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Applied and Environmental Microbiology, March 2003, p. 1791-1796, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1791-1796.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Enhanced Accumulation of Cd2+ by a Mesorhizobium sp. Transformed with a Gene from Arabidopsis thaliana Coding for Phytochelatin Synthase

Rutchadaporn Sriprang,1 Makoto Hayashi,1 Hisayo Ono,1 Masahiro Takagi,2 Kazumasa Hirata,3 and Yoshikatsu Murooka1*

Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita-shi,1 Environmental Bioengineering Laboratory, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871,3 Advanced Institute of Science and Technology, Tatsunokuchi, Nomi, Ishikawa 923-1292, Japan2

Received 30 July 2002/ Accepted 26 November 2002

We expressed the Arabidopsis thaliana gene for phytochelatin synthase (PCSAt) in Mesorhizobium huakuii subsp. rengei B3, a microsymbiont of Astragalus sinicus, a legume used as manure. The PCSAt gene was expressed under the control of the nifH promoter, which regulates the nodule-specific expression of the nifH gene. The expression of the PCSAt gene was demonstrated in free-living cells under low-oxygen conditions. Phytochelatin synthase (PCS) was expressed and catalyzed the synthesis of phytochelatins [({gamma}-Glu-Cys)n-Gly; PCs] in strain B3. A range of PCs, with values of n from 2 to 7, was synthesized by cells that expressed the PCSAt gene, whereas no PCs were found in control cells that harbored the empty plasmid. The presence of CdCl2 activated PCS and induced the synthesis of substantial amounts of PCs. Cells that contained PCs accumulated 36 nmol of Cd2+/mg (dry weight) of cells. The expression of the PCSAt gene in M. huakuii subsp. rengei B3 increased the ability of cells to bind Cd2+ approximately 9- to 19-fold. The PCS protein was detected by immunostaining bacteroids of mature nodules of A. sinicus containing the PCSAt gene. When recombinant M. huakuii subsp. rengei B3 established the symbiotic relationship with A. sinicus, the symbionts increased Cd2+ accumulation in nodules 1.5-fold.

* Corresponding author. Mailing address: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita-shi, Osaka 565-0871, Japan. Phone: 81-6-6879-7416. Fax: 81-6-6879-7418. E-mail: murooka{at}bio.eng.osaka-u.ac.jp.

Applied and Environmental Microbiology, March 2003, p. 1791-1796, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1791-1796.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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