Identification And Functional Study Of Envelope-related Genes In Deinococcus Radiodurans

Deinococcus radiodurans?DR?is one of the most tenacious organisms on the Earth.D.radiodurans exhibits exceptional strong tolerance to environmental stresses,including ionizing radiation,oxidative stress,drought,ultraviolet radiation and mitomycin C?MMC?.D.radiodurans has an unusual multi-layered cell envelope which shows the cell envelope characteristics of both Gram-negative bacteria and Gram-positive bacteria.Thus,D.radiodurans is an ideal model for studying the cell envelope and stress resistance of organisms.However,the composition,structural maintenance mechanisms of cell envelope and its roles in bacterial resistance is not clear.In this study,the genes related to DR cell envelope were identified by means of bioinformatics,gene sequencing,proteomics and molecular biology.The functions of these genes in cell envelope integrity and stability,as well as the relationship of them with surface layer?S-layer?protein were investigated.The gene loci containing the tamB?translocation and assembly module B?motif in DR genome was re-sequenced and the corrected ORF was predicted.We found that there were some sequencing errors in the original genome database.The sequences encoding DR₁462,DR₁461 and DR₁460 actually act as an ORF to encode DR₁46T,which is the homologue of TamB.DR₁46T was verified to be an intact cell envelope protein by MS analysis of the cell envelope fraction from the wild type strain.The DR₁46T contains two conserved TamB domains and a C-terminal DUF490 domain,it is different from the reported TamB that containing only one TamB domain and one C-terminal DUF490 domain,such as the E.coli TamB.The whole gene and segmentation of the DR₁46T were knockout to study the phenotypic changes and the function of individual domains.Deficiency of DR₁46T gene leads to the peeling of S layer and the carbohydrate layer,and damage of outer membrane.The bacterial growth rate decreased in DR₁46T mutant??DR₁46T?,indicating that DR₁46T is an important component to maintain the integrity and stability of DR cell envelope.Moreover,the C-terminal DUF490 domain is not crucial for the functions of DR₁46T.Tolerance of ?DR₁46T to osmotic pressure and shear stress was substantially decreased compared with the wild type,indicating that DR₁46T was involved in the rigid structure and resistance of DR.The resistance of ?DR₁46T to H2O2 was enhanced obviously.The content of Mn ion was significantly increased in?DR₁46T cells compared with the wild type,indicating that DR₁46T might be involved in ion transporter system.The Mn/Fe ratio increased more than 2 times in?DR₁46T cells than the wild type,which may be contribute to the enhanced oxidative stress resistance of the mutant.Proteomes of the cell envelope of D.radiodurans wild type and the peeling fraction of ?DR₁46T were identified.The S-layer protein SlpA mutant strain??DR₂577?was constructed.By comparison of subcellular structure and phenotype of the wild type,?DR₁46T,DR₁46T segmentation mutant and ?DR₂577.Proteomic analysis of the cell envelope and peeling fractions in these strains using MS.We found that the 123 kDa SlpA was absent and only its fragments were present in the cell envelope of DR₁46T mutant,indicating that the deletion of DR₁46T could lead to the disruption of the proper assembly of S-layer protein and DR₁46T might be involved in maintenance of the S-layer.In addition,DUF490 domain mutation??DR₁46T-DUF490?had no effect on the expression of SlpA.The cell envelope of ?DR₁46T-DUF490 was still formed,but it cannot be stably attached to the cell surface,suggesting that DUF490 is not the important functional domain for DR₁46T protein.Homologue of TamA,another transport and assembly module component,was not detected in D.radiodurans,however,a BamA??-barrel assembly machinery A?homologue DR₀379 was detected,which was evolutionarily close to the TamA.The DR₀379 knockout mutant strain was constructed and its function was investigated.We found that DR₀379 knockout led to abnormal cell division with the cells clustered into groups.The mutant cell envelope peel off and exhibit extremely sensitive to osmotic pressure.The results indicate that DR₀379 might be one of the important proteins in cell envelope biosynthesis pathway.The relationship between BamA and TamB in DR remains to be studied.In this study,we have identified the genes encoding two cell envelope proteins of D.radiodurans,and investigated their roles in maintenance of cell envelope integrity and the relationships with surface layer.The results of the study not only deepen the understanding of the multi-layered cell envelope structure of the extreme bacterium D.radiodurans as well as the diversity and functions of TAMs in bacteria,but also provide the basis for the further study of bacterial cell envelope proteins.