Biological Characteristics And Genomics Study Of Three Plague Bacteriophages Isolated From Yunnan Squirrel

ObjectiveSquirrel,the host animal of plague in Yunnan Province,was captured and its plague phage was isolated.The biological characteristics and genomics of the isolated plague phage were studied.To provide a basis for exploring the law of synergetic genetic evolution between plague phage and Yersinia pestis,and to provide scientific support for plague prevention and control in Yunnan in the future.MethodsFrom 2015 to 2018,at 5 plague monitoring sites in Mile City,Xinping County,Jianchuan County,Eryuan County and Yongshan County in Yunnan Province,the mouse trapping method and the 5m wire method were used to collect the pine and mouse samples.After anatomy A specimen of the cecum was taken,and the plague phage in the specimen was separated by a double-layer plate method.Take the isolated plague phage as the research object,use 10-fold dilution method and double-layer agar plate method to separate and purify it,and record its plaque morphology;use transmission electron microscope to observe its morphological characteristics;determine its host spectrum and one-step growth Curve,optimal multiplicity of infection,and biological characteristics such as sensitivity to chloroform,ethanol,temperature,p H,and ultraviolet light.Extract the DNA with the λ phage nucleic acid extraction kit and submit it to the sequencing company for gene sequencing,and perform genomic analysis on the gene sequence such as protein function annotation and kinship comparison through NCBI,RAST and other websites and CG View,MEGA and other software.Results1.A total of 4 strains of plague bacteriophage were isolated from the 10 captured Matsutake books,named MLS140,XPS6,YSS153 and YSS157.Since YSS153 was found to be the same as YSS157 in the follow-up study,three plague phages of MLS140,XPS6 and YSS153 were selected for research.2.Through transmission electron microscopy,it was found that the plague bacteriophage MLS140,XPS6 and YSS153 belong to the Myoviridae family.3.The plague bacteriophage MLS140,XPS6 and YSS153 are broad-spectrum phages,and the EV76 plague vaccine strain can be lysed at room temperature(18-22°C),28°C and 37°C,but they are lysed at 28°C and 37°C It is stronger than the cleavage at room temperature(18-22℃).In addition to the above three bacteriophages that can lyse Yersinia pestis,Yersinia pestis phage MLS140 can also lyse Shigella Soxhlete and Shigella flexneri type 2a;and Yersinia pestis phage XPS6 can also lyse type 2a and X variants of flexneria flexneri Gergella and Escherichia coli(ATCC25922);Yersinia pestis phage YSS153 can also lyse part of Escherichia coli and Shigella flexneri X variants.4.As the results of preliminary experiments showed that the basic biological characteristics of the plague bacteriophage MLS140,XPS6 and YSS153 were similar and relatively common,one strain of plague bacteriophage(MLS140)was randomly selected to conduct a relatively complete study of the basic biological characteristics.The results showed that the plaque growth status of MLS140 was better,with a diameter of about 1.5-2mm;the optimal multiplicity of infection was 0.1;the lysis period was about 30 minutes;it was more sensitive to ultraviolet rays and ethanol,but not to chloroform;when the temperature was higher than 70 When the temperature is too acid or too alkaline,the phage will be inactivated.5.The genome sizes of plague phage MLS140,XPS6 and YSS153 are relatively close,with an average size of 31875 bp and an average GC content percentage of 51.5%.The GC content percentage of the non-coding region is significantly lower than that of the coding region.The coding region is rich in AT sequences.6.Since no lysogenic module was found in the gene prediction results of bacteriophages MLS140 and YSS153,these two phages were preliminarily determined to be lytic phages;and the gene prediction results of phage XPS6 showed that its predicted gene g22 encodes a lysogenic switch protein,This is one of the evidences suggesting that XPS6 is a lysogenic phage.7.The Yersinia pestis phage MLS140,XPS6 and YSS153 are closely related to the pestilence phage L-413 C.Based on the results of the host profile determination,the above three phage strains are preliminarily deduced as the pestilence phage.In addition,the homology between the two is high(>90%),so it is believed that the three strains of plague bacteriophages are closely related.ConclusionThis study found that the bacteriophages MLS140,XPS6 and YSS153 are all broad-spectrum Yersinia pestis phages of the Myoviridae family,and temperature has a certain influence on their lysis characteristics,and they are considered to be more suitable for living in the intestine of the host animal;and it is preliminarily deduced that XPS6 is Lysogenic phages,MLS140 and YSS153 are lytic phages.This enriches the plague phage gene pool,provides important clues for the study of the conservation mechanism of the Yersinia pestis phage in the ecological environment,and provides indirect evidence for the discovery of the law of cooperative genetic evolution between the plague phage and the Yersinia pestis,which will provide future development for Yunnan The plague prevention and control work of the People’s Republic of China provides scientific guarantee and has the value of continuing in-depth research.