Study On Mscl Gene Function Of Strain Deinococcus Radiodurans And Polyphasic Taxonomy Analysis Of Strain S14-83~T

The bacterium Deinococcus radiodurans has been extensively studied by biologists due to its strong resistance to stress.And the mechanosensitive channel(MS channel)is considered to be the main channel protein of bacteria to respond to changes in membrane tension caused by hypotonic stress.But the relationship between MS channel and stress resistant mechanism of Deinococcus radiodurans has not been studied so far,so we have studied how MscL protein act on influencing resistance stress mechanism of Deinococcus radiodurans.First,since open state of single-channel can be measured by the patch clamp technique,which is a main tool to research mechanosensitive channel,we performed a patch clamp technique study on the MJF465 spheroplast carry recombinant plasmid with D-MscL.And we found that the current of D-MscL channel can be detected with a pressure threshold of 53 mmHg and its channel conductance was 3.37 nS.In order to ensure that the channel can be directly studied in D.radiodurans in future research by patch clamp,we optimized the preparation conditions of DR1 spheroplasts,it showed that after the cells cultured in LB medium to OD600=0.45 and treated it with lysozyme at a concentration of 1200 ug/mL would formed large and smooth spheroplasts.Secondly,we successfully constructed the mscl mutant of Deinococcus radiodurans R1(mDR1)and it showed a decrease on growth in alkali(pH 8.0)and hypertonicity(1%sodium chloride or 0.6 M glucose)environment after lacking mscl gene.And we found that Deinococcus radiodurans changed its morphology from multimeric to monomeric under hypotonic treatment,which is significantly different from the cell death of other bacteria with the same treatment,and the lack of mscl gene seems to contribute to this transformation.In addition,we also found that the tolerance to desiccation of strain mDR1 has also decreased.On this basis,the UV radiation tolerance of strain mDR1 also decreased significantly.A Gram-stain-negative,non-motile,strictly aerobic,coccus-shaped bacterium,designated S14-83^T,was isolated from a soil sample collected from South Shetland Islands of Antarctica.Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain is a novel member of the genus Deinococcus,with Deinococcus alpinitundrae as its closest relative(96.1%similarity).The strain has a genome size of4.4 M,it contains 2 looped nuclear genomes and 5 plasmids.The DNA G+C content of the strain was 61.09 mol%and the major respiratory quinone was MK-8.Major cellular fatty acids were summed feature 3(C_16:1?7c/C_16:1?6c)and C_16:0.Containing aminophospholipids,glycophospholipid and glycolipid as the major polar lipids and there were also some unknown polar lipids.The diagnostic diamino acid in the cell-wall peptidoglycan was ornithine,corroborating the assignment of the strain to the genus Deinococcus.Strain S14-83^T was shown to be extremely resistant to gamma radiation(>10 kGy)and UV light(460 Jm^-2).On the basis of phylogenetic,chemotaxonomic and phenotypic data presented,strain S14-83^T represents a novel species of the genus Deinococcus,for which the name Deinococcus psychrotolerans sp.nov.is proposed.The type strain is S14-83^T(=CCTCC AB 2015449^T=DSM 105285 ^T).A possible mscl gene sequence was found in the longer nuclear genome of the bacterium,and the speculatived MscL channel were consistent with those of other sources.The strain could be a new material for study the function of MscL channel of the genus Deinococcus.