Metal Resistance Determinants In Endophytic/Symbiotic Bacteria Isolated From Robinia Pseudoacacia And Phytoremediation Of Metal Aided By Robinia Plant-rhizobacteria Symbiosis

Due to the ability of nitrogen fixation by rhizobia, rhizobia-legumes symbiosis plays animportant and potential role in aiding phytoremediation of some metal contaminated soils. Toobtain the maximum benefits from legumes assisted by rhizobia for phytoremediation ofmetals, it is critical to have a good understanding of the metal resistant machenism of rhizobiaand the symbiotic plant-rhizobia relationship with metals. In this study, analysis of thegenome sequence and of transcription via RT-PCR combined with transposon genedisruptions revealed the copper or zinc resistance of Agrobacterium tumefaciensCCNWGS0286and Mesorhizobium amorphae CCNWGS0123isolated from the nodules ofRobinia pseudoacacia growing in zinc-lead mine tailing. The phytoremediation assisted bythe two strains was also estimated in this study.The MICs of CCNWGS0286and CCNWGS0123to copper were2.8mM and2.5mM,while to zinc were3.0mM and2.0mM, respectively. Biosorptions of zinc by livingbiomasses of CCNWGS0286and CCNWGS0123were investigated under optimal conditionsat pH5.0, initial metal concentrations of100mg/L and a dose of1.0g/L. Kinetics modelssuggested there was more than one step involved in the Zn²⁺sorption process, while apseudo-second-order model was more suitable to describe the kinetic behavior accurately. Thefittings of Langmuir and Freundlich isotherms to experimental data for Zn²⁺sorption of thetwo strains were not exactly the same. However, they all belonged to the chemisorptionprocess. Moreover, more than70%Zn²⁺could be recovered from Zn²⁺-loaded biomasses atpH1.0. Carbonyl, amino, carboxyl and aromatic groups were responsible for the biosorptionof Zn²⁺by both CCNWGS0286and CCNWGS0123. In addition, cellular deformation,precipitate and damage might be involved in Zn²⁺resistance of the two strains.Plant growth promoting bacterium Agrobacterium tumefaciens CCNWGS0286（176）displayed both high metal resistance and enhanced the growth of Robinia plants in metalcontaminated environment. Genes putatively conferring zinc resistance were identified in thedraft genome of A. tumefaciens176. Among the7,600Tn5insertional mutants of A. tumefaciens176generated by transposon mutagenesis, nine zinc sensitive isolates werescreened individually, most of which showed great and specific sensitivity to Zn²⁺and Cd²⁺.In addition, interruption of a putative oligoketide cyclase/lipid transport protein in mutant15-6reduced IAA synthesis and also showed reduced Zn²⁺and Cd²⁺resistance. Analysis ofthe genome sequence and of transcription via RT-PCR combined with transposon genedisruptions revealed ZntA-4200and transcriptional regulator ZntR1played an important rolein zinc homeostasis of A. tumefaciens176. In greenhouse studies, R. pseudoacacia inoculatedwith A. tumefaciens176displayed a significant increase in biomass production compared toplant biomass without inoculation even in a zinc-contaminated environment. Moreover, IAAoverproduction by A. tumefaciens176with little affected by Zn²⁺was proved to be the mainreason to enhance plant growth under zinc contaminated environment. Interestingly, thedifferences in plant biomass improvement among A. tumefaciens176（ZnRIAA+）, A.tumefaciens C58（ZnRIAA-）, zinc sensitive mutants12-2（ZnSIAA+） and15-6（ZnSIAA-）revealed phytohormones were the dominant factor in enhancing plant growth in contaminatedsoil when compared to the influence of genes encoding zinc resistance determinants.Genes putatively conferring copper resistance were identified in the draft genome of M.amorphae CCNWGS0123（186）.10,000Tn5insertional mutants were generated bytransposon mutagenesis and eight copper sensitive isolates were obtained. Tn5insertion siteswere located on the whole genome of M. amorphae186, which were sorted into threecategories as P-type ATPase, hypothetical protein and others including GTP-binding proteinand ribosomal protein. Analysis of the genome sequence and of transcription via RT-PCRcombined with transposon gene disruptions revealed CopA-6910and CusB played animportant role in copper homeostasis of M. amorphae186. CopA-6910predominantly carriedout Cu+efflux from the cytoplasm to the periplasm, while CusAB was responsible for Cu+detoxification of the periplasm by export to extracellular space. Symbiotic nitrogen fixationwas proved to be the key point that helping plant growth ever under copper contaminatedenvironment. Moreover, M. amorphae186and its two mutants3-42（copA::Tn5） and34-28（ccmX::Tn5） were able to form symbiosis with R. pseudoacacia. However, nodule numbers,total N content, plant biomass and leghemoglobin expression were all reduced wheninoculated with mutant34-28（ccmX::Tn5）. Therefore, mutagenesis of hytothetical gene（ccmX） which is likely related with cbb3-Cox not only affected copper resistance, but alsoaffected symbiotic relationship with R. pseudoacacia. In greenhouse studies, R. pseudoacaciainoculated with M. amorphae186displayed a significant increase in biomass production, totalN content and copper accumulation compared to plant biomass without inoculation even in acopper-contaminated environment. Moreover, copper accumulation in roots was much higher than the accumulation in shoots, indicating the potential and safety when applyingrhizobia-legumes symbiosis for phytoremediation of metals.