesting that PaYy-2 can be a new member of the Pakpunavirus genus within the Myoviridae family. Terminase large subunit protein shows homologous to those of the phages of the Pakpunavirus genus including Pseudomonas phages PAK P1, PAK P2, PAP1 and YS35. The identities of the terminase large subunit proteins of PaYy-2 compared to Pseudomonas phages PAK P1, PAP1, and YS35 of the Pakpunavirus genus is 96%, indicating it may be a new species of the Pakpunavirus genus.
Nucleotide sequence accession numbers The annotated genome sequence for the phage is being submitted to the NCBI nucleotide database as GenBank: MH725810.1.