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Methylobacterium is a bacterial group tied to plants. Despite the ubiquity of methylobacteria and the importance to their hosts, little is known about the processes driving Methylobacterium community dynamics. Methylobacterium is a prevalent bacterial genus of the phyllosphere. Despite its ubiquity, little is known about the extent to which its diversity reflects neutral processes like migration and drift, versus environmental filtering of life history strategies and adaptations. In two temperate forests, we investigated how phylogenetic diversity within Methylobacterium is structured by biogeography, seasonality, and growth strategies. Using deep, culture-independent barcoded marker gene sequencing coupled with culture-based approaches, we uncovered a considerable diversity of Methylobacterium in the phyllosphere. We cultured different subsets of Methylobacterium lineages depending upon the temperature of isolation and growth (20°C or 30°C), suggesting long-term adaptation to temperature. To a lesser extent than temperature adaptation, Methylobacterium diversity was also structured across large (>100 km; between forests) and small (<1.2 km; within forests) geographical scales, among host tree species, and was dynamic over seasons. By measuring the growth of 79 isolates during different temperature treatments, we observed contrasting growth performances, with strong lineage- and season-dependent variations in growth strategies. Finally, we documented a progressive replacement of lineages with a high-yield growth strategy typical of cooperative, structured communities in favor of those characterized by rapid growth, resulting in convergence and homogenization of community structure at the end of the growing season. Together, our results show how Methylobacterium is phylogenetically structured into lineages with distinct growth strategies, which helps explain their differential abundance across regions, host tree species, and time. This work paves the way for further investigation of adaptive strategies and traits within a ubiquitous phyllosphere genus. IMPORTANCE Methylobacterium is a bacterial group tied to plants. Despite the ubiquity of methylobacteria and the importance to their hosts, little is known about the processes driving Methylobacterium community dynamics. By combining traditional culture-dependent and -independent (metabarcoding) approaches, we monitored Methylobacterium diversity in two temperate forests over a growing season. On the surface of tree leaves, we discovered remarkably diverse and dynamic Methylobacterium communities over short temporal (from June to October) and spatial (within 1.2 km) scales. Because we cultured different subsets of Methylobacterium diversity depending on the temperature of incubation, we suspected that these dynamics partly reflected climatic adaptation. By culturing strains under laboratory conditions mimicking seasonal variations, we found that diversity and environmental variations were indeed good predictors of Methylobacterium growth performances. Our findings suggest that Methylobacterium community dynamics at the surface of tree leaves results from the succession of strains with contrasting growth strategies in response to environmental variations.

ABSTRACT Phylosymbiosis refers to a congruent pattern between the similarity of microbiomes of different species and the branching pattern of the host phylogeny. Phylosymbiosis has been detected in a variety of vertebrate and invertebrate hosts, but has only been assessed in geographically isolated populations. We tested for phylosymbiosis in eight (sub)species of western chipmunks with overlapping ranges and ecological niches; we used a nuclear (Acrosin) and a mitochondrial (CYTB) phylogenetic marker because there are many instances of mitochondrial introgression in chipmunks. We predicted that similarity among microbiomes increases with: (1) increasing host mitochondrial relatedness, (2) increasing host nuclear genome relatedness and (3) decreasing geographic distance among hosts. We did not find statistical evidence supporting phylosymbiosis in western chipmunks. Furthermore, in contrast to studies of other mammalian microbiomes, similarity of chipmunk microbiomes is not predominantly determined by host species. Sampling site explained most variation in microbiome composition, indicating an important role of local environment in shaping microbiomes. Fecal microbiomes of chipmunks were dominated by Bacteroidetes (72.2%), followed by Firmicutes (24.5%), which is one of the highest abundances of Bacteroidetes detected in wild mammals. Future work will need to elucidate the effects of habitat, ecology and host genomics on chipmunk microbiomes. Gut microbiomes of western chipmunks species did not show the same pattern as would be expected based on their species phylogeny

To effectively reduce vision loss due to age-related macular generation (AMD) on a global scale, knowledge of its genetic architecture in diverse populations is necessary. A critical element, AMD risk profiles in African and Hispanic/Latino ancestries, remains largely unknown. We combined data in the Million Veteran Program with five other cohorts to conduct the first multi-ancestry genome-wide association study of AMD and discovered 63 loci (30 novel). We observe marked cross-ancestry heterogeneity at major risk loci, especially in African-ancestry populations which demonstrate a primary signal in a major histocompatibility complex class II haplotype and reduced risk at the established CFH and ARMS2/HTRA1 loci. Dissecting local ancestry in admixed individuals, we find significantly smaller marginal effect sizes for CFH risk alleles in African ancestry haplotypes. Broadening efforts to include ancestrally distinct populations helped uncover genes and pathways that boost risk in an ancestry-dependent manner and are potential targets for corrective therapies. Multi-ancestry genome-wide analyses identify new risk loci for age-related macular degeneration. Ancestry-specific analyses identify distinct effects at major risk loci, including smaller effect sizes for CFH risk alleles in haplotypes of African ancestry.

