Mechanisms Involved In The Evolution Of Bacteriophage VMY22 With Temperatures Variation

A strain of Bacillus cereus 41-22 with a wide growth temperature range and its bacteriophage VMY22 were used in this study.The phage P₂₈ isolated at 28?was used as the initial phage to infect host bacteria that cultivated at different temperatures,the result phages with high infectivity were isolated and enriched at corresponding temperature.Based on comparison of physiological and biochemical characteristics and genetic changes of isolated phages at different temperatures,we try to illustrate the general law involved in phage temperature-adaptive evolutions.It was found that the phage VMY22 could infect host bacteria and form plaque at temperatures ranging from4?to 32?.We try to use this model to explain the general law of temperature-adaptive evolution of microorganisms.The results showed that the phage VMY22 could infect host bacteria and form plaque at temperatures ranging from 4?to 32?.After continuous domesticated,phage P₄,P₁₅,P₂₈?and P₃₂ with high infectivity at 4,15,28 and 32?were obtained and their titers increased by 1.3×10²,7×10²,1.1×10⁴ and 1.8 folds compared with initiate phage P28,respectively.When the same amount of P₄,P₁₅,P₂₈ and P ₃₂ were infected with the host bacteria cultured at 28°C,the titers of P₄,P₁₅ and P₃₂ decreased by 4.8,3.0 and 666.7 times.In addition,part of the structural and functional genes of the four phages were re-sequenced and analyzed.No any changes can be found in the genes of tail filar,capsid protein and transcription?factor,while the genes encoding ATPase and endolysin were significantly changed.The primary structure of protein was changed by 17%(P₄),7%(P₁₅),5%(P₂₈?),6%(P₃₂)and 30%(P₄),18%(P₁₅),7%(P₂₈?),20%(P₃₂),respectively.This result indicated that the temperature-adaptive evolution of phage VMY22 mainly occurs in the functional protein in the replication and release stages,but not at the adsorption stage.The 3D structure analysis of the protein showed that the?-helix at the C-terminus of the mutant ATPase changed significantly,which in turn changed the hydrophobicity of the enzyme to improve its stability at different temperatures.The mutation of endolysin mainly occurs in the irregularly coiled part,which can respond to the external temperature changes randomly,and improve its stability at different temperatures through the transition from disorder to order.The study also analyzed the transcriptome of the host bacteria at different cultural temperatures,and the results showed that the host bacterias genes showed significant differences in transcription levels at different culture temperatures,especially the difference between 4?and 37?.Meanwhile,the phage infection experiment showed that the phage can almost completely lyse the host bacteria at 4°C(shaking clear);while the host bacteria cultured at 37°C cannot be infected by the phage.This result indicated that bacteriophages can use the host bacteria's metabolic system to serve themselves at low temperatures optimally;while the host bacteria's energy metabolism and pyrimidine synthesis-related genes are down-regulated and the phages which led the phage can not infect the host bacteria at 37°C.This study revealed the evolutionary laws of B.cereus bacteriophage VMY22under temperature stress,and laid a foundation for further exploration of the adaptive evolution of bacteria under temperatures variation and the general laws of host-phage interaction.