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ABSTRACT Leptothrix ochracea creates distinctive iron-mineralized mats that carpet streams and wetlands. Easily recognized by its iron-mineralized sheaths, L. ochracea was one of the first microorganisms described in the 1800s. Yet it has never been isolated and does not have a complete genome sequence available, so key questions about its physiology remain unresolved. It is debated whether iron oxidation can be used for energy or growth and if L. ochracea is an autotroph, heterotroph, or mixotroph. To address these issues, we sampled L. ochracea-rich mats from three of its typical environments (a stream, wetlands, and a drainage channel) and reconstructed nine high-quality genomes of L. ochracea from metagenomes. These genomes contain iron oxidase genes cyc2 andmtoA, showing that L. ochracea has the potential to conserve energy from iron oxidation. Sox genes confer potential to oxidize sulfur for energy. There are genes for both carbon fixation (RuBisCO) and utilization of sugars and organic acids (acetate, lactate, and formate). In silico stoichiometric metabolic models further demonstrated the potential for growth using sugars and organic acids. Metatranscriptomes showed a high expression of genes for iron oxidation; aerobic respiration; and utilization of lactate, acetate, and sugars, as well as RuBisCO, supporting mixotrophic growth in the environment. In summary, our results suggest that L. ochracea has substantial metabolic flexibility. It is adapted to iron-rich, organic carbon-containing wetland niches, where it can thrive as a mixotrophic iron oxidizer by utilizing both iron oxidation and organics for energy generation and both inorganic and organic carbon for cell and sheath production. IMPORTANCEWinogradsky's observations of L. ochracea led him to propose autotrophic iron oxidation as a new microbial metabolism, following his work on autotrophic sulfur-oxidizers. While much culture-based research has ensued, isolation proved elusive, so most work on L. ochracea has been based in the environment and in microcosms. Meanwhile, the autotrophic Gallionella became the model for freshwater microbial iron oxidation, while heterotrophic and mixotrophic iron oxidation is not well-studied. Ecological studies have shown that Leptothrix overtakes Gallionella when dissolved organic carbon content increases, demonstrating distinct niches. This study presents the first near-complete genomes of L. ochracea, which share some features with autotrophic iron oxidizers, while also incorporating heterotrophic metabolisms. These genome, metabolic modeling, and transcriptome results give us a detailed metabolic picture of how the organism may combine lithoautotrophy with organoheterotrophy to promote Fe oxidation and C cycling and drive many biogeochemical processes resulting from microbial growth and iron oxyhydroxide formation in wetlands. 

ABSTRACT The iron-oxidizing Gallionellaceae drive a wide variety of biogeochemical cycles through their metabolisms and biominerals. To better understand the environmental impacts of Gallionellaceae, we need to improve our knowledge of their diversity and metabolisms, especially any novel iron oxidation mechanisms. Here, we used a pangenomic analysis of 103 genomes to resolve Gallionellaceae phylogeny and explore their genomic potential. Using a concatenated ribosomal protein tree and key gene patterns, we determined Gallionellaceae has four genera, divided into two groups: iron-oxidizing bacteria (FeOB)Gallionella,Sideroxydans, andFerriphaseluswith iron oxidation genes (cyc2,mtoA) and nitrite-oxidizing bacteria (NOB)CandidatusNitrotoga with the nitrite oxidase genenxr. The FeOB and NOB have similar electron transport chains, including genes for reverse electron transport and carbon fixation. Auxiliary energy metabolisms, including S oxidation, denitrification, and organotrophy, were scattered throughout the FeOB. Within FeOB, we found genes that may represent adaptations for iron oxidation, including a variety of extracellular electron uptake mechanisms. FeOB genomes encoded more predictedc-type cytochromes than NOB genomes, notably more multihemec-type cytochromes (MHCs) with >10 CXXCH motifs. These include homologs of several predicted outer membrane porin-MHC complexes, including MtoAB and Uet. MHCs efficiently conduct electrons across longer distances and function across a wide range of redox potentials that overlap with mineral redox potentials, which can expand the range of usable iron substrates. Overall, the results of pangenome analyses suggest that the Gallionellaceae generaGallionella,Sideroxydans, andFerriphaselushave acquired a range of adaptations to succeed in various environments but are primarily iron oxidizers. IMPORTANCENeutrophilic iron-oxidizing bacteria (FeOB) produce copious iron (oxyhydr)oxides that can profoundly influence biogeochemical cycles, notably the fate of carbon and many metals. To fully understand environmental microbial iron oxidation, we need a thorough accounting of iron oxidation mechanisms. In this study, we show the Gallionellaceae FeOB genomes encode both characterized iron oxidases as well as uncharacterized multiheme cytochromes (MHCs). MHCs are predicted to transfer electrons from extracellular substrates and likely confer metabolic capabilities that help Gallionellaceae occupy a range of different iron- and mineral-rich niches. Gallionellaceae appear to specialize in iron oxidation, so it would be advantageous for them to have multiple mechanisms to oxidize various forms of iron, given the many iron minerals on Earth, as well as the physiological and kinetic challenges faced by FeOB. The multiple iron/mineral oxidation mechanisms may help drive the widespread ecological success of Gallionellaceae. 

