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ABSTRACT Methylothon is an inquiry-based high school learning module in microbial ecology, molecular biology, and bioinformatics that centers around pink-pigmented plant-associated methylotrophic bacteria. Here, we present an overview of the module’s learning goals, describe course resources (available for public use at http://methylothon.com ), and relate lessons learned from adapting Methylothon for remote learning during the pandemic in spring of 2021. This curriculum description is intended not only for instructors but also for microbial ecology researchers with an interest in conducting K-12 outreach. The original in-person version of the module allows students to isolate their own strains of methylotrophic bacteria from plants they sample from the environment, to identify these using PCR, sequencing, and phylogenetic analysis, and to contribute their strains to original research in a university lab. The adapted version strengthens the focus on bioinformatics and increases its flexibility and accessibility by making the lab portion optional and adopting free web-based tools. Student feedback and graded assignments from spring 2021 revealed that the lesson was especially effective at introducing the concepts of BLAST and phylogenetic trees and that students valued and felt inspired by the opportunity to conduct hands-on work and to participate in community science.

Earth structure beneath the Antarctic exerts an important control on the evolution of the ice sheet. A range of geological and geophysical data sets indicate that this structure is complex, with the western sector characterized by a lithosphere of thickness ∼50–100 km and viscosities within the upper mantle that vary by 2–3 orders of magnitude. Recent analyses of uplift rates estimated using Global Navigation Satellite System (GNSS) observations have inferred 1-D viscosity profiles below West Antarctica discretized into a small set of layers within the upper mantle using forward modelling of glacial isostatic adjustment (GIA). It remains unclear, however, what these 1-D viscosity models represent in an area with complex 3-D mantle structure, and over what geographic length-scale they are applicable. Here, we explore this issue by repeating the same modelling procedure but applied to synthetic uplift rates computed using a realistic model of 3-D viscoelastic Earth structure inferred from seismic tomographic imaging of the region, a finite volume treatment of GIA that captures this complexity, and a loading history of Antarctic ice mass changes inferred over the period 1992–2017. We find differences of up to an order of magnitude between the best-fitting 1-D inferences and regionally averaged depth profilesmore »through the 3-D viscosity field used to generate the synthetics. Additional calculations suggest that this level of disagreement is not systematically improved if one increases the number of observation sites adopted in the analysis. Moreover, the 1-D models inferred from such a procedure are non-unique, that is a broad range of viscosity profiles fit the synthetic uplift rates equally well as a consequence, in part, of correlations between the viscosity values within each layer. While the uplift rate at each GNSS site is sensitive to a unique subspace of the complex, 3-D viscosity field, additional analyses based on rates from subsets of proximal sites showed no consistent improvement in the level of bias in the 1-D inference. We also conclude that the broad, regional-scale uplift field generated with the 3-D model is poorly represented by a prediction based on the best-fitting 1-D Earth model. Future work analysing GNSS data should be extended to include horizontal rates and move towards inversions for 3-D structure that reflect the intrinsic 3-D resolving power of the data.

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Abstract Methylobacterium is a group of methylotrophic microbes associated with soil, fresh water, and particularly the phyllosphere, the aerial part of plants that has been well studied in terms of physiology but whose evolutionary history and taxonomy are unclear. Recent work has suggested that Methylobacterium is much more diverse than thought previously, questioning its status as an ecologically and phylogenetically coherent taxonomic genus. However, taxonomic and evolutionary studies of Methylobacterium have mostly been restricted to model species, often isolated from habitats other than the phyllosphere and have yet to utilize comprehensive phylogenomic methods to examine gene trees, gene content, or synteny. By analyzing 189 Methylobacterium genomes from a wide range of habitats, including the phyllosphere, we inferred a robust phylogenetic tree while explicitly accounting for the impact of horizontal gene transfer (HGT). We showed that Methylobacterium contains four evolutionarily distinct groups of bacteria (namely A, B, C, D), characterized by different genome size, GC content, gene content, and genome architecture, revealing the dynamic nature of Methylobacterium genomes. In addition to recovering 59 described species, we identified 45 candidate species, mostly phyllosphere-associated, stressing the significance of plants as a reservoir of Methylobacterium diversity. We inferred an ancient transition from a free-livingmore »lifestyle to association with plant roots in Methylobacteriaceae ancestor, followed by phyllosphere association of three of the major groups (A, B, D), whose early branching in Methylobacterium history has been heavily obscured by HGT. Together, our work lays the foundations for a thorough redefinition of Methylobacterium taxonomy, beginning with the abandonment of Methylorubrum.« less

