David Emerson

The Sphaerotilus-Leptothrix group of bacteria includes one of the first described microorganisms, Leptothrix ochracea , an uncultured type strain, plus isolates of Leptothrix and Sphaerotilus . This group is unified by the ability to form sheaths and oxidize metals, although L. ochracea exhibits obvious ecological, morphological, and functional differences from the rest of Sphaerotilus-Leptothrix . Recently, there have been calls to combine the group into one genus, Sphaerotilus ; however, these studies lacked adequate genomic representation of L. ochracea . Here, we present a comprehensive comparative genomic analysis of the Sphaerotilus-Leptothrix group, including expanded representation of L. ochracea , a closely related novel species, Leptothrix toolikensis , and two new isolates ( Leptothrix mechoopdaensis ). Analysis of 38 genomes resolves three phylogenetic and functional groups: the ochracea -type Leptothrix (Group 1), the mobilis -type Leptothrix (Group 2), and Sphaerotilus (Group 3). Group 1 genomes form a separate genus based on average nucleotide identity and alignment fraction. The genomes clearly diverge from the rest of Sphaerotilus-Leptothrix in phylogeny, size, and metabolic potential. Group 1 genomes are much smaller (2.59–3.04 Mb) than those of Groups 2 (4.55–6.06 Mb) and 3 (3.94–5.07 Mb), while encoding more metal oxidases and fewer carbohydrate-active enzymes. Group 2 clusters with Group 3 phylogenetically and is similar in organic carbon metabolisms but maintains more metal oxidation genes. Group 2 members lack homogeneity in phenotype and genotype, suggesting that additional isolates and genomes are needed for confident classification. However, Group 1 genomes ( L. ochracea and L. toolikensis ) show clear divergence, precluding their inclusion in Sphaerotilus and supporting the retention of the genus Leptothrix . IMPORTANCE: Researchers have long noted differences in metal oxidation, morphology, and ecology among Sphaerotilus-Leptothrix , but longstanding confusion over phylogeny and genus boundaries led to inconsistent taxonomic classification between the two genera. This confusion stems from previous work that used isolates that are unavailable or lost distinguishing traits in culture, and from limited genomic data. Furthermore, the Leptothrix type strain L. ochracea has never been isolated. This study provides molecular evidence that substantiates calls to reassign some Leptothrix members to the genus Sphaerotilus but adds to an emerging body of evidence that Group 1 L. ochracea and now L. toolikensis represent a functionally distinct lineage. While genomic similarity metrics left taxonomic divisions unclear, integrating metabolic potential with phylogeny resolved genus boundaries based on clear functional groupings. This polyphasic approach for delineating genera clarifies longstanding taxonomic confusion and refines our understanding of functional diversity both across and within Sphaerotilus-Leptothrix lineages.

Long‐term river monitoring of the Kuparuk River (North Slope, Alaska, USA) confirms significant increases in solutes that are indicative of active layer thickening due to thawing permafrost. However, there is no evidence of an increase in total dissolved phosphorus (TDP) or soluble reactive phosphorus (SRP), the nutrient that limits primary production in this and similar rivers in the region. Here, we show that Mehlich‐3 extractable iron (Fe) and aluminum (Al) in active layer soils impart high P geochemical sorption capacities across a range of landscape features that we would expect to promote lateral movement of water and solutes to headwater streams in our study watershed. Reanalysis of a recently published pan‐Arctic soils database that includes active layer and permafrost soil samples suggests that this high P sorption capacity could be common in other parts of the Arctic region. We conclude that soil minerals enhance P retention on hillslopes and propose pedogenic secondary Fe and Al minerals may continue to retain P in these soils and limit biological productivity in the adjacent river even as active layer thickening increases potential P mobility in the watershed. We suggest that similar interactions may occur in other areas of the Arctic where comparable geochemical conditions prevail.

Using environmental DNA (eDNA)-based tools, we examined sediments underlying a ~ 1.25 hectare commercial kelp farm in the Gulf of Maine growing sugar kelp (Saccharina latissima) for two farming seasons, post-harvest. Two eDNA methods were used: a newly designed S. latissima-specific digital polymerase chain reaction (dPCR) assay targeting the cytochrome oxidase subunit I (COI) mitochondrial gene, as well as metabarcoding for the 16S and 18S ribosomal RNA (rRNA) genes, to examine overall bacterial, archaeal, and eukaryotic diversity. Sediment carbon and nitrogen content was analyzed using isotope ratio mass spectrometry (IRMS) as more traditional indicators of potential kelp biomass-derived nutrient enrichment in the benthos. When targeted sampling sites were added inside the footprint of the farm lease area in year two of the study, dPCR data showed subtle but significant differences between sediment samples inside and outside of the farm, with mean S. latissima COI gene copies from cores taken inside the farm being ~8% greater than mean values outside the farm. The highest COI copy numbers in marine sediments were from sites with observed accumulation of kelp biomass, while there was no conclusive difference in carbon and nitrogen content of those same sediment samples. Metabarcoding data also revealed subtle differences in taxa associated with sediments inside and outside the farm. For example, microbial taxa that correlated with kelp eDNA from cores within the farm included the families Rhodothermaceae, Rubritaleaceae, Flavobacteriaceae, Prolixibacteraceae, Nitrosomonadaceae, Nitrincolaceae and Rubinisphaeraceae. However, the majority of the above taxa were low in relative abundance, with only Flavobacteriaceae ranking among the top 30 most abundant and prevalent families in these sediments. In summary, this study demonstrates the sensitivity and specificity of eDNA tools to detect potential ecological and anthropogenic effects in marine sediments, beyond that of bulk nutrient and stable isotope analyses.

