To gain insight into the diversity and origins of antibiotic resistance genes, we identified resistance genes in the soil in an apple orchard using functional metagenomics, which involves inserting large fragments of foreign DNA into Escherichia coli and assaying the resulting clones for expressed functions. Among 13 antibioticresistant clones, we found two genes that encode bifunctional proteins. One predicted bifunctional protein confers resistance to ceftazidime and contains a natural fusion between a predicted transcriptional regulator and a ␤-lactamase. Sequence analysis of the entire metagenomic clone encoding the predicted bifunctional ␤-lactamase revealed a gene potentially involved in chloramphenicol resistance as well as a predicted transposase. A second clone that encodes a predicted bifunctional protein confers resistance to kanamycin and contains an aminoglycoside acetyltransferase domain fused to a second acetyltransferase domain that, based on nucleotide sequence, was predicted not to be involved in antibiotic resistance. This is the first report of a transcriptional regulator fused to a ␤-lactamase and of an aminoglycoside acetyltransferase fused to an acetyltransferase not involved in antibiotic resistance.

Pyrite (FeS ) is the most abundant sulfide mineral on Earth and represents a significant reservoir of reduced iron and sulfur both today and in the geologic past. In modern environments, oxidative transformations of pyrite and other metal sulfides play a key role in terrestrial element partitioning with broad impacts to contaminant mobility and the formation of acid mine drainage systems. Although the role of aerobic micro-organisms in pyrite oxidation under acidic-pH conditions is well known, to date there is very little known about the capacity for aerobic micro-organisms to oxidize pyrite at circumneutral pH. Here, we describe two enrichment cultures, obtained from pyrite-bearing subsurface sediments, that were capable of sustained cell growth linked to pyrite oxidation and sulfate generation at neutral pH. The cultures were dominated by two Rhizobiales species (Bradyrhizobium sp. and Mesorhizobium sp.) and a Ralstonia species. Shotgun metagenomic sequencing and genome reconstruction indicated the presence of Fe and S oxidation pathways in these organisms, and the presence of a complete Calvin-Benson-Bassham CO fixation system in the Bradyrhizobium sp. Oxidation of pyrite resulted in thin (30-50 nm) coatings of amorphous Fe(III) oxide on the pyrite surface, with no other secondary Fe or S phases detected by electron microscopy or X-ray absorption spectroscopy. Rates of microbial pyrite oxidation were approximately one order of magnitude higher than abiotic rates. These results demonstrate the ability of aerobic microbial activity to accelerate pyrite oxidation and expand the potential contribution of micro-organisms to continental sulfide mineral weathering around the time of the Great Oxidation Event to include neutral-pH environments. In addition, our findings have direct implications for the geochemistry of modern sedimentary environments, including stimulation of the early stages of acid mine drainage formation and mobilization of pyrite-associated metals.

Chocolate Pots hot springs (CP) is a unique, circumneutral pH, iron-rich, geothermal feature in Yellowstone National Park. Prior research at CP has focused on photosynthetically driven Fe(II) oxidation as a model for mineralization of microbial mats and deposition of Archean banded iron formations. However, geochemical and stable Fe isotopic data have suggested that dissimilatory microbial iron reduction (DIR) may be active within CP deposits. In this study, the potential for microbial reduction of native CP Fe(III) oxides was investigated, using a combination of cultivation dependent and independent approaches, to assess the potential involvement of DIR in Fe redox cycling and associated stable Fe isotope fractionation in the CP hot springs. Endogenous microbial communities were able to reduce native CP Fe(III) oxides, as documented by most probable number enumerations and enrichment culture studies. Enrichment cultures demonstrated sustained DIR driven by oxidation of acetate, lactate, and H2 . Inhibitor studies and molecular analyses indicate that sulfate reduction did not contribute to observed rates of DIR in the enrichment cultures through abiotic reaction pathways. Enrichment cultures produced isotopically light Fe(II) during DIR relative to the bulk solid-phase Fe(III) oxides. Pyrosequencing of 16S rRNA genes from enrichment cultures showed dominant sequences closely affiliated with Geobacter metallireducens, a mesophilic Fe(III) oxide reducer. Shotgun metagenomic analysis of enrichment cultures confirmed the presence of a dominant G. metallireducens-like population and other less dominant populations from the phylum Ignavibacteriae, which appear to be capable of DIR. Gene (protein) searches revealed the presence of heat-shock proteins that may be involved in increased thermotolerance in the organisms present in the enrichments as well as porin-cytochrome complexes previously shown to be involved in extracellular electron transport. This analysis offers the first detailed insight into how DIR may impact the Fe geochemistry and isotope composition of a Fe-rich, circumneutral pH geothermal environment.