ABSTRACT On the roots of wetland plants such as rice, Fe(II) oxidation forms Fe(III) oxyhydroxide-rich plaques that modulate plant nutrient and metal uptake. The microbial roles in catalyzing this oxidation have been debated and it is unclear if these iron-oxidizers mediate other important biogeochemical and plant interactions. To investigate this, we studied the microbial communities, metagenomes, and geochemistry of iron plaque on field-grown rice, plus the surrounding rhizosphere and bulk soil. Plaque iron content (per mass root) increased over the growing season, showing continuous deposition. Analysis of 16S rRNA genes showed abundant Fe(II)-oxidizing and Fe(III)-reducing bacteria (FeOB and FeRB) in plaque, rhizosphere, and bulk soil. FeOB were enriched in relative abundance in plaque, suggesting FeOB affinity for the root surface. Gallionellaceae FeOBSideroxydanswere enriched during vegetative and early reproductive rice growth stages, while aGallionellawas enriched during reproduction through grain maturity, suggesting distinct FeOB niches over the rice life cycle. FeRBAnaeromyxobacterandGeobacterincreased in plaque later, during reproduction and grain ripening, corresponding to increased plaque iron. Metagenome-assembled genomes revealed that Gallionellaceae may grow mixotrophically using both Fe(II) and organics. TheSideroxydansare facultative, able to use non-Fe substrates, which may allow colonization of rice roots early in the season. FeOB genomes suggest adaptations for interacting with plants, including colonization, plant immunity defense, utilization of plant organics, and nitrogen fixation. Taken together, our results strongly suggest that rhizoplane and rhizosphere FeOB can specifically associate with rice roots, catalyzing iron plaque formation, with the potential to contribute to plant growth. IMPORTANCEIn waterlogged soils, iron plaque forms a reactive barrier between the root and soil, collecting phosphate and metals such as arsenic and cadmium. It is well established that iron-reducing bacteria solubilize iron, releasing these associated elements. In contrast, microbial roles in plaque formation have not been clear. Here, we show that there is a substantial population of iron oxidizers in plaque, and furthermore, that these organisms (SideroxydansandGallionella) are distinguished by genes for plant colonization and nutrient fixation. Our results suggest that iron-oxidizing and iron-reducing bacteria form and remodel iron plaque, making it a dynamic system that represents both a temporary sink for elements (P, As, Cd, C, etc.) as well as a source. In contrast to abiotic iron oxidation, microbial iron oxidation results in coupled Fe-C-N cycling, as well as microbe-microbe and microbe-plant ecological interactions that need to be considered in soil biogeochemistry, ecosystem dynamics, and crop management. 

We present an efficient, effective, and economical approach, named E3technology, for proteomics sample preparation. By immobilizing silica microparticles into the polytetrafluoroethylene matrix, we develop a robust membrane medium, which could serve as a reliable platform to generate proteomics-friendly samples in a rapid and low-cost fashion. We benchmark its performance using different formats and demonstrate them with a variety of sample types of varied complexity, quantity, and volume. Our data suggest that E3technology provides proteome-wide identification and quantitation performance equivalent or superior to many existing methods. We further propose an enhanced single-vessel approach, named E4technology, which performs on-filter in-cell digestion with minimal sample loss and high sensitivity, enabling low-input and low-cell proteomics. Lastly, we utilized the above technologies to investigate RNA-binding proteins and profile the intact bacterial cell proteome. 

Abstract Sideroxydans sp. CL21 is a microaerobic, acid-tolerant Fe(II)-oxidizer, isolated from the Schlöppnerbrunnen fen. Since the genome size of Sideroxydans sp. CL21 is 21% larger than that of the neutrophilic Sideroxydans lithotrophicus ES-1, we hypothesized that strain CL21 contains additional metabolic traits to thrive in the fen. The common genomic content of both strains contains homologs of the putative Fe(II) oxidation genes, mtoAB and cyc2. A large part of the accessory genome in strain CL21 contains genes linked to utilization of alternative electron donors, including NiFe uptake hydrogenases, and genes encoding lactate uptake and utilization proteins, motility and biofilm formation, transposable elements, and pH homeostasis mechanisms. Next, we incubated the strain in different combinations of electron donors and characterized the fen microbial communities. Sideroxydans spp. comprised 3.33% and 3.94% of the total relative abundance in the peatland soil and peatland water, respectively. Incubation results indicate Sideroxydans sp. CL21 uses H2 and thiosulfate, while lactate only enhances growth when combined with Fe, H2, or thiosulfate. Rates of H2 utilization were highest in combination with other substrates. Thus, Sideroxydans sp. CL21 is a mixotroph, growing best by simultaneously using substrate combinations, which helps to thrive in dynamic and complex habitats. 

