Cellular reductase activity in uncultivated Thiomargarita spp. assayed using a redox-sensitive dye

Abstract

The largest known bacteria, Thiomargarita spp., have yet to be isolated in pure culture, but their large size allows for individual cells to be followed in time course experiments, or to be individually sorted for omics-based investigations. Here we report a novel application of a tetrazolium-based dye that measures the flux of reductase production from catabolic pathways to investigate the metabolic activity of individual cells of Thiomargarita spp. When coupled to microscopy, staining of the cells with a tetrazolium-formazan dye allows for metabolic responses in Thiomargarita spp. to be to be tracked in the absence of observable cell division. Additionally, the metabolic activity of Thiomargarita spp. cells can be differentiated from the metabolism of other microbes in specimens that contain adherent bacteria. The results of our redox-dye-based assay suggests that Thiomargarita is the most metabolically versatile under anoxic conditions where it appears to express cellular reductase activity in response to the electron donors succinate, acetate, citrate, formate, thiosulfate, H2, and H2S. Under hypoxic conditions, formazan staining results suggest the metabolism of succinate, and likely acetate, citrate, and H2S. Cells incubated under oxic conditions showed the weakest formazan staining response, and then only to H2S, citrate, and perhaps succinate. These results provide experimental validation of recent genomic studies of Ca. Thiomargarita nelsonii that suggest metabolic plasticity and mixotrophic metabolism. The cellular reductase response of bacteria attached to the exteriors of Thiomargarita also supports the possibility of trophic interactions between these largest of known bacteria and attached epibionts.\n\nIMPORTANCEThe metabolic potentials of many microorganisms that cannot be grown in the laboratory are known only from genomic data. Genomes of Thiomargarita spp. suggest that these largest of known bacteria are mixotrophs, combining lithotrophic metabolisms with organic carbon degradation. Our use of a redox-sensitive tetrazolium dye to query the metabolism of these bacteria provides an independent line of evidence that corroborates the apparent metabolic plasticity of Thiomargarita observed in recently produced genomes. Finding new cultivation- independent means of testing genomic results is critical to testing genome-derived hypotheses on the metabolic potentials of uncultivated microorganisms.