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1. Gallionellaceae in rice root plaque: metabolic roles in iron oxidation, nutrient cycling, and plant interactions

   https://doi.org/10.1128/aem.00570-23  

   Chan, Clara S.; Dykes, Gretchen E.; Hoover, Rene L.; Limmer, Matt A.; Seyfferth, Angelia L. (December 2023, Applied and Environmental Microbiology)

   Glass, Jennifer B. (Ed.)

   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. 

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2. An organotrophic Sideroxydans reveals potential iron oxidation marker genes

   https://doi.org/10.1128/aem.00395-25  

   Hoover, Rene L; Küsel, Kirsten; Chan, Clara S (September 2025, Applied and Environmental Microbiology)

   Bose, Arpita (Ed.)

   ABSTRACT To understand the ecophysiology and the role of iron-oxidizing bacteria (FeOB) in various ecosystems, we need to identify marker genes of the iron oxidation pathway to track activity in situ. The Gallionellaceae Sideroxydanssp. CL21, an autotrophic iron-oxidizing bacterium isolated from a peatland, is unusual among FeOB isolates in its genomic potential to utilize organic compounds as energy sources. Therefore, it offers the unique opportunity to determine genes expressed under litho- vs organotrophic conditions. We demonstrated the growth of Sideroxydanssp. CL21 on organic substrates (lactate and pyruvate) and inorganic substrates [Fe(II), magnetite, thiosulfate, and S(0)]. Thus, cells were capable of lithoautotrophic, organotrophic, and potentially organoheterotrophic growth. Surprisingly, when lactate-grown cells were given Fe(II), mid-log phase cells were unable to oxidize iron, while late-log phase cells oxidized iron. To elucidate iron oxidation pathways, we compared gene expression between mid-log (non-iron-oxidizing) and late-log (iron-oxidizing) lactate-grown cells. Genes for iron oxidases (cyc2, mtoA) were highly expressed at both time points but did not correspond to iron oxidation capability, making them unsuitable marker genes of iron oxidation activity by themselves. However, genes encoding periplasmic and inner membrane cytochromes were significantly upregulated in cells capable of iron oxidation. These genes include mtoD, cymA/imoA, and a cluster of Fe(II)-responsive genes (ircABCD). These findings suggest that Gallionellaceae regulate their iron oxidation pathways in multiple stages, with iron oxidase-encoding genes proactively expressed. Other genes encoding electron carriers are upregulated only when iron oxidation is needed, which makes these genes (i.e.,ircABCD) good prospective indicators of iron oxidation ability.IMPORTANCEIron-oxidizing bacteria (FeOB) are widespread in the environment, and we suspect that they play key roles in nutrient and other elemental cycles. However, with no isotopic marker, we lack the ability to monitor FeOB activity, prompting us to search for genetic markers. Previous work suggests that expression of iron oxidase genes does not directly correspond to iron oxidation activity in Gallionellaceae, and little was known about the other genes in the pathway. Here, we study a unique FeOB isolate that possesses organotrophic capabilities and demonstrate its potential for mixotrophic growth on lactate and Fe(II). Its ability to oxidize iron is regulated, allowing us to discover potential iron oxidation pathway genes with expression that corresponds to iron oxidation activity. If these genes can be further validated as iron oxidation marker genes, they will enable us to delineate autotrophic and organoheterotrophic FeOB impacts on carbon cycling in wetlands and other natural and engineered environments. 

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3. Microbial magnetite oxidation via MtoAB porin-multiheme cytochrome complex in Sideroxydans lithotrophicus ES-1

   https://doi.org/10.1128/aem.01865-24  

   Keffer, Jessica L; Zhou, Nanqing; Rushworth, Danielle D; Yu, Yanbao; Chan, Clara S (April 2025, Applied and Environmental Microbiology)

   Buan, Nicole R (Ed.)

