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Applied and Environmental Microbiology, April 2009, p. 2266-2274, Vol. 75, No. 8
0099-2240/09/$08.00+0     doi:10.1128/AEM.02142-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Different Patterns of Regulation for the Copper and Cadmium Metallothioneins of the Ectomycorrhizal Fungus Hebeloma cylindrosporum{triangledown}

G. Ramesh,1 G. K. Podila,2 G. Gay,3 R. Marmeisse,3 and M. S. Reddy1*

Thapar University, Department of Biotechnology, Bhadson Road, Patiala 147 004, India,1 Department of Biological Sciences, University of Alabama in Huntsville, Huntsville, Alabama 35899,2 Université de Lyon, Université Lyon 1, Ecologie Microbienne, UMR CNRS 5557, USC INRA 1193, Bâtiment Lwoff, 43 Boulevard du 11 Novembre 1918, F-69 622 Villeurbanne Cedex, France3

Received 16 September 2008/ Accepted 8 February 2009

Metallothioneins (MTs) are small cysteine-rich peptides involved in metal homeostasis and detoxification. We have characterized two MT genes, HcMT1 and HcMT2, from the ectomycorrhizal fungus Hebeloma cylindrosporum in this study. Expression of HcMT1 and HcMT2 in H. cylindrosporum under metal stress conditions was studied by competitive reverse transcription-PCR analysis. The full-length cDNAs were used to perform functional complementation in mutant strains of Saccharomyces cerevisiae. As revealed by heterologous complementation assays in yeast, HcMT1 and HcMT2 each encode a functional polypeptide capable of conferring increased tolerance against Cd and Cu, respectively. The expression levels of HcMT1 were observed to be at their maximum at 24 h, and they increased as a function of Cu concentration. HcMT2 was also induced by Cu, but the expression levels were lower than those for HcMT1. The mRNA accumulation of HcMT1 was not influenced by Cd, whereas Cd induced the transcription of HcMT2. Zn, Pb, and Ni did not affect the transcription of HcMT1 or of HcMT2. Southern blot analysis revealed that both of these genes are present as a single copy in H. cylindrosporum. While the promoters of both HcMT1 and HcMT2 contained the standard stress response elements implicated in the metal response, the numbers and varieties of potential regulatory elements were different in these promoters. These results show that ectomycorrhizal fungi encode different MTs and that each of them has a particular pattern of expression, suggesting that they play critical specific roles in improving the survival and growth of ectomycorrhizal trees in ecosystems contaminated by heavy metals.

* Corresponding author. Mailing address: Department of Biotechnology, Thapar University, Bhadson Road, Patiala 147 004, India. Phone: 91-175-2393743. Fax: 91-175-2393738. E-mail: vasu70{at}yahoo.com

{triangledown} Published ahead of print on 20 February 2009.

Applied and Environmental Microbiology, April 2009, p. 2266-2274, Vol. 75, No. 8
0099-2240/09/$08.00+0     doi:10.1128/AEM.02142-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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