The early receptor current (ERC) is the charge redistribution occurring in plasma membrane rhodopsin during light activation of photoreceptors. Both the molecular mechanism of the ERC and its relationship to rhodopsin conformational activation are unknown. To investigate whether the ERC could be a time-resolved assay of rhodopsin structure-function relationships, the distinct sensitivity of modern electrophysiological tools was employed to test for flash-activated ERC signals in cells stably expressing normal human rod opsin after regeneration with 11- cis-retinal. ERCs are similar in waveform and kinetics to those found in photoreceptors. The action spectrum of the major R 2 charge motion is consistent with a rhodopsin photopigment. The R 1 phase is not kinetically resolvable and the R 2 phase, which overlaps metarhodopsin-II formation, has a rapid risetime and complex multiexponential decay. These experiments demonstrate, for the first time, kinetically resolved electrical state transitions during activation of expressed visual pigment in a unicellular environment (single or fused giant cells) containing only 6 × 10 6-8 × 10 7 molecules of rhodopsin. This method improves measurement sensitivity 7 to 8 orders of magnitude compared to other time-resolved techniques applied to rhodopsin to study the role particular amino acids play in conformational activation and the forces that govern those transitions.

We discovered an enhanced functionality hammerhead ribozyme (EhhRz), designed to act in trans against human rod opsin (RHO) mRNA, with turnover activity >300 nM min−1 under substrate-excess conditions and physiological Mg2+ levels (1 mM). We developed a real-time moderate-throughput fluorescence quantitative hhRz kinetic assay, which is linear with substrate and product moles and supported by gel-based measures. The EhhRz targets a CUC↓ cleavage site in a substrate with no predicted secondary/tertiary structure and demonstrates classic Michaelis-Menten turnover behavior when the substrate is in 10-fold excess (Vmax/Km up to 1.60 x 10(8) min−1 M−1 ), which is comparable to RNase A. EhhRzs show cooperative titration with a Kd of 0.73 ± 0.02 mM at cellular Mg2+ concentrations and a Hill coefficient of 1.73 ± 0.07. The upstream EhhRz antisense flank (bound to a downstream substrate flank) interacts with stem-loop II, and examinations of different variants revealed that a U7 residue in the downstream flank of the substrate is not essential for enhanced activity. Under single-turnover conditions with substrate pre-annealed to enzyme, reaction rates exceeded 1,000 min−1. These findings show that RNA catalysis approaches the efficiency of the ribosome and suggests EhhRz in trans is a druggable nucleic acid therapeutic. Competing Interest Statement The authors have declared no competing interest.

Methylobacterium is a prevalent bacterial genus of the phyllosphere. Despite its ubiquity, little is known about the extent to which its diversity reflects neutral processes like migration and drift, versus environmental filtering of life history strategies and adaptations. In two temperate forests, we investigated how phylogenetic diversity within Methylobacterium was structured by biogeography, seasonality, and growth strategies. Using deep, culture-independent barcoded marker gene sequencing coupled with culture-based approaches, we uncovered a considerable diversity of Methylobacterium in the phyllosphere. We cultured different subsets of Methylobacterium lineages depending upon the temperature of isolation and growth (20 °C or 30 °C), suggesting long-term adaptation to temperature. To a lesser extent than temperature adaptation, Methylobacterium diversity was also structured across large (>100km; between forests) and small geographical scales (<1.2km within forests), among host tree species, and was dynamic over seasons. By measuring growth of 79 isolates at different temperature treatments, we observed contrasting growth performances, with strong lineage- and season-dependent variations in growth strategies. Finally, we documented a progressive replacement of lineages with a high-yield growth strategy typical of cooperative, structured communities, in favor of those characterized by rapid growth, resulting in convergence and homogenization of community structure at the end of the growing season. Together our results show how Methylobacterium is phylogenetically structured into lineages with distinct growth strategies, which helps explain their differential abundance across regions, host tree species, and time. This works paves the way for further investigation of adaptive strategies and traits within a ubiquitous phyllosphere genus. Methylobacterium is a bacterial group tied to plants. Despite its ubiquity and importance to their hosts, little is known about the processes driving Methylobacterium community dynamics. By combining traditional culture-dependent and independent (metabarcoding) approaches, we monitored Methylobacterium diversity in two temperate forests over a growing season. On the surface of tree leaves, we discovered remarkably diverse and dynamic Methylobacterium communities over short temporal (from June to October) and spatial scales (within 1.2 km). Because we cultured different subsets of Methylobacterium diversity depending on the temperature of incubation, we suspected that these dynamics partly reflected climatic adaptation. By culturing strains in lab conditions mimicking seasonal variations, we found that diversity and environmental variations were indeed good predictors of Methylobacterium growth performances. Our findings suggest that Methylobacterium community dynamics at the surface of tree leaves results from the succession of strains with contrasted growth strategies in response to environmental variations.