   ABSTRACT Most of Earth’s iron is mineral-bound, but it is unclear how and to what extent iron-oxidizing microbes can use solid minerals as electron donors. A prime candidate for studying mineral-oxidizing growth and pathways is Sideroxydans lithotrophicus ES-1, a robust, facultative iron oxidizer with multiple possible iron oxidation mechanisms. These include Cyc2 and Mto pathways plus other multiheme cytochromes and cupredoxins, and so we posit that the mechanisms may correspond to different Fe(II) sources. Here, S. lithotrophicus ES-1 was grown on dissolved Fe(II)-citrate and magnetite. S. lithotrophicus ES-1 oxidized all dissolved Fe²⁺ released from magnetite and continued to build biomass when only solid Fe(II) remained, suggesting it can utilize magnetite as a solid electron donor. Quantitative proteomic analyses of S. lithotrophicus ES-1 grown on these substrates revealed global proteome remodeling in response to electron donor and growth state and uncovered potential proteins and metabolic pathways involved in the oxidation of solid magnetite. While the Cyc2 iron oxidases were highly expressed on both dissolved and solid substrates, MtoA was only detected during growth on solid magnetite, suggesting this protein helps catalyze oxidation of solid minerals inS. lithotrophicusES-1. A set of cupredoxin domain-containing proteins were also specifically expressed during solid iron oxidation. This work demonstrated that the iron oxidizer S. lithotrophicus ES-1 utilized additional extracellular electron transfer pathways when growing on solid mineral electron donors compared to dissolved Fe(II). IMPORTANCEMineral-bound iron could be a vast source of energy to iron-oxidizing bacteria, but there is limited physiological evidence of this metabolism, and it has been unknown whether the mechanisms of solid and dissolved Fe(II) oxidation are distinct. In iron-reducing bacteria, multiheme cytochromes can facilitate iron mineral reduction, and here, we link a multiheme cytochrome-based pathway to mineral oxidation, expanding the known functionality of multiheme cytochromes. Given the growing recognition of microbial oxidation of minerals and cathodes, increasing our understanding of these mechanisms will allow us to recognize and trace the activities of mineral-oxidizing microbes. This work shows how solid iron minerals can promote microbial growth, which, if widespread, could be a major agent of geologic weathering and mineral-fueled nutrient cycling in sediments, aquifers, and rock-hosted environments. 

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4. An abundant bacterial phylum with nitrite-oxidizing potential in oligotrophic marine sediments

   https://doi.org/10.1038/s42003-024-06136-2  

   Zhao, Rui; Jørgensen, Steffen L.; Babbin, Andrew R. (December 2024, Communications Biology)

   Abstract Nitrite-oxidizing bacteria (NOB) are important nitrifiers whose activity regulates the availability of nitrite and dictates the magnitude of nitrogen loss in ecosystems. In oxic marine sediments, ammonia-oxidizing archaea (AOA) and NOB together catalyze the oxidation of ammonium to nitrate, but the abundance ratios of AOA to canonical NOB in some cores are significantly higher than the theoretical ratio range predicted from physiological traits of AOA and NOB characterized under realistic ocean conditions, indicating that some NOBs are yet to be discovered. Here we report a bacterial phylumCandidatusNitrosediminicolota, members of which are more abundant than canonical NOBs and are widespread across global oligotrophic sediments.Ca. Nitrosediminicolota members have the functional potential to oxidize nitrite, in addition to other accessory functions such as urea hydrolysis and thiosulfate reduction. While one recovered species (Ca. Nitrosediminicola aerophilus) is generally confined within the oxic zone, another (Ca. Nitrosediminicola anaerotolerans) additionally appears in anoxic sediments. CountingCa. Nitrosediminicolota as a nitrite-oxidizer helps to resolve the apparent abundance imbalance between AOA and NOB in oxic marine sediments, and thus its activity may exert controls on the nitrite budget. 

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5. Unraveling Fe(II)-Oxidizing Mechanisms in a Facultative Fe(II) Oxidizer, Sideroxydans lithotrophicus Strain ES-1, via Culturing, Transcriptomics, and Reverse Transcription-Quantitative PCR

   https://doi.org/10.1128/AEM.01595-21  

   Zhou, Nanqing; Keffer, Jessica L.; Polson, Shawn W.; Chan, Clara S. (January 2022, Applied and Environmental Microbiology)

   Buan, Nicole R. (Ed.)

   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. 

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