| How life adapts to extreme and fluctuation conditions with small genome is one the key obstacles for further understanding serious biological processes,such as the origin of life,the evolution,the boundary of deep biosphere and the robust of synthetic cells.One order of archaea,namely Thermococcales,is abundant and dominant in the deep-sea hydrothermal vent,which is among the most extreme environments on Earth and is supposed to be the window to the early Earth.Moreover,Thermococcales has successful in adapted to the dramatic environmental fluctuations in situ.Most of Thermococcales have growth ranges exceeding the typical ones of normal microorganisms,but relatively small genome size(<2.3 Mbp).One classical research strategy on the adaptation mechanism of Thermococcales is to target the specific responding genes to individual stress.However,hardly any specific gene identified since the gene that responds to one environmental stress often responds to other stresses as well.In this research,we took an alternative approach and have reported a series of responding proteins under different environmental stresses including extreme temperature,pressure,salinity and pH.Our results proved that a common adaptation strategy does exist in Thermococcales.Thermococcales strain A501 with wide-growth-range of temperature,pH and pressure,was isolated from the chimney of a deep-sea hydrothermal vent in Guaymas Basin.Cells of strain A501 are highly motile,irregular cocci occurring singly under light microscope.This isolate is anaerobic chemoheterotroph and utilizes complex carbon sources like yeast extract,peptone and tryptone.Elemental sulfur and cysteine stimulated growth..Compared with most of the other microorganisms,strain A501 had a wider growth ranges of temperature,pH and pressure.An obvious growth was detected over the range of temperature 50-100 °C(optimum growth at 85 °C),pH 4-9(optimum pH 7.0),pressure of 0.1-70 MPa(optimum pressure 0.1-30 MPa).Under the optimal condition(85 °C,atmospheric pressure,pH 7,NaCl concentration of 2.3 %),the doubling time of strain A501 was 48 min approximately.Results of phylogenetically analysis indicated that strain A501 represents a novel species,Thermococcus eurythermalis sp.Nov.To acquire further information about strain A501,complete genome of T.eurythermalis A501 was sequenced using Paired-End strategy with Illumina sequencing platform.The complete genome of T.eurythermalis A501 consists of a circular chromosome of 2.1 Mbp and a plasmid of 3.6 kbp.Based on the genome analysis,A501 was identified as the only Thermococcales isolate containing all the 4 energetic membrane bond complexes,higher copy number of ferrous ion transporter genes and biosynthesis of various compatible solutes including trehalose.These special characters in metabolism could be essential to its environmental adaptation.Global comparative proteomic analysis with iTRAQ(isobaric tags for relative and absolute quantitation)was used to study multi-extreme(heat,cold,acid,alkali,hypo-and hyper-osmotic)adaptation of T.eurythermalis A501.Obtained proteomic data covered 79.8 % of genome-predicted proteins.Statistic result of significant differentially expressed proteins(DEPs)demonstrated that approximately 61.5 % of DEPs may be involved in common adaptation because each of them participated in the responses to more than one stresses.This provided strong evidence to hypothesis that microorganisms have evolved certain common adaptation strategy.Stress responses to extreme pH,temperature,salinity and high hydrostatic pressure(HHP)were closely correlated,while responses to low temperature were most distinct from others.Enrichment of gene ontology(GO)terms showed that T.eurythermalis A501 uses both common and unique strategies to respond to multiple environmental stresses.Biosynthesis and protection of macromolecules,biosynthesis and metabolism of amino acids,and ion transport and binding activity were the most crucial common processes when common adaptation strategy was elucidated in functional level.Moreover,the experimental data have revealed that the cells changed their require on amino acids under different stresses.Compared to other Thermococcales strains,efficient ion transport system of T.eurythermalis A501 may be the key to its wide growth range.Moreover,this study also reported that proton or sodium dependent energetic conversion may play a critical role in limiting the growth range.I discovered that energetic processes enriched in hyper-/hypo-osmotic conditions and further found that sodium-dependent energy conversion could be the key limiting factor for adaptation of T.eurythermalis A501 to salinity and other sodium-depend energetic archaea.Last but not least,unique processes responding to cold,acid,alkali and HHP stresses were identified and this has highlighted the major challenges and limitations cells were facing.Cellular adaptation to HHP is another focuse in this thesis.Multi-omics analysis was conducted to understand the obligate piezophilie,using obligate piezophilic archaea strain P.yayanosii CH1(only 1.7 Mbp of genome)and a series of facultative piezophilic derivative strains of it.Entirely different proteomics responses at both low and high HHP stresses were observed between CH1 and its one derivative namely A1.No large fragments deletion,insertion,or significant epigenetic differences were observed in derivatives.Only 27 site mutations detected in genome and enabled facultative piezophilic mutants to grow under ambient pressure.It showed an easy way to adapt to atmosphere of the hyperthermophilic obligate piezophlie.Major functional differences between facultative piezophilic mutant and CH1 were in transport system,amino acids utilization,redox and DNA repair,as indicated by multi-omics analysis.Further analysis revealed disadvantages of obligate piezophiles living at atmosphere and the absent of the “obligate piezophilic genes”.Survival of CH1 under low pressure was limited by insufficient supply of amino acids,nucleotides and energy,which may due to insufficient transport system.Mutations occurring in ATP synthase,transport,amino acids utilization,phosphate group related genes and defense system in A1 solved the problem of insufficient supply,which leads to the growth under ambient pressure.Moreover,the up-expression of redox enzymes and DNA repair system of A1 also facilitated its growth under atmosphere.Up-expression of redox enzymes helps providing intracellular energy and maintaining intracelluar redox balance while up-expression of DNA repairing related enzymes helps dealing with DNA damage under atmosphere.In addition,the construction of all facultative piezophilic mutants have gone through the cold processes,which indicated that HHP adaptation was accompanied by cold adaptation.This is the first study that examines the global cross-stress response in a wide-growth-range archaea T.eurythermalis A501 based on gene expression,which is entirely different from previous adaptation researches which were based on single stress response.We proposed that the microorganisms have developed certain common adaptation strategy to cope with various environmental stresses and our findings have provided solid evidence to support.Metabolic pathways were distinguished into common adaptation strategy,specifice pathways and unique responding processes according to cross-stress response.The results explained the potential strategy used in multi-extreme adaptation of such an ancient microorganism,with wide growth range and small genome.Multi-omics analysis were conducted to understand the obligate piezophilie P.yayanosii CH1 and its facultative piezophilic derivative mutants,which exposed the potential mechanism on how deep-sea obligate piezophilies could be able to survive on surface.This research has revealed the molecular basis of co-evolution between life and Earth environment,and has provided innovative approach to isolate novel microorganism from deep biosphere. |
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