ABSTRACT Sideroxydans lithotrophicus ES-1 grows autotrophically either by Fe(II) oxidation or by thiosulfate oxidation, in contrast to most other isolates of neutrophilic Fe(II)-oxidizing bacteria (FeOB). This provides a unique opportunity to explore the physiology of a facultative FeOB and constrain the genes specific to Fe(II) oxidation. We compared the growth of S. lithotrophicus ES-1 on Fe(II), thiosulfate, and both substrates together. While initial growth rates were similar, thiosulfate-grown cultures had higher yield with or without Fe(II) present, which may give ES-1 an advantage over obligate FeOB. To investigate the Fe(II) and S oxidation pathways, we conducted transcriptomics experiments, validated with reverse transcription-quantitative PCR (RT-qPCR). We explored the long-term gene expression response at different growth phases (over days to a week) and expression changes during a short-term switch from thiosulfate to Fe(II) (90 min). The dsr and sox sulfur oxidation genes were upregulated in thiosulfate cultures. The Fe(II) oxidase gene cyc2 was among the top expressed genes during both Fe(II) and thiosulfate oxidation, and addition of Fe(II) to thiosulfate-grown cells caused an increase in cyc2 expression. These results support the role of Cyc2 as the Fe(II) oxidase and suggest that ES-1 maintains readiness to oxidize Fe(II), even in the absence of Fe(II). We used gene expression profiles to further constrain the ES-1 Fe(II) oxidation pathway. Notably, among the most highly upregulated genes during Fe(II) oxidation were genes for alternative complex III, reverse electron transport, and carbon fixation. This implies a direct connection between Fe(II) oxidation and carbon fixation, suggesting that CO 2 is an important electron sink for Fe(II) oxidation. IMPORTANCE Neutrophilic FeOB are increasingly observed in various environments, but knowledge of their ecophysiology and Fe(II) oxidation mechanisms is still relatively limited. Sideroxydans isolates are widely observed in aquifers, wetlands, and sediments, and genome analysis suggests metabolic flexibility contributes to their success. The type strain ES-1 is unusual among neutrophilic FeOB isolates, as it can grow on either Fe(II) or a non-Fe(II) substrate, thiosulfate. Almost all our knowledge of neutrophilic Fe(II) oxidation pathways comes from genome analyses, with some work on metatranscriptomes. This study used culture-based experiments to test the genes specific to Fe(II) oxidation in a facultative FeOB and refine our model of the Fe(II) oxidation pathway. We gained insight into how facultative FeOB like ES-1 connect Fe, S, and C biogeochemical cycling in the environment and suggest a multigene indicator would improve understanding of Fe(II) oxidation activity in environments with facultative FeOB. 

Twisted stalks are morphologically unique bacterial extracellular organo-metallic structures containing Fe(III) oxyhydroxides that are produced by microaerophilic Fe(II)-oxidizers belonging to the Betaproteobacteria and Zetaproteobacteria. Understanding the underlying genetic and physiological mechanisms of stalk formation is of great interest based on their potential as novel biogenic nanomaterials and their relevance as putative biomarkers for microbial Fe(II) oxidation on ancient Earth. Despite the recognition of these special biominerals for over 150 years, the genetic foundation for the stalk phenotype has remained unresolved. Here we present a candidate gene cluster for the biosynthesis and secretion of the stalk organic matrix that we identified with a trait-based analyses of a pan-genome comprising 16 Zetaproteobacteria isolate genomes. The “ s talk f ormation in Z etaproteobacteria” (sfz) cluster comprises six genes ( sfz1-sfz6 ), of which sfz1 and sfz2 were predicted with functions in exopolysaccharide synthesis, regulation, and export, sfz4 and sfz6 with functions in cell wall synthesis manipulation and carbohydrate hydrolysis, and sfz3 and sfz5 with unknown functions. The stalk-forming Betaproteobacteria Ferriphaselus R-1 and OYT-1, as well as dread-forming Zetaproteobacteria Mariprofundus aestuarium CP-5 and Mariprofundus ferrinatatus CP-8 contain distant sfz gene homologs, whereas stalk-less Zetaproteobacteria and Betaproteobacteria lack the entire gene cluster. Our pan-genome analysis further revealed a significant enrichment of clusters of orthologous groups (COGs) across all Zetaproteobacteria isolate genomes that are associated with the regulation of a switch between sessile and motile growth controlled by the intracellular signaling molecule c-di-GMP. Potential interactions between stalk-former unique transcription factor genes, sfz genes, and c-di-GMP point toward a c-di-GMP regulated surface attachment function of stalks during sessile growth. 

