Biological Characterization And Genomic Analysis Of A Novel Bacteriophage Infecting Halomonas Sp.

Viruses are the most abundant biological entities on the earth,and it is estimated that the concentration of the virus in hypersaline environment is as high as 10¹⁰ VLP/mL of water.Bacteriophage is the most dominant group of viruses in all kinds of environment,which can not only regulate the number and community structure of hosts,but also play an important role in the biogeochemical cycle of elements and the flow of food and energy.Especially,there are no predators,such as protozoa,in the hypersaline environment,the virus has become the main biological factor to regulate the host community.Therefore,understanding the virus in hypersaline environment is of great significance for understanding the formation and maintenance of microbial connunication,energy flow and element circulation,host ecological function and so on.Salt mine as a kind of special hypersaline environment,the most research is focused on the understanding prokaryotes in saltmine,but there is a limit to the understanding of the virus.In this study,a novel Halomonas titanicae phage was isolated,which provides a theoretical basis for the development of halovirus resources.Halomonas titanicae H5 preserved in our laboratory was used as the host bacteria,and a lytic bacteriophage was isolated from Yipinglang Salt Mine.The bacteriophage was named YPHTV-1,and its biological characteristics and genome were studied.Transmission electron microscope showed that YPHTV-1 had an icosahedral head with a diameter of 49.12±0.15 nm and a tail with a length of 141.7±0.58 nm,which is a typical Siphoviridae virus.YPHTV-1 has high host specificity and only infects the host strain H5.The one-step growth curve showed that the incubation period of YPHTV-1 was 30 min,the lytic period was 30min,and the burst size was 69 PFU/cell.Virus YPHTV-1 is more suitable to live in a neutral environment,and the optimum pH is 7.0.The survival rate of the virus is almost zero after maintained 20 min at 50?.It was not sensitive to chloroform,ionic and nonionic detergents,but sensitive to protease K,indicating that there was no lipid layer outside the capsid of YPHTV-1.The salinity range of virus YPHTV-1 infecting the host is 2%-12%,and the best infection salinity is 2%-5%.The higher the salinity,the smaller the plaque and the longer the plaque appears.The adsorption rate of YPHTV-1 decreases when the salinity exceeds 25%,so the adsorption rate is affected by salinity.It can maintain stability for a long time at 2%-10%salt concentration,but it is not tolerant to pure water.YPHTV-1 genome is 37980 bp with the GC content of 36.16%and 57 putative open reading frames(ORFs).The total gene coding length is 35412 bp,accounting for 93.24%of the whole genome.By comparing with multiple databases,33 ORFs have known function in the database.All the predicted ORFs were arranged according to modularization,and the genome was divided into five functional parts,consisting of structure,host lytic and lysogen,DNA replication/regulation and nucleotide metabolism,packaging and transcription.Lysogen-related genes in the YPHTV-1 genome may be able to integrate the genome into the host genome under specific conditions.Sequence alignment of the whole genome showed that there was no significant similarity to YPHTV-1,and only a few partial sequences of the virus were similar to YPHTV-1.The similarity between YPHTV-1 and Virgibacillus phage mimir 87 was 75.27%coverage only 26%,and the similar regions were genes encoding DNA replication/regulation and nucleotide metabolism and transcription.secondly,the similarity with Bacillus phage vB_BtS_BMBtp15 was 74.16%(coverage only 25%),the similar regions were genes encoding packaging and structure.The phylogenetic tree of four conserved proteins was constructed.The results showed that HNH endonuclease,terminase large subunit,portal protein and major capsid protein of YPHTV-1 were closely related to Bacillus phage,but far away from Halomonas virus,indicating that YPHTV-1 may have a common ancestor with these phage in evolution.Through the study of the biological characterization and genome of this bacteriophage,it enriches the diversity of viruses in hypersaline environment,deepens the understanding of haloviruses,and provides a virus-host system for understanding interaction between halophiles and haloviruses.