To achieve this, we predicted genes for all co-assembled contigs greater than 1,000bp in length and assigned functional categories to all genes using the Clusters of Orthologous Groups (COG) categories, which resulted in 203,620 bacterial genes with COG annotations for downstream analysis. The .gov means its official. Science 2015;348. Raw sequence libraries deposited in the Sequence Read Archive (accession no. The O2 pretreatment involved either a 5% or 10% vol/vol addition of O2 in the incubation headspace via flushing with a mixture of analytical grade air and 100% N2. 1973;113:52939. As part of this study, we recovered four Actinobacterial genomes classified within the class Thermoleophilia, a recently proposed lineage with very few species cultivated and characterized54; to date, there are only 18 reference genomes that belong to this class on the NCBI database (Taxonomy ID: 1497346), and none are closely related to the MAGs identified in this study. de Vries, F. T., Griffiths, R. I., Knight, C. G., Nicolitch, O. 2020 Mar 3;21(5):1734. doi: 10.3390/ijms21051734. Xu, L. & Coleman-Derr, D. Causes and consequences of a conserved bacterial root microbiome response to drought stress. In total, we retrieved 49,816,847 contigs with 29,639,429,852 base pairs. 6a), and that little change is observed between tom1 and wild type under drought conditions (PERMANOVA, F-statistic=0.9656, R2=0.0645, p-value = 0.5373, Fig. Annotations for genes of interest were compared across the various annotation tool outputs and double-checked by BLASTP searches against the NCBI nonredundant protein, RefSeq, and UniprotKB/Swiss-port databases. Particularly intriguing is the presence of alternative nitrogenase systems in several MAGs, which use a vanadium-iron (V-Fe) or an iron-iron (Fe-Fe) cofactor instead of the canonical molybdenum-iron (Mo-Fe) cofactor. Characterization of the gut microbiome using 16S or shotgun metagenomics. Several hypotheses regarding the underlying causes of this conserved pattern in microbiome development have recently been proposed16, based primarily on putative genetic properties and metabolic capabilities of the enriched taxonomic groups and collective activity of these drought-stressed communities as assayed by metatranscriptomics6. Functional traits whose presence significantly (P<0.05) differs between GCD-MAGs and non-GCD-MAGs. & Green, J. L. Incorporating 16S gene copy number information improves estimates of microbial diversity and abundance. Becklin, K. M. et al. To fulfill their needs for iron, all living organisms have highly specialized, and in some cases lineage-specific, systems to solubilize, transport, and store ferric (Fe2+) or ferrous (Fe3+) iron. Proc. Therefore, fluctuating O2 levels due to changes in water table position can be a major control on peat carbon stability. By submitting a comment you agree to abide by our Terms and Community Guidelines. To directly measure the effect of the drought treatments on plant performance, measurements of the Crop Water Stress Index (CSWI), which serves as an approximation for reductions in the levels of active leaf transpiration22,23, were taken at a subset of time points throughout the experiment (Supplementary Fig. The microbial degradation of the relatively nutrient-poor sphagnum litter yields little nitrogen (17), and the external input of nitrogen into acidic peatlands is primarily from atmospheric deposition (47). 11, 28642868 (2017). A. Metagenomic and 16S rRNA gene data previously indicated an enrichment of phenol oxidase-harboring Novosphingobium in peat incubations following oxygenation (26). Newberry EA, Bhandari R, Minsavage GV, Timilsina S, Jibrin MO, Kemble J, Sikora EJ, Jones JB, Potnis N. Appl Environ Microbiol. GhostKOALA (74) and eggNOG-mapper (75, 76) were used to obtain KO annotations, which were then used for metabolic reconstructions via the Reconstruct Pathway tool in KEGG Mapper (77). Google Scholar. XJW, JJP, and WL troubleshooted the methods and wrote the paper. Here we employ genome-resolved metagenomics and comparative genomics to demonstrate that carbohydrate and secondary metabolite transport functionalities are overrepresented within drought-enriched taxa. Received 2022 Jan 17; Accepted 2022 Aug 1. Published by Oxford University Press. Values are means SD (n=7) from seven independent biological replicates. Bold text represents genomes, Selected metabolic features across the assembled genomes. 