To develop the potential of plants to sequester and accumulate mercurials from the contaminated sites, we engineered a tobacco (Nicotiana tabacum) plant to express a bacterial ppk gene, encoding polyphosphate kinase (PPK), under control of a plant promoter. The designated plant expression plasmid pPKT116 that contains the entire coding region of ppk was used for Agrobacterium-mediated gene transfer into tobacco plants. A large number of independent transgenic tobacco plants were obtained, in some of which the ppk gene was stably integrated in the plant genome and substantially translated to the expected PPK protein in the transgenic tobacco. The presence of Hg2+ did not cause considerable morphological abnormalities in the transgenic tobacco, which grew, flowered, and set seed similarly to the wild-type tobacco on the medium containing normally toxic levels of Hg2+. The ppk-transgenic tobacco showed more resistance to Hg2+ and accumulated more mercury than its wild-type progenitors. These results suggest that ppk-specified polyphosphate has abilities to reduce mercury toxicity, probably via chelation mechanism, and also to accumulate mercury in the transgenic tobacco. Based on the results obtained in the present study, the expression of ppk gene in transgenic tobacco plants might provide a means for phytoremediation of mercury pollution.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Archibald FS, Fridovich I (1982) Investigations of the state of the manganese in Lactobacillus plantarum. Arch Biochem Biophys 215:589–596
Bizily SP, Pugh CL, Summers AO, Meagher RB (1999) Phytoremediation of methylmercury pollution: merB expression in Arabidopsis thaliana confers resistance to organomercurials. Proc Natl Acad Sci USA 96:6808–6813
Bizily SP, Pugh CL, Meagher RB (2000) Phytodetoxification of hazardous organomercurials by genetically engineered plants. Nat Biotechnol 18:213–217
Bizily SP, Kim T, Kandasamy MK, Meagher RB (2003) Subcellular targeting of methylmercury lyase enhances its specific activity for organic mercury detoxification in plants. Plant Physiol 131:463–471
Brunke M, Deckwer WD, Frischmuth A, Horn JM, Lunsdorf H, Rhode M, Rohricht M, Timmis KN, Weppen P (1993) Microbial retention of mercury from waste streams in a laboratory column containing merA gene bacteria. FEMS Microbiol Rev 11:145–152
Dunn T, Gable K, Beeler T (1994) Regulation of cellular Ca2+ by yeast vacuoles. J Biol Chem 269:7273–7278
Gatz C, Frohberg C, Wendenburg R (1992) stringent repression and homogenous de-repression by tetracycline of a modified CaMV 35S promoter in intact transgenic tobacco plants. Plant J 40:397–404
Horsche RB, Rogers SG, Fraley RT (1985) Transgenic plants. Cold Spring Harb Symp Quant Biol 50:433–437
Kulaev IS, Vagabov VM (1983) Polyphosphate metabolism in micro-organisms. Adv Microb Physiol 24:83–171
Kärenlampi S, Schat H, Vangronsveld J, Verkleiji JAC, Lelie D, Mergeay M, Tervahauta AI (2000) Genetic engineering in the improvement of plants for phytoremediation of metal polluted soils. Environ Pollut 107:225–231
Kiyono M, Omura H, Omura T, Murata S, Pan-Hou H (2003) Removal of inorganic mercurials by immobilizied bacteria having mer–ppk fusion plasmids. Appl Microbiol Biotechnol 62:274–278
Kornberg A (1995) Inorganic polyphosphate: toward making a forgotten polymer unforgettable. J Bacteriol 177:491–496
Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18:100–127
Nakano T, Murakami S, Shoji T, Yoshida S, Yamada Y, Sato F (1997) A novel protein with DNA binding activity from tobacco chloroplast nucleotide. The Plant Cell 27:1673–1682
Pan-Hou H, Kiyono M, Kawase T, Omura T, Endo G (2001) Evaluation of ppk-specified polyphosphate as a mercury remedial tool. Biol Pharm Bull 24:1423–1426
Pan-Hou H, Kiyono M, Omura H, Omura T, Endo G (2002) Polyphosphate produced in recombinant Escherichia coli confers mercury resistance. FEMS Microbiol Lett 207:159–164
Rugh CL, Wilde HD, Stack NM, Thompson DM, Summers AO, Meagher RB (1996) Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene. Proc Natl Acad Sci USA 93:3182–3187
Rugh CL, Senecoff JF, Meagher RB, Merkle SA (1998) Development of transgenic yellow poplar for mercury phytoremediation. Nat Biotechnol 16:925–928
Ruiz ON, Hussein HS, Terry N, Daniell H (2003) Phytoremediation of organomercurial compounds via chloroplast genetic engineering. Plant Physiol 132:1344–1352
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Takeda S, Sato F, Ida K, Yamada Y (1990) Characterization of polypeptides that accumulate in cultured Nicotiana tabacum cells. Plant Cell Physiol 31:215–221
von Canstein H, Li Y, Timmis KN, Deckwer W-D, Wagner-Dobler I (1999) Removal of mercury from chloralkali electrolysis wastewater by a mercury-resistant Pseudomonas putida strain. Appl Environ Microbiol 65:5279–5284
von Canstein H, Li Y, Wagner-Dobler I (2001) Long-term performance of bioreactors cleaning mercury-contaminated wastewater and their response to temperature and mercury stress and mechanical perturbation. Biotechnol Bioeng 74:212–219
Wagner-Döbler I, von Canstein H, Li Y, Timmis KN, Deckwer W-D (2000) Removal of mercury from chemical wastewater by microorganisms in technical scale. Environ Sci Technol 34:4628–4634
We are grateful to Prof. K. Yazaki of Kyoto University for the gift of binary vector pHM6 and A. tumefaciens LBA4404 strain, and for valuable suggestions on the construction of genetically engineered tobacco plants. The wild-type tobacco (N. tabacum cv. Samsun NN) was a generous gift from Japan Tobacco. We also thank C. Ishikawa, R. Isekawa, T. Fuke, H. Hirata, and T. Kawahara of this university for their technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research (B) (No. 13450216 and 16360267) and Grant-in-Aid for Young Scientists (B) (No. 17790103) from the Ministry of Education, Science, and Culture, Japan.
About this article
Cite this article
Nagata, T., Kiyono, M. & Pan-Hou, H. Engineering expression of bacterial polyphosphate kinase in tobacco for mercury remediation. Appl Microbiol Biotechnol 72, 777–782 (2006). https://doi.org/10.1007/s00253-006-0336-3
- Transgenic Tobacco
- Mercury Resistance
- Polyphosphate Kinase