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 transfers. We showed that Methylobacterium contains four evolutionary 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 of 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-living lifestyle to association with plant roots in Methylobacteriaceae ancestor, followed by phyllosphere association of three of the major groups (A, B, D), which early branching in Methylobacterium history was heavily obscured by HGT. Together, our work lays the foundations for a thorough redefinition of Methylobacterium taxonomy, beginning with the abandon of Methylorubrum. Competing Interest Statement The authors have declared no competing interest. Footnotes * https://github.com/JBLED/methylobacterium-phylogenomics.git

Late-onset ataxia is common, often idiopathic, and can result from cerebellar, proprioceptive, or vestibular impairment; when in combination, it is also termed cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS). We used non-parametric linkage analysis and genome sequencing to identify a biallelic intronic AAGGG repeat expansion in the replication factor C subunit 1 ( RFC1 ) gene as the cause of familial CANVAS and a frequent cause of late-onset ataxia, particularly if sensory neuronopathy and bilateral vestibular areflexia coexist. The expansion, which occurs in the poly(A) tail of an AluSx3 element and differs in both size and nucleotide sequence from the reference (AAAAG) 11 allele, does not affect RFC1 expression in patient peripheral and brain tissue, suggesting no overt loss of function. These data, along with an expansion carrier frequency of 0.7% in Europeans, implies that biallelic AAGGG expansion in RFC1 is a frequent cause of late-onset ataxia. Biallelic expansion of an intronic AAGGG repeat in RFC1 is identified here as a common cause of late-onset ataxia. This expansion occurs in the poly(A) tail of an AluSx3 element and is observed at a carrier frequency of 0.7% in populations of European ancestry.

Bacteriophages largely impact bacterial communities via lysis, gene transfer, and metabolic reprogramming and thus are increasingly thought to alter nutrient and energy cycling across many of Earth’s ecosystems. However, there are few model systems to mechanistically and quantitatively study phage-bacteria interactions, especially in soil systems. Here, we isolated, sequenced, and genomically characterized 24 novel phages infecting Klebsiella sp. M5al, a plant growth-promoting, nonencapsulated rhizosphere-associated bacterium, and compared many of their features against all 565 sequenced, dsDNA Klebsiella phage genomes. Taxonomic analyses revealed that these Klebsiella phages belong to three known phage families ( Autographiviridae , Drexlerviridae , and Straboviridae ) and two newly proposed phage families (Candidatus Mavericviridae and Ca. Rivulusviridae ). At the phage family level, we found that core genes were often phage-centric proteins, such as structural proteins for the phage head and tail and DNA packaging proteins. In contrast, genes involved in transcription, translation, or hypothetical proteins were commonly not shared or flexible genes. Ecologically, we assessed the phages’ ubiquity in recent large-scale metagenomic datasets, which revealed they were not widespread, as well as a possible direct role in reprogramming specific metabolisms during infection by screening their genomes for phage-encoded auxiliary metabolic genes (AMGs). Even though AMGs are common in the environmental literature, only one of our phage families, Straboviridae , contained AMGs, and the types of AMGs were correlated at the genus level. Host range phenotyping revealed the phages had a wide range of infectivity, infecting between 1–14 of our 22 bacterial strain panel that included pathogenic Klebsiella and Raoultella strains. This indicates that not all capsule-independent Klebsiella phages have broad host ranges. Together, these isolates, with corresponding genome, AMG, and host range analyses, help build the Klebsiella model system for studying phage-host interactions of rhizosphere-associated bacteria.