Purpose: The objectives of this study were to sequence a novel lytic V. parahaemolyticus phage (VPp1) and determine the antibacterial activity of recombinant endolysin (LysVPp1) derived from this phage.
Methods: The phage DNA was extracted from a purified bacteriophage VPp1 and sequenced. Putative endolysin LysVPp1 (gp33) was predicted via nucleotide collection database by BLAST and amplified by PCR with phage VPp1 DNA as the template. The purified PCR products were cloned into vector pET-28a (+) and then transformed into competent cells Rosetta (DE3). Next, the transformants were induced with IPTG and purified. The antibacterial spectrum of bacteriophage VPp1 and LysVPp1 was determined by overlay method and gel diffusion assays, respectively, whereas the antibacterial activity of LysVPp1 was detected with the turbidity reduction method.
Results: The complete VPp1 genome sequence contained a circularly double-stranded DNA of 50,431bp with a total GC content of 41.35%. The genome was predicted to encode 67 gene products and two tRNAs. The average length of the gene is 752bp. In addition, LysVPp1 could lyse 9 out of 12 V. parahaemolyticus strains, showing its relatively broader host spectrum than 3 out of 12 of its preliminary bacteriophage VPp1. Furthermore, in the presence of LysVPp1, the optical density of bacterial cells treated with EDTA decreased by 0.4, showing hydrolysis properties for degrading the outer-membrane of cell envelope.
Significance: Our results lay a significant foundation for the development of novel enzybiotics for inhibiting V. parahaemolyticus.