Insights into the fundamental physiology of the uncultured Fe-oxidizing bacterium Leptothrix ochracea .

Leptothrix ochracea is known for producing large volumes of iron-oxyhydroxide sheaths that alter wetland biogeochemistry. For over a century, these delicate structures have fascinated microbiologists and geoscientists. Because L. ochracea still resists long-term in vitro culture, the debate regarding its metabolic classification dates back to 1885. We developed a novel culturing technique for L. ochracea using in situ natural waters, and coupled this with single cell genomics and nanoscale secondary ion mass spectrophotometry (nanoSIMS) to probe L. ochracea's physiology. In micro-slide cultures L. ochracea doubled every 5.7 hrs, had an absolute growth requirement for ferrous iron, had the genomic capacity for iron-oxidation, and a branched electron transport chain with cytochromes putatively involved in lithotrophic iron-oxidation. Additionally, its genome encoded several electron transport chain proteins including, a molybdopterin ACIII complex, a cytochrome bd oxidase reductase, and several terminal oxidase genes. L. ochracea contained two key autotrophic proteins in the Calvin Benson Bassham cycle, a Form II ribulose-bis-phosphate carboxylase and a phosphoribulose kinase. L. ochracea also assimilated bicarbonate, although calculations suggest bicarbonate assimilation is a small fraction of its total carbon assimilation. Finally, L. ochracea's fundamental physiology is a hybrid of the chemolithotrophic Gallionella- type iron-oxidizing bacteria and the sheathed, heterotrophic filamentous metal-oxidizing bacteria of the Leptothrix-Sphaerotilus genera. This allows L. ochracea to inhabit a unique niche within the neutrophilic iron seeps. Importance Leptothrix ochracea was one of three groups of organisms Sergei Winogradsky used in the 1880s to develop the hypothesis on chemolithotrophy. L. ochracea continues to resist cultivation and appears to have an absolute requirement for organic rich waters suggesting its true physiology remains unknown. Further, L. ochracea is an ecological engineer, a few L. ochracea cells can generate prodigious volumes of iron-oxyhydroxides changing the ecosystem geochemistry and ecology. Therefore, to determine L ochracea 's basic physiology, we employed new single-cell techniques to demonstrate: L. ochracea oxidizes iron to generate energy and, despite predicted genes for autotrophic growth, L. ochacea assimilates a fraction of the total CO2 that autotrophs do. Although not a true chemolithoautotroph, L. ochracea 's physiological strategy allows it to be flexible, extensively colonize iron-rich wetlands.