Biological Characteristics And Genome Analysis Of Two Cold-active Phages

Napahai wetland is a unique low-latitude, high-altitude and seasonal plateau marsh wetland, which has rich microbial resources and complex diversity composition. The cold-active bacteriophages, VW-6S and VW-6B, infecting the same Pseudomonas fluorescence host, were isolated from Napahai wetland in previous study. In this study, the biological features, genomic analysis and lysis mechanism were characterized.Firstly, the biological characteristics were studied. They belong to the Siphoviridae family based on morphological observation, and were typical cold-active phages which could infect and lyse the host cell at 4℃. Their optimum infection pH were 8.0 (VW-6S) and 10.0 (VW-6B), respectively. The one-step growth curve of VW-6S and VW-6B showed that the latent period were 60 min and 30 min, the rise period were 30 min and 30 min, respectively. The optimal MOI of VW-6S and VW-6B were determined to be 0.1 and 0.01. They were not sensitive to chloroform, but very sensitive to Triton X-100 and SDS. Genomic restriction endonuclease map and SDS-PAGE analysis of capsid protein showed that the genome sizes were about 30-50 kb, but they had obvious different on major structural in size and quantity.Genome analysis of VW-6S and VW-6B revealed that the genome sizes were 37,917 bp and 35,306 bp, G+C content were 56.9%　and 56.76%, and encoded 58 and 52 genes, respectively. More than half of the predicted functional proteins were hypothetical protein. In known function proteins, VW-6S was higher thanVW-6B in both species and quantity, so it was presumed that VW-6B was more dependent on the host than VW-6S. The number of the predicted promoter was two times that of the functional gene, and indicated that the two phages were active. The sequences encoding tRNA were not found, indicating that the expression systems mainly depended on the host. It indicated that some genes may be obtained from the host by gene transfer based on CpG island analysis.To explore the lysis mechanism of the cold-active bacteriophage, the biological information analysis of four proteins (holin, endolysin, i-spanin and o-spanin) involving phage lysis in VW-6S was carried out. Finally, the function of these proteins was identified, and the possible lysis pathway was established. It would provide an important basis for the further study by gene knockout.In conclusion, this study provides a good theoretical and practical basis for the further research on the mechanism of low temperature adaptation, the co-evolution mechanism between phage and host, and the development and utilization of low temperature protein.