PCR detection of a putative manganese oxidation gene (mofA) in environmental samples and assessment of mofA gene homology among diverse manganese‐oxidizing bacteria

Abstract

Nondegenerate PCR primers were designed to amplify a 706‐bp portion of the putative manganese oxidizing factor gene, mofA, from several Leptothrix strains (“L. discophora” SS‐1 and SP‐6, L. cholodnii IMG 7171. and Leptothrix sp. NC‐1). Elec‐trophoresis of amplification products revealed significant amplification only in the strain from which mofA was originally cloned (“L. discophora” SS‐1). Very weak amplification (as witnessed by faint bands in ethidium bromide‐stained agarose gels) was observed with both L. cholodnii IMG 7171 and Leptothrix sp. NC‐1, while no amplification was observed with “L. discophora” SP‐6. In order to determine whether the manganese‐oxidizing genes of Leptothrix spp. were closely related, these primers were used to synthesize a 706‐bp digoxigenin‐labeled mofA probe from “L. discophora” SS‐1 for use in variable stringency hybridization analyses. These analyses showed that the manganese oxidation genes of Leptothrix spp. were closely related to one another, but they are not homologous to the unidentified presumptive manganese oxidation genes from other genera. Degenerate PCR primers were then synthesized that permitted strong amplification of a 200 bp portion of the mofA gene from all of the Leptothrix strains just mentioned. Both sets of primers were used in attempts to amplify the mofA gene from nucleic acids separately extracted from both water and sediment samples from a location in the Sapsucker Woods wetland in Ithaca, NY, near the site of origin of “L. discophora” SS‐1. Only the nondegenerate primers allowed PCR detection of the mofA gene in nucleic acids extracted from wetland water samples. No amplification was seen in nucleic acids extracted from the sediment samples using either set of primers. These results are consistent with previous observations that the growth of Leptothrix spp. was restricted to the ferromanganese surface film and the root zone of Lemna spp. in the water column.