Characterization And Functional Studies Of A Genomic Island PYG1 In The Piezophilic Hyperthermophilic Archaeon Pyrococcus Yayanosii

Deep-sea hydrothermal vents environments characterized by a steep temperature gradient,high pressure,and dark.In these environments,a unique ecosystem has been built on the basis of microbial chemoautotrophy.Heterotrophic Thermococcales is a dominant archaeal group in the deep-sea hydrothermal ecosystems.Pyrococcus yayanosii CH1 is a culturable strain with strictly piezophilic hyperthermophilic characteristics.This study mainly focuses on identifying the role of an integrative genomic island PYG1 in the chromosome of P.yayanosii,in adaptation to high temperature and high pressure.Main results of this work are as follows:1.Functional prediction of the genome island:15 putative GIs were identified using bioinformatics approaches in the genome of P.yayanosii.The biggest genomic island PYG1(21.4 kb)can be divided into 5 putative functional modules:The integrase gene is carried by module I which participates in the integration and excision of PYG1;module II carries a putative type IV restriction-modification(RM)system;module III might involve in the metabolisms of amino acids,nucleotides or post-translational modification of protein;A transposase is encoded in module IV and a putative toxin-antitoxin(TA)system contained in module V.2.The mobility of PYG1:PYG1 was specifically integrated at the 3'terminus of the t RNA^Gln gene,which can generate a 43 bp direct repeats(DRs)in its flanking sequence.PYG1 could spontaneously excise from the chromosome at very low frequencies(1.2×10^-8).Integrase PYCH?15110 can accurately mediate the integration and excision of PYG1 at the 43 bp DR sequence.A site-specific integrative genetic tool for P.yayanosii A1,p LMO033,could be utilized for gene complementation experiment.3.PYG1 carrying a type IV TA system:The toxin PYCH?15330(Pyg T)can inhibit the growth of E.coli,and the antitoxin PYCH?15320(Pyg A)can counteract the effects of toxins on the cells.The pyg A and pyg T constitute an Abi E TA system and are organized into an operon.It has been found that pyg T can significantly prolong the lag phase of?pyg A under high pressure.EMSA and DNase I footprinting experiments confirmed that the Pyg A can specifically bind to the Pyg TA promoter region and regulate the transcriptional level of the pyg T gene in vivo.Pyg TA can promote the genetic stability of plasmid in E.coli.4.Adaptability:There is no effect on the growth of?PYG1 under many stress conditions.But high temperature(100?)could inhibit the growth of?PYG1,whereas,the high pressure(80 MPa)could promote the growth of?PYG1.It was further confirmed through genetic analysis that the mutant strain?1526 shared a similar physiological phenotype with?PYG1 under high temperature and high pressure.PYCH?15260 is an unknown functional Yaf J protein.In a synthetic medium which contains 20 amino acids,a huge quantity of Trp can be consumed in?1526.Hypoxanthine,xanthine,guanine and AMP can promote the growth of?1526.We speculated that PYCH?15260 has adenine phosphoribosyltransferase activity involved in purine nucleotide salvage pathways.5.iTRAQ-based proteomics revealed 881 proteins were identified in?1526 and A2 under 80 MPa,of which,178 showed significant differentially expression.These proteins are mainly enriched in the energy metabolism and substance transport and metabolism process.Combined with the function of PYCH?15260,we proposed an explanation:the deletion of PYCH?15260 leads to the disruption of AMP synthesis in the purine nucleotide salvage pathway,which affects ATP and the purines de novo synthesis,and changes in amino acid metabolism associated with these pathways.In this study,the mobility and physiological function of a genomic island PYG1 of the hyperthermophilic piezophilic archaea P.yayanosii from deep-sea hydrothermal vent has been studied for the first time.The modular structure of PYG1 imply that its formation has undergone multiple gene recombination.It was confirmed that the integrase could mediate the spontaneously excision and integration of PYG1.A site-specific integration vector has enriched the genetic operation tools for hyperthermophilic archaea.It was found that the two coding genes of the type IV TA system carried by PYG1 constituted a co-transcribed operon,and the antitoxin protein can specifically bind to the promoter region of the operon.The TA system can regulate the growth of P.yayanosii and improve the genetic stability of plasmids in E.coli.PYG1 and its carrying amindotransferase gene have an effect on the biomass of P.yayanosii under high temperature and high hydrostatic pressure.This study provides a new clue for studying the environmental adaptation mechanisms of piezophilic hyperthermophilic archaea.