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

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

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

^* To whom correspondence should be addressed. Email: nk{at}dmu.dk.

	Abstract

Mercury resistant bacteria may be important players in the mercury biogeochemistry. To assess the potential for mercury reduction of two subsurface microbial communities, resistant subpopulations and their merA genes were characterized by a combined molecular and cultivation-dependent approach. The cultivation method simulated the natural conditions by using polycarbonate membranes as growth support and nonsterile soil slurry as culture medium. Resistant bacteria were pre-grown to microcolonies (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. DGGE analysis of DNA extracted from membranes suggested stimulation of growth of hard-to-culture bacteria during the pre-incubation. A total of 25 different 16S rDNA sequences were observed including , , and -Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. The diversity of isolates obtained by direct plating included 8 different 16S rDNA sequences (- and -Proteobacteria, and Actinobacteria). Partial sequencing of merA of selected isolates led to the discovery of new merA sequences. Using phylum specific merA primers, PCR products were obtained for - and -Proteobacteria 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.