Copper And Zinc Resistance Mechanisms Of Sinorhizobium Meliloti And Its Role In Promoting Metal Uptake By Medicago Lupulina

Sinorhizobium meliloti CCNWSX0020,isolated from root nodules of Medicago lupulina growing in mine tailings of Fengxian in the northwest of China,displayed tolerance to high levels of multiple metals and growth promotion of M.lupulina in Cu contaminated soil.Thus,this rhizobium-legume symbiosis has been considered as an efficient tool for bioremediation of heavy metal polluted soils.In this study,RNA-Seq technology and gene deletion mutagenesis were used to analysis of the Cu and Zn resistance mechanisms of S.meliloti CCNWSX00200.The effects of Cu/Zn resistance determinants on the rhizobium-legume symbiosis and subsequently bioremediation of Cu/Zn contaminated soils were also estimated.The gene expression patterns of S.meliloti CCNWSX0020 strain under four conditions were illustrated by transcriptome analysis.Irrespective of the differences of the transcript expressions between Cu-exposed and Zn-exposed samples,there were more transcripts induced by short-term?30 min?metal exposure than by long-term?24 h?metal exposure,suggesting immediate-early response genes play a role in the first line of protection against the heavy metal stress while continuous expression of genes that might play vital roles in the metal resistance mechanisms of S.meliloti CCNWSX0020.Genes encoding P_1B-ATPase,multicopper oxidase,out membrane protein and metal binding protein were significantly up-regulated by Cu or Zn exposure for 24 h,indicating that S.meliloti CCNWSX0020 could employ efflux transport,intracellular sequestration,or transformation for detoxification.Moreover,the effects of different metals stresses on the gene expression patternsof S.meliloti CCNWSX0020 were analyzed.The results showed that 54% of the up-regulated genes of the Zn-treated strain were also up-regulated in the Cu-treated strain,and most of them involved in ion transport,energy production,oxidation-reduction process,carbohydrate and amino acid metabolism,suggesting that S.meliloti CCNWSX0020 defenses against non-specific metal injury through multiple avenues or adjust self-metabolism to the global stress response.In addition,some transcriptional regulators,transporter or molecular proteins were induced specifically by Cu or Zn.These results revealed that the regulation of intracellular Cu and Zn homeostasis are complicated and refined,and the Cu and Zn detoxification or resistance mechanisms of S.meliloti CCNWSX0020 are also different.P_1B-type ATPases are very import for metal ions efflux in the bacteria.There are five P_1B-type ATPases in S.meliloti CCNWSX0020,and two of them?CopA1b and ZntA?were confirmed to be involved in Zn/Cd/Pb resistance by gene deletion and functional complementation.Based on sequence homology analysis,CopA1 b belonged to P_1B-1 ATPases and ZntA belonged to P_1B-2 ATPases.The expression of zntA in S.meliloti CCNWSX0020 of was induced by Zn2+/Cd2+/Pb2+,and zntA deletion led to the hypersensitivity to Zn/Cd/Pb and the increased intracellular concentrations of Zn/Cd/Pb,suggesting S.meliloti CCNWSX0020 Znt A is a typical Zn2+-ATPase playing a crucial role in the efflux of Zn/Cd/Pb.In contrast,the expression of copA1 b was induced by Cu+/Ag+,and copA1 b deletion led to the sensitivity to high levels of Zn/Cd/Pb but not Cu and the increased intracellular concentrations of Zn/Cd/Pb,suggesting S.meliloti CCNWSX0020 CopA1 b belonging to P_1B-1 ATPases could be required for Zn/Cd/Pb homeostasis and detoxification.Six highly up-regulated transcripts involved in Cu and Zn resistance were identified through deletion mutagenesis.?cueO showed great and specific sensitivity to Cu while other mutants showed differing sensitivity to Cu,Zn,Cd and Pb,suggesting that cueO played a specific role in Cu resistance while other genes were all involved in Cu,Zn,Cd and Pb tolerance of S.meliloti CCNWSX00200.The wild-type strain,Cu-sensitive mutant??cueO?,and three both Cu and Zn sensitive mutants??yedYZ,?cusA-like and ?fixH-like?were selected for further study the effects of these metal resistance determinants on bioremediation.Results showed that inoculation with ?cusA-like almost led to loss of symbiotic capacity of M.lupulina to even grow in uncontaminated soil;deletion of cueO severely inhibited infection establishment and nodulation formation with M.lupulina under Cu stress condition;deletion of yedYZ or fixH-like decreased the earlier infection frequency and nodulation with M.lupulina under Cu and Zn stress,but had no influence on the final nodulation and nitrogen fixation.Moreover,the antioxidant enzyme activity and Cu and Zn contents of M.lupulinainoculated with all mutants were lower than those with wild-type strain.These results suggest that heavy metal resistance determinants could promote metal uptake by directly or indirectly influencing the rhizobium-legume symbiosis in metal contaminated soils.