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1. Gallionellaceae pangenomic analysis reveals insight into phylogeny, metabolic flexibility, and iron oxidation mechanisms

   https://doi.org/10.1128/msystems.00038-23  

   Hoover, Rene L. ; Keffer, Jessica L. ; Polson, Shawn W. ; Chan, Clara S. ( December 2023 , mSystems)

   Newton, Ryan J. (Ed.)

   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.

   Neutrophilic 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.

    

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2. Mixotrophy broadens the ecological niche range of the iron oxidizer Sideroxydans sp. CL21 isolated from an iron-rich peatland

   https://doi.org/10.1093/femsec/fiac156  

   Cooper, Rebecca E. ; Finck, Jessica ; Chan, Clara ; Küsel, Kirsten ( January 2023 , FEMS Microbiology Ecology)

   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.

    

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3. Evidence for Quinol Oxidation Activity of ImoA, a Novel NapC/NirT Family Protein from the Neutrophilic Fe(II)-Oxidizing Bacterium Sideroxydans lithotrophicus ES-1

   https://doi.org/10.1128/mbio.02150-22  

   Jain, Abhiney ; Coelho, Anaísa ; Madjarov, Joana ; Paquete, Catarina M. ; Gralnick, Jeffrey A. ( September 2022 , mBio)

   Newman, Dianne K. (Ed.)

   ABSTRACT Sideroxydans species are important chemolithoautotrophic Fe(II)-oxidizing bacteria in freshwater environments and play a role in biogeochemical cycling of multiple elements. Due to difficulties in laboratory cultivation and genetic intractability, the electron transport proteins required for the growth and survival of this organism remain understudied. In Sideroxydans lithotrophicus ES-1, it is proposed that the Mto pathway transfers electrons from extracellular Fe(II) oxidation across the periplasm to an inner membrane NapC/NirT family protein encoded by Slit_2495 to reduce the quinone pool. Based on sequence similarity, Slit_2495 has been putatively called CymA, a NapC/NirT family protein which in Shewanella oneidensis MR-1 oxidizes the quinol pool during anaerobic respiration of a wide range of substrates. However, our phylogenetic analysis using the alignment of different NapC/NirT family proteins shows that Slit_2495 clusters closer to NirT sequences than to CymA. We propose the name ImoA (inner membrane oxidoreductase) for Slit_2495. Our data demonstrate that ImoA can oxidize quinol pools in the inner membrane and is able to functionally replace CymA in S. oneidensis. The ability of ImoA to oxidize quinol in vivo as opposed to its proposed function of reducing quinone raises questions about the directionality and/or reversibility of electron flow through the Mto pathway in S. lithotrophicus. IMPORTANCE Fe(II)-oxidizing bacteria play an important role in biogeochemical cycles. At circumneutral pH, these organisms perform extracellular electron transfer, taking up electrons from Fe(II) outside the cell, potentially through a porin-cytochrome complex in the outer membrane encoded by the Mto pathway. Electrons from Fe(II) oxidation would then be transported to the quinone pool in the inner membrane via periplasmic and inner membrane electron transfer proteins. Directly demonstrating the functionality of genes in neutrophilic iron oxidizers is challenging due to the absence of robust genetic methods. Here, we heterologously expressed a NapC/NirT family tetraheme cytochrome ImoA, encoded by Slit_2495, an inner membrane protein from the Gram-negative Fe(II)-oxidizing bacterium Sideroxydans lithotrophicus ES-1, proposed to be involved in extracellular electron transfer to reduce the quinone pool. ImoA functionally replaced the inner membrane c-type cytochrome CymA in the Fe(III)-reducing bacterium Shewanella oneidensis. We suggest that ImoA may function primarily to oxidize quinol inS. lithotrophicus. 

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4. 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.)

   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.

   In 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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5. Validating the Cyc2 Neutrophilic Iron Oxidation Pathway Using Meta-omics of Zetaproteobacteria Iron Mats at Marine Hydrothermal Vents

   https://doi.org/10.1128/mSystems.00553-19  

   McAllister, Sean M. ; Polson, Shawn W. ; Butterfield, David A. ; Glazer, Brian T. ; Sylvan, Jason B. ; Chan, Clara S. ; Lloyd, Karen G. ( February 2020 , mSystems)

   ABSTRACT Zetaproteobacteria create extensive iron (Fe) oxide mats at marine hydrothermal vents, making them an ideal model for microbial Fe oxidation at circumneutral pH. Comparison of neutrophilic Fe oxidizer isolate genomes has revealed a hypothetical Fe oxidation pathway, featuring a homolog of the Fe oxidase Cyc2 from Acidithiobacillus ferrooxidans . However, Cyc2 function is not well verified in neutrophilic Fe oxidizers, particularly in Fe-oxidizing environments. Toward this, we analyzed genomes and metatranscriptomes of Zetaproteobacteria , using 53 new high-quality metagenome-assembled genomes reconstructed from Fe mats at Mid-Atlantic Ridge, Mariana Backarc, and Loihi Seamount (Hawaii) hydrothermal vents. Phylogenetic analysis demonstrated conservation of Cyc2 sequences among most neutrophilic Fe oxidizers, suggesting a common function. We confirmed the widespread distribution of cyc2 and other model Fe oxidation pathway genes across all represented Zetaproteobacteria lineages. High expression of these genes was observed in diverse Zetaproteobacteria under multiple environmental conditions and in incubations. The putative Fe oxidase gene cyc2 was highly expressed in situ , often as the top expressed gene. The cyc2 gene showed increased expression in Fe(II)-amended incubations, with corresponding increases in carbon fixation and central metabolism gene expression. These results substantiate the Cyc2-based Fe oxidation pathway in neutrophiles and demonstrate its significance in marine Fe-mineralizing environments. IMPORTANCE Iron oxides are important components of our soil, water supplies, and ecosystems, as they sequester nutrients, carbon, and metals. Microorganisms can form iron oxides, but it is unclear whether this is a significant mechanism in the environment. Unlike other major microbial energy metabolisms, there is no marker gene for iron oxidation, hindering our ability to track these microbes. Here, we investigate a promising possible iron oxidation gene, cyc2 , in iron-rich hydrothermal vents, where iron-oxidizing microbes dominate. We pieced together diverse Zetaproteobacteria genomes, compared these genomes, and analyzed expression of cyc2 and other hypothetical iron oxidation genes. We show that cyc2 is widespread among iron oxidizers and is highly expressed and potentially regulated, making it a good marker for the capacity for iron oxidation and potentially a marker for activity. These findings will help us understand and potentially quantify the impacts of neutrophilic iron oxidizers in a wide variety of marine and terrestrial environments. 

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