A novel bioremediation system for heavy metals using the symbiosis between leguminous plant and genetically engineered rhizobia

A novel plant-bacterial remediation system for heavy metals (HM) was developed by expression of tetrameric human metallothionein (MTL4) in Mesorhizobium huakuii subsp. rengei B3, a strain which infects and forms nodules on a green manure, Astragalus sinicus. The MTL4 gene was fused to the nifH and nolB promoters, which generated nodule- specific expression of the MTL4 gene. The expression analysis of the MTL4 gene was demonstrated in free-living cells in the presence of Cd²⁺ and Cu²⁺, under the low oxygen condition. The MTL4 under the nifH and nolB promoters was expressed and increased the accumulation of Cd²⁺, but not Cu²⁺ in free-living cells. The expression of the integrated nifH-MTL4 gene in the chromosome of strain B3 was also expressed stably and accumulated Cd²⁺ in the bacterial cells. The MTL4 transcripts were detected by in situ hybridization in bacteroids of mature nodules of A. sinicus containing nifH-MTL4 and nolB-MTL4 fusion gene. Moreover the MTL4 protein was detected by immunostaining. By infection of the recombinant B3, A. sinicus established symbiosis with the recombinant B3 that was grown in Cd²⁺ and Cu²⁺-polluted soils. The symbionts increased Cd²⁺ accumulation in nodules 1.7-2.0-fold, whereas, no significantly increase in Cu²⁺ accumulation was noted.