2). CWSI has been described as a relative indicator of the levels of crop water stress, with values closer to 0.0 indicating non-water-stressed plants that transpire at levels indicating nonlimiting soil water availability, and a value of 1.0 indicating maximum water stress, with essentially no transpiration. Plant 8, 439453 (2015). Segregating individual plants from a test-cross family were genotyped, and homozygous wild-type and mutant siblings were isolated for the rhizosphere sampling. L.R. 2017. We observed similar patterns in gene and transcript abundance between two geochemically . This may indicate that in phosphorus-limited environments, non-GCD-MAGs compete more effectively for phosphorus resources with other biota than GCD-MAGs. Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS. The Stordalen Mire genomes (21), which constitute the majority of the presently available Terracidiphilus reference genomes (Fig. Download FIGS3, TIF file, 2.6 MB. Crits-Christoph A, Olm MR, Diamond S, Bouma-Gregson K, Banfield JF. Kim, S. A., LaCroix, I. S., Gerber, S. A. Sixty-four MAGs satisfied the conditions of medium-quality genome (completeness > 50%, contamination < 10%) and seven MAGs showed high quality (completeness > 90% and contamination < 10%). Loper, J. E. & Buyer, J. S. Siderophores in microbial interactions on plant surfaces. In the second cluster (n=114), gene expression generally increased under drought stress and decreased upon re-watering (Fig. Yang C, Chowdhury D, Zhang Z, Cheung WK, Lu A, Bian Z, Zhang L. Comput Struct Biotechnol J. PubMed Central 8a), iron is oxidized to iron hydroxide (Fe(OH)3), which is poorly soluble in water, and not available for assimilation by both plants and microbes70,71. Though iron is one of the most abundant elements in the crust of the earth, its availability to living organisms is limited by its oxidation state, solubility, and other environmental factors. Sci. Examining plant physiological responses to climate change through an evolutionary lens. 3B). We performed two types of assembly. Methods 11, 11441146 (2014). Greening C, Biswas A, Carere CR, Jackson CJ, Taylor MC, Stott MB, Cook GM, Morales SE. Google Scholar. (B) Average nucleotide identity (ANI) comparison among the MAGs assembled here. Jez, J. M., Lee, S. G. & Sherp, A. M. The next green movement: plant biology for the environment and sustainability. Increasing evidence shows that the chicken gastrointestinal microbiota has a major effect on the modulation of metabolic functions and is correlated . Brumfield, K. D., Huq, A., Colwell, R. R., Olds, J. L. & Leddy, M. B. Microbial resolution of whole genome shotgun and 16S amplicon metagenomic sequencing using publicly available NEON data. The control plants were watered every five days with 350ml of water/pot. Recovery of genomes from metagenomes via a dereplication, aggregation and scoring strategy. These two genera fall within SDs 1 and 8, classes Acidobaceriia and Holophagae, respectively. On this front, genome-resolved metagenomics has emerged as a highly effective way to identify genomes directly from the metagenomic assembly outputs, bypassing cultivation to explore the gene content of individual microbial populations. Sci. Changes in microbial functional potential and metabolism. The vials were either incubated under continuously anoxic conditions for 232days, or exposed to O2 for 98days, followed by anoxic incubation for 134days. Moreover, genes involved in phosphate metabolism, polysaccharide metabolism, cell motility and microbial competition were markedly enriched in GCD-MAGs (P<0.05; Fig. Nat. In this study, metagenome data helped validate broad compositional shifts obtained through amplicon techniques in the same study6, corroborating previously described enrichment of Actinobacteria in drought-stressed roots observed in a variety of plant systems6,9,10,11,14,15. Microbiol. Li, D., Liu, C.