| Antarctic ecosystem is one of several major ecosystems on the earth.Antarctic psychrophilic bacteria is one of the important members of microbial community.As decomposers,they are capable of acting as a decomposer to drive multi-element biogeochemical cycles and transformations in Antarctic ecosystems.In the long-term genetic evolution process,Antarctic psychrophiles rely on a unique cold adaptation mechanism to adapt to the cold environment,which plays an important role in maintaining the balance of the ecosystem.However,the adaptation mechanism of psychrophiles to low temperature environment still lacks systematic research,which largely limits the understanding of the relationship between the physiological functions of psychrophiles and the environment.Focusing on the above problems,Psychrobacter sp.ANT206 from Antarctic sea-ice,a new Antarctic psychrophilic bacterium,was selected as the research strain,and its complete genome was sequenced to analyze the genomic components and functional information.Comparative genomics was used to analyze the genetic relationship,metabolic pathway and cold adapted genes.Furthermore,the structure and activity of glutaredoxin system protein in psychrophilic bacterium ANT206 were studied,its function in microbial adaptation to low temperature environment was analyzed,a new way to participate in the regulation of oxidative stress was put forward,and revealed the low temperature adaptation mechanism of Psychrobacter from a new perspective,which can and deepen the understanding of the impact of extreme environmental microorganisms on the ecological environment and promote the development and application of marine environmental microbial resources.In this paper,the complete genome of psychrophilic ANT206 from Antarctica was sequenced and its cold-adaptive genes were analyzed.The results showed that the gene size of ANT206 was 3.00 Mbp,the GC content was 44.17%.KEGG pathway analysis showed that ANT206 had multiple genes compared to pentose phosphate metabolic pathway,pyruvate metabolic pathway,butyrate metabolic pathway,glyoxal and dicarboxylic acid metabolic pathway,oxidative phosphorylation pathway,methane metabolic pathway and glutathione metabolic pathway,indicating that it may play an important role in carbon cycle and energy transformation in the ecological environment.According to the annotation of CAZy database,1,4-benzoquinone reductase of AA6family in ANT206 was involved in the biodegradation of aromatic compounds.It was speculated that ANT206 played an important role in the degradation of toxic substances in the Antarctic ecosystem.The results of comparative genomics showed that the cold-adapted genes in ANT206 include Csp,Hsp,glutaredoxin（Grx）and glutathione reductase（GR）composed of glutaredoxin system and thioredoxin system.Since the enzymes in Grx system can respond to environmental stress,the GR of Grx system in strain ANT206 was studied firstly.gr gene（psgr）was amplified by PCR and analyzed by bioinformatics.PsGR possessed the flavin adenine dinucleotide（FAD）and nicotinamide adenine dinucleotide phosphate（NADPH）binding motifs.Recombinant PsGR（rPsGR）was found to have a molecular mass of approximately 53.5kDa.rPsGR was found to be optimally active at 25℃,it was found to be a cold-adapted enzyme,with 42.2%of its optimal activity remaining at 0℃.Thus,rPsGrx was a novel cold-adapted enzyme.The results of rPsGR kinetics and thermodynamics showed that the substrate affinity of rpsgr for NADPH was higher than that of GSSG,and it was speculated that rPsGR adapted to the low temperature environment by increasing kcat.The results of disk diffusion assay demonstrated that could inhibit oxidative stress induced by H2O2.The important component protein Grx in Grx system was further studied.A gene,encoding a rPsGrx,was cloned and purified.The results of site-directed mutagenesis indicated that C13,P14,and C16 were likely to be crucial for catalysis.CD-spectroscopy was used to ensure that the loss or weakening of the activity of the mutant enzyme was indeed due to point mutations in key amino acid residues.The enzymatic properties of rPsGrx were analyzed.rPsGrx exhibited optimal activity against HED at 25℃,it retained more than 40%of its activity when treated at 0～10℃and was thermal instability.It still had 78%initial activity with 1.0 M Na Cl treatment,indicating that PsGrx had salt tolerance,which was related to the high salinity environment of Antarctic sea ice where the psychrophilic bacteria were located.Furthermore,it was capable of protecting supercoiled DNA from oxidation-induced damage at low temperatures.Compared with Ec Grx,PsGrx had fewer hydrophobic interactions,hydrogen bonds and salt bridges,which increased the conformational flexibility to improve the catalytic ability at low temperature.In the molecular dynamics simulation results,the RMSD and RMSF values of PsGrx were higher at low temperature,indicating PsGrx increased the flexibility of protein structure and maintain the catalytic activity to adapt to low temperature environment.An in-frame deletion mutant of psgrx（Δpsgrx-ANT206）was constructed successfully using allelic replacement method,and the effect of psgrx gene on strain ANT206 was further studied.The mutantΔpsgrx-ANT206 exhibited slower growth rate under low temperature than strain ANT206.Furthermore,the content of malondialdehyde（MDA）and protein carbonylation content were higher in the deletion mutantΔpsgrx-ANT206.Taken together,these results indicated that psgrx was beneficial to the growth of strain ANT206 at low temperature.In order to study the role of psgrx in the resistance to low temperature stress,the yeast two hybrid（Y2H）system was used to screen its interacting proteins,and 26 target proteins interacting with PsGrx were identified.These proteins were involved in stress response,protein secretion,RNA metabolism and protein synthesis.Furthermore,BiFC and Co-IP assays were used to verify the interaction of 6 target proteins include GR,glutathione peroxidase,AhpC,GspI and FtsK with PsGrx.The results showed that PsGrx interacted with GR and AhpC respectively.Moreover,PsGrx participated in glutathione metabolism by enhancing the activity and expression of GR at low temperature.Besides,PsGrx interacted with AhpC and increased trxR expression,and then participated in the reactive oxygen species（ROS）scavenging pathway.That is,PsGrx can resist oxidative stress damage caused by ROS by regulating the above two ways,and strain ANT206 can maintain normal growth and metabolic activities at low temperature.These results provide a new theoretical basis for understanding the adaptation mechanism of Antarctic psychrophilic bacteria to low temperature environment from a new perspective of PsGrx regulating metabolic pathways. |