Iron (Fe) oxidation is one of Earth’s major biogeochemical processes, key to weathering, soil formation, water quality, and corrosion. However, our understanding of microbial contribution is limited by incomplete knowledge of microbial iron oxidation mechanisms, particularly in neutrophilic iron oxidizers. The genomes of many diverse iron oxidizers encode a homolog to an outer membrane cytochrome (Cyc2) shown to oxidize iron in two acidophiles. Phylogenetic analyses show Cyc2 sequences from neutrophiles cluster together, suggesting a common function, though this function has not been verified in these organisms. Therefore, we investigated the iron oxidase function of heterologously expressed Cyc2 from a neutrophilic iron oxidizer Mariprofundus ferrooxydans PV-1. Cyc2 PV-1 is capable of oxidizing iron, and its redox potential is 208 ± 20 mV, consistent with the ability to accept electrons from Fe2+ at neutral pH. These results support the hypothesis that Cyc2 functions as an iron oxidase in neutrophilic iron-oxidizing organisms. The results of sequence analysis and modeling reveal that the entire Cyc2 family shares a unique fused cytochrome-porin structure, with a defining consensus motif in the cytochrome region. On the basis of results from structural analyses, we predict that the monoheme cytochrome Cyc2 specifically oxidizes dissolved Fe2+, in contrast to multiheme iron oxidases, which may oxidize solid Fe(II). With our results, there is now functional validation for diverse representatives of Cyc2 sequences. We present a comprehensive Cyc2 phylogenetic tree and offer a roadmap for identifying cyc2/Cyc2 homologs and interpreting their function. The occurrence of cyc2 in many genomes beyond known iron oxidizers presents the possibility that microbial iron oxidation may be a widespread metabolism. IMPORTANCE Iron is practically ubiquitous across Earth’s environments, central to both life and geochemical processes, which depend heavily on the redox state of iron. Although iron oxidation, or “rusting,” can occur abiotically at near-neutral pH, we find neutrophilic iron-oxidizing bacteria (FeOB) are widespread, including in aquifers, sediments, hydrothermal vents, pipes, and water treatment systems. FeOB produce highly reactive Fe(III) oxyhydroxides that bind a variety of nutrients and toxins; thus, these microbes are likely a controlling force in iron and other biogeochemical cycles. There has been mounting evidence that Cyc2 functions as an iron oxidase in neutrophiles, but definitive proof of its function has long eluded us. This work provides conclusive biochemical evidence of iron oxidation by Cyc2 from neutrophiles. Cyc2 is common to a wide variety of iron oxidizers, including acidophilic and phototrophic iron oxidizers, suggesting that this fused cytochrome-porin structure is especially well adapted for iron oxidation. 

Abstract In principle, iron oxidation can fuel significant primary productivity and nutrient cycling in dark environments such as the deep sea. However, we have an extremely limited understanding of the ecology of iron-based ecosystems, and thus the linkages between iron oxidation, carbon cycling, and nitrate reduction. Here we investigate iron microbial mats from hydrothermal vents at Lōʻihi Seamount, Hawaiʻi, using genome-resolved metagenomics and metatranscriptomics to reconstruct potential microbial roles and interactions. Our results show that the aerobic iron-oxidizing Zetaproteobacteria are the primary producers, concentrated at the oxic mat surface. Their fixed carbon supports heterotrophs deeper in the mat, notably the second most abundant organism, Candidatus Ferristratum sp. (uncultivated gen. nov.) from the uncharacterized DTB120 phylum. Candidatus Ferristratum sp., described using nine high-quality metagenome-assembled genomes with similar distributions of genes, expressed nitrate reduction genes narGH and the iron oxidation gene cyc2 in situ and in response to Fe(II) in a shipboard incubation, suggesting it is an anaerobic nitrate-reducing iron oxidizer. Candidatus Ferristratum sp. lacks a full denitrification pathway, relying on Zetaproteobacteria to remove intermediates like nitrite. Thus, at Lōʻihi, anaerobic iron oxidizers coexist with and are dependent on aerobic iron oxidizers. In total, our work shows how key community members work together to connect iron oxidation with carbon and nitrogen cycling, thus driving the biogeochemistry of exported fluids.