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

Abstract

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.

Widespread Occurrence of Bacterial Human Virulence Determinants in Soil and Freshwater Environments

“…Total numbers of bacteria in the soils were determined by acridine orange (AO) direct counting (27). Briefly, a 1:10,000 dilution of 1 g soil in 9 ml sterile Milli-Q water was filtered onto 0.2-m polycarbonate membranes (25 mm in diameter; Nuclepore), and at least 400 bacteria per sample were counted.…”

Mercury Reduction and Methyl Mercury Degradation by the Soil Bacterium Xanthobacter autotrophicus Py2

“…It was long thought that Hg was relatively immobile in the subsurface due to sorption on sediment components such as iron oxides (1), but this assumption has come under scrutiny, since Hg has been unexpectedly found in groundwater at numerous sites, such as the Kirkwood-Cohansey aquifer in New Jersey (15, 16), the Waquoit Bay near Cape Cod, MA (17), and the coasts of California, northern France,. One attractive hypothesis to explain the mobilization of Hg in the subsurface is that bacteria reduce sediment-bound Hg(II) to Hg(0), possibly catalyzed by MerA.Several lines of evidence have indicated that Alphaproteobacteria are active in Hg-and uranium-contaminated sediments (22)(23)(24)(25). We have developed Xanthobacter autotrophicus Py2 (26) as a model system to study mer function in soil Alphaproteobacteria.…”

Presencia de bacterias rizosféricas resistentes a mercurio en suelos del sur de Bolívar, Colombia

“…Esta situación ocurre debido a que relativamente la baja diversidad genética de las comunidades microbianas se aclimata a la presencia del mercurio e induce un cambio en la composición de las mismas comunidades (RASMUSSEN et al, 2008). Además, la tolerancia al mercurio en las comunidades microbianas aclimatadas, varía en respuestas a las diferentes concentraciones del metal en los diferentes ambientes.…”