Elucidation Of The Biological Characteristics,Molecular Identification Of Compatible Solutes And Its Mechanism In Response To Salt Stress From Moderately Halophilic Bacteria Brachybacterium Muris

The halophilic microorganism is capable of growing and metabolizing under high salt conditions.Several salt-resistant mechanisms have been found such as the cell morphology,compatible solute,and the transmembrane transport of Na⁺and K⁺in order to cope with the salt stress.With the continuous improvement in the application of compatible solutes and microbial active enzymes in various fields,halophilic microorganisms have attracted widespread attention.Halophilic microorganisms have a wide range of use and are of great significance in the fields of food and pharmaceutical industries,cosmetics industry,heavy metal wastewater treatment and soil bioremediation.Especially as the chassis cells in synthetic biology,halophilic microorganisms have unique advantages in the industrial production of various biopolymers,compatible solutes,hydrolytic enzymes,antioxidants and biosurfactants.Before the research of this paper,a moderately halophilic bacterium Brachybacterium muris(B.muris)from Chaka Salt Lake adapted to the environment containing 10%Na Cl was successfully isolated which can maintain great physiological activities under high concentration of Mg²⁺.However how B.muris can tolerate high concentrations of Mg²⁺has not been explained so far.Therefore,this research focuses on the compatible solutes of B.muris to respond to salt stress and its mechanism of systemic research.The main results acquired are as follows:(1)Compare the cell morphology and cell membrane structure and composition of B.muris under different salinities to explore their relationship with salt stress adaptation.Transmission electron microscopy observations revealed that B.muris cells changed their shape,such as thinning of the cell wall,lengthening of the cells or tending to be elliptical,to cope with the high osmotic pressure environment.In addition,it was found that the proportion of unsaturated fatty acids in the cell membrane of B.muris increased,the content of zymosterol,fecosterol and ergosterol decreased,the content of phosphatidylethanolamine and phosphatidylinositol increased significantly.And the fluidity of the cell membrane of B.muris was also improved under high salinity situation.(2)The intracellular compatible solute molecular structure of the moderately halophilic bacteria B.muris was detected by lactation process and LC-MS.It is inferred that the main compatible solute in B.muris may be L-glutamate.Then the salt-sensitive mutants were constructed by chemical reagent-?-ray combined mutagenesis to verify the speculation.It showed that 2 mmol/L L-glutamate can significantly improve the growth of salt-sensitive mutants under high salt conditions.The results of the intracellular amino acid analysis also showed that the synthesis of L-glutamate in the original strain under high salinity was significantly higher than that under low salinity condition,and the synthesis of L-glutamate in the mutant strain was significantly lower than that of the original strain.It is concluded that L-glutamic acid is one of the main compatible solute of B.muris.(3)Through whole genome scanning and sequencing,the relevant information of the B.muris genome was obtained.GO annotations found that the molecular functions of the B.muris genome had the highest gene abundance.In KEGG analysis,the CDS in the B.muris genome was classified into 40 KEGG functional categories,mainly focusing on global and overview diagrams,carbohydrate metabolism,amino acid metabolism and cell membrane transport functions.It was also found that the B.muris genome contains abundant carbohydrate-active enzymes and two secondary metabolite gene clusters.One of the gene cluster may be related to carotene synthesis,and the function of the other gene cluster is not yet clear,and it is worthy of further excavation and analysis.In addition,specific genes in the B.muris genome that may be related to the regulation of compatible solutes were also further explored and analyzed,including the related metabolic pathways of L-glutamate and L-proline.(4)Using transcriptomics research methods to study the differentially expressed genes of B.muris under high and low salinity conditions,the study found that with low salinity as the control group,several KEGG pathways such as the ribosome in translation process,photosynthesis in energy metabolism and ABC transporter in cell membrane transport were significantly enriched in differentially expressed genes in the high salinity group.There are 128genes of which expression levels are upregulated under high salt stress,and the main functions involved translation and ribosome structure synthesis,inorganic ion transport and metabolism,amino acid transport and metabolism,etc.There are 174 down-regulated genes,and their main functions include amino acid transport and metabolism,inorganic ion transport and metabolism,energy production and transformation and so on.The changes in salt tolerance-related metabolic pathways under different salinities were studied in the meantime,and the results showed that L-glutamate synthetase encoding genes glt B,glt D,ABC transporting ATP binding protein encoding gene glu and sodium cotransporter gene glt S were up-regulated by 2.197,1.491,2.850and 1.301-fold,respectively,which indicated that the synthesis and transport of glutamate are significantly enhanced under high salt stress.The real-time fluorescent quantitative PCR results are also consistent with the results above,proving that L-glutamate is the main compatible solute of B.muris.(5)Finally,this research utilized the hydroponic co-cultivation model to explore the application prospect of this halophilic bacteria in biological repair.The results found that in a high-copper forced water environment,B.muris can alleviate the inhibitory effect of Cu²⁺on wheat roots and buds,which means B.muris mitigated copper toxicity from wheat.It reduced the H₂O₂content in wheat seedling roots by 30.14%,increased the GSH content by 45.96%,and the POD activity was significantly increased by 83.86%.Studies have shown that B.muris may inhibit the toxic effects of copper by increasing the absorption of Mg²⁺in wheat seedlings or promoting the antioxidant effects of wheat,such as stimulating the production of GSH.