| Bacteriophages can be detected in almost every biological niche and represent ahuge source of biodiversity and possibly the largest part of the biomass on the planet.Therefore,phages are thought to play major roles in the ecological balance ofmicrobial life and in microbial diversity.This view has gained strong support from thework in the past 30 years or so on viruses in extreme ecosystems.With the discoveryof deep-sea hydrothermal vents and their attendant communities,researches onhydrothermal vent communities have become attractive interests in the field ofoceanography and biology.The isolation and characterization of viruses often lead tonew insights into virus relationships and to a more detailed understanding of thebiochemical environment of their host cells.The increasing number of viral (andcellular) genomes that have been sequenced has directed our attention to utilize thisinformation to rationalize the organization of viral life forms.In general,ourunderstanding of the deep-sea thermophilic bacteriophages is far behind ourknowledge of the terrestrial,mesophilic bacteriophages.The recent discovery of manynovel extremely thermophilic bacteriophage,especially among members isolatedfrom deep-sea hydrothermal vents,is likely to lead to a more complete understandingof not only thermophiles,but also the biochemical adaptations required for the life inextreme environments,and to new insights into both host and virus evolution.Genome sequencing has revealed the important role of horizontal gene transfer inprokaryotes.Comparative viral genomics has created a wealth of information that hasmade it possible to construct gene and genome phylogenies as well as to observecomplex relationships among virus genomes.However,phages that infectthermophilic eubacteria have remained mostly unexplored.Genomes of only a few ofsuch phages have been sequenced completely.The only phage infecting thermophilicbacteria which identified from deep-sea hydrothermal vent that has been characterizedat the molecular level is GVE2,a siphovirus that infects Gebacillus sp.The functionalanalysis of the GVE2 genome and its gene expression strategy has revealed a wealthof new data about transcription and replication regulation,indicating that furtherstudies of phages infecting thermophilic bacteria are warranted.Our studies focus on the molecular biology of host-phage interactions especially at protein level using biochemistry,molecular microbiology and molecular biologyapproaches.In this investigation,several thermophilic bacteriophages were isolatedfrom deep-sea hydrothermal vents in east pacific.Among them,the thermophilicbacteriophage D6E was characterized.D6E was an atypical myovirus with anicosahedral capsid (60 nm in diameter),a tail (16 nm in width,60 in length) and a tailfiber (4 nm in width,60 nm in length).An accurate mode of DNA pyrosequencingwas used to sequence the genome and mass spectrometry was used to accomplish thecomprehensive virion protein survey.Based on sequencing,the phage contained a49335-bp double-stranded genomic DNA.The genome encoded 49 putative openreading frames (ORFs).Functions for D6E gene products were predicted on the basisof similarity to proteins of known functions from diverse phages and bacteria.D6Eencoded four clusters of proteins involved in DNA packaging and headmorphogenesis,lysis and lysogeny,DNA replication and transcription.Advancedbioinformatics techniques were used to identify classical morphogenesis genes.Thestructural genes of D6E,most of which had no similarity to sequences in publicdatabases,were identified by mass spectrometric analysis of purified virions.Therewas also evidence that the capsid protein and portal protein could generate tertiaryand quaternary structures similar to corresponding proteins of other bacteriophages,despite the lack of significant sequence similarity.A comparative analysis of D6Esequence with GVE2,isolated from east pacific,was conducted.The comparisonsconfirmed that these phages were genetic mosaics,with mosaic segments separated bysharp transition in the sequence.The mosaicism provided clear evidence thathorizontal exchange of genetic material myoviruse and siphovirus was a resource forfuture studies of vertical gene transmission.Based on proteomic analysis of thepurified D6E virions,10 structural proteins were revealed,including capsid protein,portal protein,etc.To reveal the protein-protein interaction between bacteriophage and its host,thethermophilic bacteriophage GVE2 was characterized.Twenty of the predicted 62GVE2 ORFs were expressed as recombinant proteins,and the purified recombinantproteins were used for antibody preparations.The expression profiles of these geneswere analyzed by Western blots.Based on GST pull down,Co-IP and nativeSDS-PAGE,several protein complexes associated with host-phage interaction wereobtained.Subsequently the interactions between ORF5 encoding capsid protein (VP371),chaperonin protein (CHG) and aspartate aminotransferase (AST) werecharacterized by Western blot and bacteria two-hybrid system.The results showedthat the VP371 was bound with CHG and CHG bound with AST.However no bindingwas observed between VP371 and AST.Northern blots and Western blots indicatedthat chg and ast genes were up-regulated after phage infection.It is well known thatCHG and AST were involved in energy metabolism,matter and energy transportation,suggesting that they might play very important roles in host immune response againstbacteriophage infection.In this paper,novel portal protein and thymidylate synthase were identifiedfrom the deep-sea thermophilic bacteriophage GVE2 for the first time.Portal protein,located asymmetrically at one of the twelve vertices of the capsid,play very importantroles in viral DNA packaging.The GVE2 portal protein (designated as VP411 protein)shared low similarity to known portal proteins from other species,but they showedhigh similarities in the predicted secondary structures,suggesting that they had thesame function in viral DNA packaging.The Northern blot and Western blot resultsdemonstrated that the vp411 gene was expressed in the late stage of GVE2 infection,implying that it might be a viral late gene.As revealed by immuno-electronmicroscopy,the gold particles were observed in the junction between the phage headand the phage tail when the anti-VP411 IgG was used as the primary antibody,indicating that it had the location in the virion expected of a portal protein.Thymidylate synthase (TS) is essential for de novo synthesis of dTMP and is a keyenzyme involved in DNA synthesis and transcriptional regulation of organisms.Dueto their biologic importance,TSs have been intensively studied.In this investigation,a thermostable TS was identified from the deep-sea thermophilic bacteriophageGeobacillus virus E2 (GVE2).It was demonstrated that the GVE2-TS was highlyhomologous to known TSs and contained five characteristic conserved domains.Thetemporal analyses by Northern and Western blots revealed that the GVE2-TS wastranscribed and expressed early after Geobacillus virus E2 infection,identifying it asan early viral gene.As shown by gel mobility shift assays,the recombinant GVE2-TSprotein had the capacity to bind its own mRNA.Our study presented the first reporton thymidylate synthase from deep-sea thermophilic bacteriophage.
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