Climate warming in the Arctic is thawing previously frozen soil (permafrost). Permafrost thaw alters landscape hydrology and increases weathering rates, which can increase the delivery of solutes to adjacent waters. Long-term river monitoring of the Kuparuk River (North Slope, Alaska, USA) confirms significant increases in solutes that are indicative of thawing permafrost. However, there is no evidence of an increase in total phosphorus (TP) or soluble reactive phosphorus (SRP), the nutrient that limits primary production in this and similar rivers in the region. Here, we show that Mehlich-3 extractable iron (Fe) and aluminum (Al) impart high P biogeochemical sorption capacities across a range of landscape features that we would expect to promote lateral movement of water and solutes to headwater streams in our study watershed. Reanalysis of a recently published pan-Arctic soils database suggests that this high P sorption capacity could be common in other parts of the Arctic region. We conclude that while warming-induced permafrost thaw may increase the potential for P mobility in our watershed, simultaneous increases in pedogenic secondary Fe and Al minerals may continue to retain P in these soils and limit biological productivity in the adjacent river. We suggest that similar interactions may occur in other areas of the Arctic where comparable biogeochemical conditions prevail.

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 and mtoA, 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. IMPORTANCE: Winogradsky'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.

We present a cost model for implementing a deployment scale effort for conducting ocean iron fertilization (OIF) for marine‐based carbon dioxide removal (CDR). The model incorporates basic oceanographic parameters critical for estimating the effective export of newly fixed CO₂ into biomass that is stimulated by Fe addition to an Fe‐limited region of the Southern Ocean. Estimated costs can vary by nearly 100‐fold between best‐case and worst‐case scenarios, with best‐case values of $7/net tonne C captured versus worst‐case $1,500/net tonne C captured, without accounting for verification costs. Primary oceanographic factors that influence cost are the net primary productivity increases achieved via OIF, the amount of C exported into the deep ocean, and the amount of CO₂ ventilated back to the atmosphere. The model compares ship‐based versus aerial delivery of Fe to the ocean, and estimates aerial delivery can be 30%–40% more cost effective; however, the specific requirements for aerial delivery require additional research and development. The model also estimates costs associated with verification and environmental monitoring of OIF. These costs increase $/net tonne C captured by 3–4‐fold. Best, intermediate, and worst cases for aerial delivery and ship delivery are $21, $83, $2,033, and $24, $94, $4,691, respectively, inclusive of verification costs. The primary goal of this model is to demonstrate the variability in cost of OIF as a CDR method, and to better understand where additional research is needed to determine the major factors that may make OIF a tractable, nature‐based CDR method.

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 “stalk formation in Zetaproteobacteria” (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.

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 homologues, 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 towards a c-di-GMP regulated surface attachment function of stalks during sessile growth.

Microorganisms and burrowing animals exert a pronounced impact on the cycling of redox sensitive metals in coastal sediments. We hypothesized that the iron biogeochemical cycle and associated sedimentary microbial community will respond to seasonal changes in a bioturbated intertidal mudflat. In this study, we monitored the spatiotemporal dynamics of porewater and highly reactive solid phase iron with the corresponding prokaryotic and eukaryotic sedimentary microbial communities over one annual cycle from November 2015 to November 2016. Continuous and seasonally variable pools of both porewater Fe(II) and highly reactive iron (Fe_HR) were observed throughout the seasons with significant increases of Fe(II) and Fe_HR in response to increased sediment temperature in summer months. Maximum concentrations of Fe(II) and Fe_HR were predominantly confined to the upper 5 cm of sediment throughout the year. Iron-oxidizing and -reducing microorganisms were present and stable temporally, and exhibited a depth-dependent stratification likely due to availability of Fe(II) and Fe_HR pools, respectively. Zetaproteobacteria, presumptive lithotrophic iron-oxidizing bacteria, were present at abundances around 0.5–1% in the top 5 cm of sediment with decline abundance with depth. As a whole the microbial community was relatively stable across the seasons, and showed strongest separation with depth, probably driven by changes in oxygen availability and organic matter. The Deltaproteobacteria, principally taxa known to be associated with sulfur and iron cycling, were prevalent, especially at >5 cm depth. Gammaproteobacteria and Bacteroidetes were also abundant, with putatively aerobic members especially prevalent in the cm of the sediment. The relative abundance of diatoms, estimated from abundance of 18S rRNA gene counts, showed evidence of a seasonal signal possibly tied to spring and fall blooms. Overall, analysis of phytoplankton found significant abundance at depth, likely due to the feeding and bio-mixing activity of marine worms. Macro-, and meiofauna, consistent with expected taxa, were detected throughout the year via 18S gene counts, and showed some seasonal variations that may influence sedimentary iron transformations by active microbial grazing. In summary, this analysis revealed relatively consistent temporal and spatial trends in iron geochemistry and microbial and macrobial community composition, while also indicating a complex dynamic of microbial and macrobial interactions are responsible for maintaining these processes.