Piezophilic Adaptive Mechanism Of Deep-sea Hyperthermophilic Piezophilic Archaeon Pyrococcus Yayanosii

Deep-sea environment is typically under high hydrostatic pressure wherepiezophiles are the dominant groups in such ecosystem. As the development ofsampling and cultivation techniques, various piezophilic microorganisms have beenisolated from deep-sea, including certain obligated piezophilic species which cannotgrow under ambient pressure. Comparative analysis of the closely related speciesfrom different depth of water, which is inefficient to reveal the hydrostatic pressureregulatory mechanism form microbial individual-level. In this study, an attractivestrategy was proposed, which compare the genomic and transcriptomic differencesand physiological character of obligate piezophilic species and its non-strictpiezophilic derivant, to study the piezophilic mechanisms from a perspective ofenergy conservation and substance metabolism.Pyrococcus yayanosii CH1is the first obligate piezophilic anaerobichyperthermophilic archaeon, which was isolated from the hydrothermal vent“Ashadze”(4,100m depth) located on the Mid-Atlantic Ridge. The optimal highhydrostatic pressure for growth of CH1was52MPa, and the maximal tolerance tohigh hydrostatic pressure surpass to120MPa. This study described the isolation offacultative-piezophilic derivative strain A1from CH1at the laboratory. A1can growwell under the0.1MPa; the optimal high hydrostatic pressure for growth was52MPaand its tolerant ranges of pressure wider than CH1. Moreover, the whole genome ofA1was sequenced, and the genome comparison of CH1and A1showed23mutationswere detected. These differential gene mainly related to cell division regulation,flagellin biosynthesis, and aromatic amino acid permease. The research onfacultative-piezophilic derivative strain A1will help us to reveal the piezophilicadaptation mechanism of deep-sea hyperthermophilic piezophilic archaeon.The growth characteristic of A1at0.1MPa make it a suitable host for thedevelopment of genetic system. We constructed a SimRcassette for overexpression of 3-hydroxy-3-methylglutaryl coenzyme A in A1under the control of strong promoterof glutamate dehydrogenase, which conferred A1the resistance against to simvastatin.A suicide plasmid was constructed (pLMO03) by using the simvastatin as positiveselectable marker and5-FOA as counterselectable marker respectively, and theendogenous pyrF of A1has been disrupted successfully, generating a second host withauxotroph marker (△pyrF) named A2. Moreover, to perform the gene disruption inA1under the limitation of selectable marker, the simR-pyrF cassette was constructed,generating a new vector named pLMO03. Using this markerless cassette, anynon-essential gene of A1could be disrupted and this vector could be used repeatedly.It is reported that single microorganism using formate as the sole carbon sourcecoupled with H2production were only found in Thermococcus onnurineus NA1. Assuch, it represents one of the simplest anaerobic respirations described so far. Amongthe reported Thermococcales, only Thermococcus onnurineus NA1, Thermococcusgammatolerans EJ3, and Pyrococcus yayanosii possess a completed gene cluster (Fmr)for formate oxidation. The whole gene cluster of A1was deleted, then theaccumulation of formate was detected and mutant can’t grow well at0.1MPa.Membrane-bound hydrogenases (Mbh) and cytoplasmic hydrogenases (SHI) arerelated to the energy conservation of Thermococcales. The accumulation of formatealso was detected after the deletion of Mbh and SHI, which indicated that theyinvolved in the formate metabolism.Formate is both as an important intermediate metabolites and a one-carbon unitwas incorporated into IMP in some hyperthermophilies. The transcriptome analysis ofA1under different pressure conditions (0.1MPa vs52MPa) also was performed. Theresults showed that genes related to the IMP biosynthetic pathway and formatemetabolism pathway were down-regulated and up-regulated under0.1MPa,respectively. The distincted transcriptional responses of A1under different pressuresuggest that A1can change the formate metabolic pathway to adapt low-pressureenvironment. Howover, the transcriptome analysis of CH1under different pressureconditions (15MPa vs52MPa) showed that it fail to switch the formate metabolicpathway from IMP biosynthesis to formate-oxidation at15MPa. To further investigate the regulation of pressure to formate oxidation pathwayand IMP biosynthetic pathway, the promoter regions of related genes were analyzed indetail. A putative conserved motif “CnTn5TGn3AAA” was detected, which located inthe promoter regions of some genes from IMP biosynthetic pathway. Using this motifas probe, a pressure induced transcription factor (PTF) encoded by PYCH1749wasisolated and identified by the magnetic bead dependent affinity purification. Moreover,the model for pressure regulation and formate metabolism were builded based ontranscriptome analysis and proteome analysis. When A1cultivated at52MPa, PTFwas expressed and binding to “CnTn5TGn3AAA”, the IMP biosynthetic pathway wasactivated and formate was incorporated into IMP; but when cultivated at0.1MPa,PTF was down-regulated and IMP pathway was inhibited. Then, the formate wasaccumulated and induced the up-regulated of Fmr cluster.In addition, Fmr gene cluster also was up-regulated when A1was cultivatedunder other forms of stress (temperature, pH, and salinity). As mentioned above, suchregulation of formate metabolism in deep-sea hyperthermophilic piezophilic archaeonmay play a key role for the adaptation of multiple forms of stress.