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Applied and Environmental Microbiology, June 2008, p. 3795-3803, Vol. 74, No. 12
0099-2240/08/$08.00+0     doi:10.1128/AEM.00049-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cultivation of Hard-To-Culture Subsurface Mercury-Resistant Bacteria and Discovery of New merA Gene Sequences

L. D. Rasmussen,^1, C. Zawadsky,¹ S. J. Binnerup,¹ G. Øregaard,^2, S. J. Sørensen,² and N. Kroer^1*

Department of Environmental Chemistry & Microbiology, National Environmental Research Institute, University of Aarhus, Frederiksborgvej 399, 4000 Roskilde, Denmark,¹ Institute of Biology, University of Copenhagen, Sølvgade 83H, 1307 Copenhagen K, Denmark²

Received 7 January 2008/ Accepted 18 April 2008

Mercury-resistant bacteria may be important players in mercury biogeochemistry. To assess the potential for mercury reduction by two subsurface microbial communities, resistant subpopulations and their merA genes were characterized by a combined molecular and cultivation-dependent approach. The cultivation method simulated natural conditions by using polycarbonate membranes as a growth support and a nonsterile soil slurry as a culture medium. Resistant bacteria were pregrown to microcolony-forming units (mCFU) before being plated on standard medium. Compared to direct plating, culturability was increased up to 2,800 times and numbers of mCFU were similar to the total number of mercury-resistant bacteria in the soils. Denaturing gradient gel electrophoresis analysis of DNA extracted from membranes suggested stimulation of growth of hard-to-culture bacteria during the preincubation. A total of 25 different 16S rRNA gene sequences were observed, including Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and Bacteroidetes. The diversity of isolates obtained by direct plating included eight different 16S rRNA gene sequences (Alpha- and Betaproteobacteria and Actinobacteria). Partial sequencing of merA of selected isolates led to the discovery of new merA sequences. With phylum-specific merA primers, PCR products were obtained for Alpha- and Betaproteobacteria and Actinobacteria but not for Bacteroidetes and Firmicutes. The similarity to known sequences ranged between 89 and 95%. One of the sequences did not result in a match in the BLAST search. The results illustrate the power of integrating advanced cultivation methodology with molecular techniques for the characterization of the diversity of mercury-resistant populations and assessing the potential for mercury reduction in contaminated environments.

Published ahead of print on 25 April 2008.

Present address: National Veterinary Institute, DTU, 1790 Copenhagen V, Denmark.

Present address: Chr. Hansen A/S, INNOVATION Strains, 2930 Hørsholm, Denmark.