Lanthanides (Ln) are a new group of life metals, and many questions remain regarding how they are acquired and used in biology. Methylotrophic bacteria can acquire, transport, biomineralize, and use Ln as part of a cofactor complex with pyrroloquinoline quinone (PQQ) in alcohol dehydrogenases. For most methylotrophic bacteria use is restricted to the light Ln, which range from lanthanum to samarium (atomic numbers 57–62). Understanding how the cell differentiates between light and heavy Ln, and the impacts of these metals on the metabolic network, will advance the field of Ln biochemistry and give insights into enzyme catalysis, stress homeostasis, and metal biomineralization and compartmentalization. We report robust methanol growth with the heavy Ln gadolinium by a genetic variant of the model methylotrophic bacterium Methylorubrum extorquens AM1, named evo -HLn, for “ evo lved for H eavy L antha n ides.” A non-synonymous single nucleotide polymorphism in a cytosolic hybrid histidine kinase/response regulator allowed for sweeping transcriptional alterations to heavy metal stress response, methanol oxidation, and central metabolism. Increased expression of genes for Ln acquisition and uptake, production of the Ln-chelating lanthanophore, PQQ biosynthesis, and phosphate transport and metabolism resulted in gadolinium hyperaccumulation of 36-fold with a trade-off for light Lnmore »accumulation. Gadolinium was hyperaccumulated in an enlarged acidocalcisome-like compartment. This is the first evidence of a bacterial intracellular Ln-containing compartment that we name the “lanthasome.” Carotenoid and toblerol biosynthesis were also upregulated. Due to its unique capabilities, evo -HLn can be used to further magnetic resonance imaging (MRI) and bioremediation technologies. In this regard, we show that gadolinium hyperaccumulation was sufficient to produce MRI contrast in whole cells, and that evo -HLn was able to readily acquire the metal from the MRI contrast agent gadopentetic acid. Finally, hyperaccumulation of gadolinium, differential uptake of light and heavy Ln, increased PQQ levels, and phosphate transport provide new insights into strategies for Ln recovery.« less

Abstract The West Antarctic Ice Sheet (WAIS) overlies a thin, variable-thickness lithosphere and a shallow upper-mantle region of laterally varying and, in some regions, very low (~1018 Pa s) viscosity. We explore the extent to which viscous effects may affect predictions of present-day geoid and crustal deformation rates resulting from Antarctic ice mass flux over the last quarter century and project these calculations into the next half century, using viscoelastic Earth models of varying complexity. Peak deformation rates at the end of a 25-yr simulation predicted with an elastic model underestimate analogous predictions that are based on a 3D viscoelastic Earth model (with minimum viscosity below West Antarctica of 1018 Pa s) by ~15 and ~3 mm yr−1 in the vertical and horizontal directions, respectively, at sites overlying low-viscosity mantle and close to high rates of ice mass flux. The discrepancy in uplift rate can be reduced by adopting 1D Earth models tuned to the regional average viscosity profile beneath West Antarctica. In the case of horizontal crustal rates, adopting 1D regional viscosity models is no more accurate in recovering predictions that are based on 3D viscosity models than calculations that assume a purely elastic Earth. The magnitude and relativemore »contribution of viscous relaxation to crustal deformation rates will likely increase significantly in the next several decades, and the adoption of 3D viscoelastic Earth models in analyses of geodetic datasets [e.g., Global Navigation Satellite System (GNSS); Gravity Recovery and Climate Experiment (GRACE)] will be required to accurately estimate the magnitude of Antarctic modern ice mass flux in the progressively warming world.« less

Lanthanide (Ln) elements are utilized as cofactors for catalysis by XoxF-type methanol dehydrogenases (MDHs). A primary assumption is that XoxF enzymes produce formate from methanol oxidation, which could impact organisms that require formaldehyde for assimilation. We report genetic and phenotypic evidence showing that XoxF1 (MexAM1_1740) fromMethylobacterium extorquensAM1 produces formaldehyde, and not formate, during growth with methanol. Enzyme purified with lanthanum or neodymium oxidizes formaldehyde. However, formaldehyde oxidation via 2,6-dichlorophenol-indophenol (DCPIP) reduction is not detected in cell-free extracts from wild-type strain methanol- and lanthanum-grown cultures. Formaldehyde activating enzyme (Fae) is required for Ln methylotrophic growth, demonstrating that XoxF1-mediated production of formaldehyde is essential. Addition of exogenous lanthanum increases growth rate with methanol by 9–12% but does not correlate with changes to methanol consumption or formaldehyde accumulation. Transcriptomics analysis of lanthanum methanol growth shows upregulation ofxox1and downregulation ofmxagenes, consistent with the Ln-switch, no differential expression of formaldehyde conversion genes, downregulation of pyrroloquinoline quinone (PQQ) biosynthesis genes, and upregulation offdh4formate dehydrogenase (FDH) genes. Additionally, the Ln-dependent ethanol dehydrogenase ExaF reduces methanol sensitivity in thefaemutant strain when lanthanides are present, providing evidence for the capacity of an auxiliary role for ExaF during Ln-dependent methylotrophy.