Molecular Mechanism Of Interaction Between Deep-sea Thermophilic Bacteriophage GVE2 And Its Host

With the discovery of deep-sea hydrothermal vents and their attendant communities in 1977,researches on hydrothermal vent communities have become attractive interests in the field of oceanography and biology.Deep-sea microbes are the important components of geobiological system.They have received more and more intensive attentions as their importance in the research and application in ecology,resources,environments,and so on. Thesethermophiles which live in vent sites may light the way to the development of new drugs,industrial processes,and other products useful to us all.In recent years,it is found that viruses play important roles in controling the existence of vent biological communities. The viruses infecting vent archeaon vents have been reported at present,but the bacterial viruses(bacteriophages) are seldom studied.Studies on thermophilic bacteriophage suggest that bacteriophages are not only helpful for understanding the control of hydrothermal vent biological communities,but also of great potential for exploring the origin of life.Therefore,this investigation explores the molecular mechanism relationship between thermophilic bacteria with its phage on the basis of our previous studies.In this investigation,the thermophilic bacteriophage GVE2 was obtained from a thermophilic bacterium isolated from deep-sea hydrothermal field in east pacific.Infection mechanism was analysed at the levels of transcription and expression.Random arbitrarily primed PCR(RAP-PCR) was used to isolate and identify differentially expressed genes.14 differential cDNA fragments in total were screened.Five different cDNAs were analyzed by Northern blot,among which 3 were confirmed to be differentially expressed at the transcriptional level.The 17 differentially expressed proteins as revealed by SDS-PAGE and 2-D electrophoresis were subjected to mass spectrometry.Northern blots indicated that the 17 genes were up-regulated or down-regulated after phage infection,which were consistent with those of protein electrophoresis.20 differential genes in total were obtained, among which 13 were confirmed to be up-regulated and 7 genes were down-regulated.Our studies revealed differential genes and proteins related to bacteriophage infection for the first time.These genes and proteins were proteins involved in energy metabolism, regulatory adaptive response,matter and energy transportation and so on.AST mutant was constructed by inserting a karnamycin-resistant gene which encoded thernal-stable kamamycin.Real-time PCR revealed that the GVE2 copies in AST mutant decreased evidently suggesting that AST might play an important role in host immune response against bacteriophage infection.Based on co-immunoprecipitation with AST antibody,chaperonin GroEL was found to interact with AST.The interaction between AST and chaperonin GroEL was confirmed by bacterial two-hybrid system.Single-stranded DNA-binding(SSB) proteins were studied in this paper.SSB proteins are indispensable for survival of almost all known living organisms.Due to their numerous applications in diverse molecular biology and analytical methods,SSB proteins have received increasing research interest.In this investigation,a novel SSB gene was identified from a deep-sea thermophilic bacteriophage Geobacillus virus E2(GVE2) for the first time. The GVE2 SSB protein shared homologies to known SSB proteins from other species. After recombinant expression in E.coli,the purified SSB protein was used for antibody preparation.The Northern and Western blots showed that the GVE2 SSB gene might be an early viral gene.As revealed by DNA binding assay,the recombinant GVE2 SSB protein had single-stranded DNA binding capacity.In this investigation,a bacterial isolate capable of producing electric current during fermentation processes was obtained from a deep-sea hydrothermal field in Atlantic for the first time.The strain,assigned to Shewanella sp.DS1 based on its 16 S rDNA sequence analysis,grew at the optimum temperature of 30℃and the optimum pH 6.5.The results showed that the electric current generated by the strain increased during the first few hours, and eventually reached the maximum(0.29 mA) at about 15 hours after inoculation.The electric current,however,decreased slowly with time increase.