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Natural attenuation of heavy metals occurs via coupled microbial iron cycling and metal precipitation in creeks impacted by acid mine drainage (AMD). Here, we describe the isolation, characterization, and genomic sequencing of two iron-oxidizing bacteria (FeOB) species: Thiomonas ferrovorans FB-6 and Thiomonas metallidurans FB-Cd, isolated from slightly acidic (pH 6.3), Fe-rich, AMD-impacted creek sediments. These strains precipitated amorphous iron oxides, lepidocrocite, goethite, and magnetite or maghemite and grew at a pH optimum of 5.5. While Thiomonas spp. are known as mixotrophic sulfur oxidizers and As oxidizers, the FB strains oxidized Fe, which suggests they can efficiently remove Fe and other metals via coprecipitation. Previous evidence for Thiomonas sp. Fe oxidation is largely ambiguous, possibly because of difficulty demonstrating Fe oxidation in heterotrophic/mixotrophic organisms. Therefore, we also conducted a genomic analysis to identify genetic mechanisms of Fe oxidation, other metal transformations, and additional adaptations, comparing the two FB strain genomes with 12 other Thiomonas genomes. The FB strains fall within a relatively novel group of Thiomonas strains that includes another strain (b6) with solid evidence of Fe oxidation. Most Thiomonas isolates, including the FB strains, have the putative iron oxidation gene cyc2, but only the two FB strains possess the putative Fe oxidase genes mtoAB The two FB strain genomes contain the highest numbers of strain-specific gene clusters, greatly increasing the known Thiomonas genetic potential. Our results revealed that the FB strains are two distinct novel species of Thiomonas with the genetic potential for bioremediation of AMD via iron oxidation.IMPORTANCE As AMD moves through the environment, it impacts aquatic ecosystems, but at the same time, these ecosystems can naturally attenuate contaminated waters via acid neutralization and catalyzing metal precipitation. This is the case in the former Ronneburg uranium-mining district, where AMD impacts creek sediments. We isolated and characterized two iron-oxidizing Thiomonas species that are mildly acidophilic to neutrophilic and that have two genetic pathways for iron oxidation. These Thiomonas species are well positioned to naturally attenuate AMD as it discharges across the landscape.

Development of genome-editing tools in diverse microbial species is an important step both in understanding the roles of those microbes in different environments, and in engineering microbes for a variety of applications. Freshwater-specific clades of Actinobacteria are ubiquitous and abundant in surface freshwaters worldwide. Here, we show that Rhodoluna lacicola and Aurantimicrobium photophilum, which represent widespread clades of freshwater Actinobacteria, are naturally transformable. We also show that gene inactivation via double homologous recombination and replacement of the target gene with antibiotic selection markers can be used in both strains, making them convenient and broadly accessible model organisms for freshwater systems. We further show that in both strains, the predicted phytoene synthase is the only phytoene synthase, and its inactivation prevents the synthesis of all pigments. The tools developed here enable targeted modification of the genomes of some of the most abundant microbes in freshwater communities. These genome-editing tools will enable hypothesis testing about the genetics and (eco)physiology of freshwater Actinobacteria and broaden the available model systems for engineering freshwater microbial communities. IMPORTANCE To advance bioproduction or bioremediation in large, unsupervised environmental systems such as ponds, wastewater lagoons, or groundwater systems, it will be necessary to develop diverse genetically amenable microbial model organisms. Although we already genetically modify a few key species, tools for engineering more microbial taxa, with different natural phenotypes, will enable us to genetically engineer multispecies consortia or even complex communities. Developing genetic tools for modifying freshwater bacteria is particularly important, as wastewater, production ponds or raceways, and contaminated surface water are all freshwater systems where microbial communities are already deployed to do work, and the outputs could potentially be enhanced by genetic modifications. Here, we demonstrate that common tools for genome editing can be used to inactivate specific genes in two representatives of a very widespread, environmentally relevant group of Actinobacteria. These Actinobacteria are found in almost all tested surface freshwater environments, where they co-occur with primary producers, and genome-editing tools in these species are thus a step on the way to engineering microbial consortia in freshwater environments. [ABSTRACT FROM AUTHOR]

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 CO2 is an important electron sink for Fe(II) oxidation. [ABSTRACT FROM AUTHOR]

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 Fe 2+ 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 in S. lithotrophicus ES-1. A set of cupredoxin domain-containing proteins were also specifically expressed during solid iron oxidation. This work demonstrated 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).