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Appl Environ Microbiol. 1993 December; 59(12): 4152-4160

Design and application of rRNA-targeted oligonucleotide probes for the dissimilatory iron- and manganese-reducing bacterium Shewanella putrefaciens.

Biology Department, Woods Hole Oceanographic Institution, Massachusetts 02543.

ABSTRACT

A 16S rRNA-targeted oligonucleotide probe specific for the iron (Fe3+)- and manganese (Mn4+)-reducing bacterium Shewanella putrefaciens was constructed and tested in both laboratory- and field-based hybridization experiments. The radioactively labeled probe was used to detect S. putrefaciens in field samples collected from the water column and sediments of Oneida Lake in New York and its major southern tributary, Chittenango Creek. S. putrefaciens was quantified by (i) hybridization of the probe to bulk RNA extracted from field samples and normalization of the S. putrefaciens-specific rRNA to total eubacterial rRNA, (ii) a colony-based probe hybridization assay, and (iii) a colony-based biochemical assay which detected the formation of iron sulfide precipitates on triple-sugar iron agar. The results of field applications indicated that the three detection methods were comparable in sensitivity for detecting S. putrefaciens in water column and sediment samples. S. putrefaciens rRNA was detected in the surficial layers of the lake and creek sediments, but the levels of S. putrefaciens rRNA were below the detection limits in the lake and creek water samples. The highest concentrations of S. putrefaciens rRNA, corresponding to approximately 2% of the total eubacterial rRNA, were detected in the surficial sediments of Chittenango Creek and at a midlake site where the Oneida Lake floor is covered by a high concentration of ferromanganese nodules. This finding supports the hypothesis that metal-reducing bacteria such as S. putrefaciens are important components in the overall biogeochemical cycling of iron, manganese and other elements in seasonally anoxic freshwater basins.

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