The Mechanisms Involved In Adaptive Evolution Of Phage VMY22 At High Temperatures

Bacteriophage is a kind of non-cellular microorganism and consists of only protein and nucleic acid.Bacteriophage can’t complete its life cycle independently due to lack of intact metabolic system.It is a strictly parasitic organism and has vital characteristics only in the host body Its proliferation process not only affected by environmental factors significantly（temperature,p H,ionic concentration）,but also affected by the host metabolic status.Among the environmental factors,temperature played a key role on life cycle of phage,which has involved in the activity,stability and evolution of phage.A strain of Bacillus cereus 41-22 with a wide growth temperatures range and its phage VMY22 were used as materials in this research.Selective enrichment and domestication were performed at 32℃and 37℃by using a P28 strain which has a optimum temperature at 28℃as start phage strain,and phages with higher infectivity at the corresponding temperatures were obtained.The physiological,biochemical characteristics and genetic genes of the higher temperature adapted phages were compared and analyzed in order to elucidating the mechanisms of high-temperature adaptive evolution of microorganisms.It has found that bacteriophage VMY22 can infect host bacteria and form plaques at 32℃,but cannot form plaques at 37℃.After continuous domestication,the phage P32 with high infectivity at 32℃was obtained,and its titer could reach at 2.83×10¹⁰PFU.The newly obtained 32℃adapted strain P32 was subsequently harvested at 37℃to infect the host bacteria.Unfortunately,the P37 was unable to form plaques at 37℃.According to quantitative real-time PCR results,the titer was a significant decrease at primary infections,with the lowest decrease at 1.12×10⁴ PFU.After continued incubation,the titer gradually increased to 1.62×10⁷ and reached a plateau phase.The results of genome re-sequencing of the phage P32 and P37 showed that no significant changes were found in P32,and few changes occurred in the P37 genome.The changed sites were mainly clustered in ORF15 gene segments which encode an upper collar protein,and it presumed to be involved in high-temperature adaptation.The research preliminarily explored the evolution of Bacillus cereus phage VMY22 under high temperature stress,which laid a foundation for further exploring the adaptive evolution of phage under high temperature stress and the general law of host-phage interaction.