Emily J. Fleming

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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 microslide cultures L. ochracea doubled every 5.7 h and had an absolute growth requirement for ferrous iron, 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 alternative complex III (ACIII), 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 bisphosphate carboxylase, and a phosphoribulose kinase. L. ochracea also assimilated bicarbonate, although calculations suggest that bicarbonate assimilation is a small fraction of its total carbon assimilation. Finally, L. ochracea 's fundamental physiology is a hybrid of those 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 that Sergei Winogradsky used in the 1880s to develop his hypothesis on chemolithotrophy. L. ochracea continues to resist cultivation and appears to have an absolute requirement for organic-rich waters, suggesting that 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's geochemistry and ecology. Therefore, to determine L. ochracea 's basic physiology, we employed new single-cell techniques to demonstrate that L. ochracea oxidizes iron to generate energy and, despite having predicted genes for autotrophic growth, assimilates a fraction of the total CO ₂ that autotrophs do. Although not a true chemolithoautotroph, L. ochracea 's physiological strategy allows it to be flexible and to extensively colonize iron-rich wetlands.

It is increasingly recognized that diffuse, hydrothermal venting is an important source of iron to the deep-sea that can influence oceanic iron dynamics and abundance. Lithotrophic Fe-oxidizing bacteria (FeOB) are dominant at diffuse hydrothermal vent sites, producing microbial iron mats that are often centimeters or more thick. At present, little is known about in situ Fe-oxidation rates, or accretion rates for iron mats. An in situ productivity chamber was developed that took advantage of the unique mineral morphotypes produced by FeOB to estimate rates of Fe-oxidation and accretion. Chambers were placed at two diffuse vents (1179 and 1300 mbsl) at Lō’ihi Seamount where they were colonized by FeOB for different amounts of time. From this analysis, it was estimated that Fe-oxidation rates could range from 8.2–51.9 × 10 ⁻⁶ mol · hr ⁻¹ , and that iron mats could accrete at around 2.2 cm · yr ⁻¹ . Molecular analysis indicated that the relative abundance of Zetaproteobacteria, a group of known FeOB, accounted for 80–90% of the bacteria colonizing the chambers. There was a distinct difference between populations at the 1179m site (Pohaku), and the 1300m site (North Hiolo Ridge). Microscope slides placed within the productivity chambers were colonized by different morphotypes of FeOB. The cells responsible for one common morphotype that produces a Y-shaped filament were identified as Zetaproteobacteria by use of a small subunit rRNA probe. This work confirms the importance of FeOB in the formation of chemosynthetic iron mats, and provides the first estimates for in situ Fe-oxidation rates and mat accretion rates. Highlights: An in-situ productivity chamber was developed to estimate rates of Fe-oxidation and understand colonization patterns at chemosynthetic iron mats at Lō’ihi Seamount. Fe-oxidation rates ranged from 8.2–51.9 × 10 ⁻⁶ mol ˙ hr ⁻¹ , and it was estimated that the iron mats could accrete at around 2.2 cm ˙ yr ⁻¹ . The iron mat community was dominated by Zetaproteobacteria, whose relative abundance accounted for up to 89% of the microbial community. The community membership that grew during short-term incubations reflected the community composition of nearby microbial mats.