The Salinity-Stress Adaptation Of Tetragenococcus Halophilus Revealed By The Regulatory Mechanism Of Glycine Betaine ABC Transporter And Arginine Metabolism Pathway

Tetragenococcus halophilus is a moderate halophilic lactic acid bacterium which has been widely used in fermentation processes,and has been isolated from environments with high-salinity.T.halophilus plays important roles in fermentation processes,for example,fermentation with T.halophilus addition can improve the flavors and amino acid components.Since T.halophilus is a bacterium that can be safely used in food product,to reveal the salt-adaptation mechanism of it can not only shed light on the life forms of halophiles,but also contribute to future biological industry.However,our current knowledge about the salt-adaptation mechanism of T.halophilus is mainly focused on the utilization of compatible solutes,while the detail mechanism such as regulatory network still remains to be illustrated.This thesis focusses on T.halophilus CICC10469,which was isolated from soy sauce,and combines omics study and molecular biology methods to elucidate its regulatory network when subjected to salinity stresses;pathways that may play important roles in the salt stress adaptation process and their regulatory mechanism were characterized;detailed results were listed as followed:The current thesis used the cutting-edge technology to sequence the whole genome of T.halophilus CICC 10469,provided a whole map of genetic information.The genome size of CICC 10469 is 2.38 Mb,and CICC 10469 do not harbor plasmid;33 transposons and 16genomic islands were predicted;the evolutional relationship of all the 20 strains of T.halophilus was analyzed based on whole genome sequences;genes that may play important roles in salinity stress adaptation,such as 19 genes involved in glycine betaine transportation,and genes involving in arginine deiminase pathway(ADI)were predicted.These results provide information about functional genes and genetic structures related to horizontal gene transfer,and contributed to further study about the how this species adapt to salinity stress adaptation.Identified the differentially expressed proteins of T.halophilus under hypo-and hyper-salinity stresses.results found 178 differentially expressed proteins and further clustered these proteins into 45 GO terms and related KEGG pathways,elucidating genes and pathways which play important roles in salinity-stress adaptation processes;for example,results indicated that when subjected to hypo-salinity stress,T.halophilus employs Opu C systems to transport various compatible solutes,while in hyper-salinity adaptation,the BusA system transport glycine betaine to protect the cell from turgor stress;also the ADI pathway may play important roles in hyper-salinity stress condition by generating citrulline,NH₃ and ATP.These results gained a whole map of how T.halophilus response to salt stresses and provided information about post-transcriptional regulation by comparing the results with previous transcriptomics data.Characterized a glycine betaine ABC transporter system BusA,which play key roles in the salt stress response of T.halophilus;its function to counteract with salt stress was verified by being expressed in Escherichia coli;by fusion with reporter genes e GFP,site direct mutation et.al,the promoter sequence and structure of busA were characterized and its transcriptional regulator BusR was identified;the binding pattens of BusR to the promoter sequence of BusA was also verified as 5?-AAA(T/G)TGAC(C/A)(G/A)T(C/A)C-3?,providing possible tools for future strain developments.Identified the arginine metabolism process of T.halophilus,which was shown to be important in salt stress adaptation by proteomic study;the ADI operon was characterized and two non-identical ArgR were found;the promoter sequences and regulatory mechanism of the arginine metabolism processes were elucidated;results showed that ArgR1 mainly act as the negative regulator of the ADI pathway while both regulator can inhibit the arginine biosynthesis;and two residues act as DNA binding domain in ArgR1 and ArgR2,Ser42-Arg43 were identified;the inhibitory effect of ArgR1 on arginine biosynthesis was found to be abolished by arginine addition;the study also provided the evidences of a nova regulatory mechanism on arginine metabolism,and illustrated the regulatory mechanism on arginine metabolism of T.halophilus.The current thesis first revealed the genetic information of T.halophilus by whole genome sequencing,and provided information about the mobile genetic elements of T.halophilus;by combining proteomic study and molecular biology,a draft map of how T.halophilus regulates its gene expression during salinity stresses was drawn,and the transcriptional regulation mechanism of two pathways which play important roles in salinity adaptation process was verified.The results of the current thesis can improve our knowledge about salinity-stress adaptation and contribute to future application on T.halophilus.