-M., Luo, R., Sadakane, K. & Lam, T.-W. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Ribeiro, M. & Simes, M. Advances in the antimicrobial and therapeutic potential of siderophores. Abbreviations refer to peat layers and are as follows: UNS, unsaturated; AWT, above water table; and BWT, below water table. Log2 fold expression changes across time (x-axis) in drought-stressed root tissue for the approximately 234 expressed genes annotated as related to iron homeostasis (see Supplementary Data3). 1A), appeared to dominate in the O2-pretreated samples (Fig. Anoxic conditions were established by flushing the serum vials that contained the peat slurries with 100% N2 gas. The tuned model differentiated between GCD-MAGs and non-GCD-MAGs with a prediction accuracy of 92.25%, thereby corroborating differences in genetic potential (Fig. I. Mediterranean grassland soil C-N compound turnover is dependent on rainfall and depth, and is mediated by genomically divergent microorganisms. Iron reduction by diverse actinobacteria under oxic and pH-neutral conditions and the formation of secondary minerals. This analysis revealed that 15 of the 19 drought-stress-enriched COGs in inorganic ion transport and metabolism were specifically related to iron metabolism (Supplementary Data1). Various hydrolytic extracellular enzymes produced by microorganisms are the key catalytic agents mediating these degradation pathways (24). 2020. Biol. The potential for aerobic respiration in these lineages therefore requires further experimental validation. Ault, T. R. On the essentials of drought in a changing climate. These population dynamics were accompanied by the corresponding type 3 secreted effector repertoires associated with the co-occurring X. perforans genotypes, indicating that metapopulation structure within fields should be considered when assessing the adaptive potential of X. perforans. 7, 384394 (2013). We only kept the taxonomy level that shows consistency between the two software packages. The tree was visualized in FigTree (http://tree.bio.ed.ac.uk/software/figtree/) and modified in Inkscape (https://inkscape.org) to highlight clusters of interest. provided background and interpretation of functional annotations of iron metabolism-related genes. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. 4b, d), which was absent in previously published soil metatranscriptomics datasets6. Methanogenesis in McLean Bog, an acidic peat bog in upstate New York: stimulation by H2/CO2 in the presence of rifampicin, or by low concentrations of acetate. 295, 1344413457 (2020). Microbiol. Microbiol. Plant Sci. 2020;14:75770. Quality trimming was performed using the phred trimming method set at Q6. -, Hu Y, Yang X, Qin J, et al. To quantify bacterial abundance in the root, we performed qPCR using bacterial lineage-specific primers as previously described6. The number of copies for each transporter gene has been normalized to the total number of protein coding sequences identified in each genome. Ivanova AA, Wegner C-E, Kim Y, Liesack W, Dedysh SN. . (1) read preprocessing, (2) metagenome assembly and QC, (3) genome binning and refinement and (4) taxonomic and functional annotation of the recovered MAGs. Solid circles indicate that the enrichment was significant (p-value of <= 0.05) in a one-sided hypergeometric test. Cantalapiedra, C. P. et al. These include group 1 respiratory hydrogen-oxidizing hydrogenases (Groups 1c and 1d), group 3 putatively bidirectional hydrogenases (3b and 3d), and group 4 respiratory hydrogenases (4b and 4d). 2). Briefly, peat samples of three distinct layers (i.e., unsaturated, below water table, and above water table) collected from the Ward Reservation (Andover, MA, USA) were ground into layer-specific slurries with filtered porewater (10% vol/vol) while continuously being flushed with nitrogen gas (N2). Geochemical and molecular evidence pointed to O2 facilitating the breakdown of complex aromatic lignin and tannin-like carbon compounds, followed by hydrogen (H2)-evolving and CO2-evolving fermentation, and eventually leading to the proliferation of the genus Methanobacterium, which utilized this higher substrate flux (i.e., H2 and CO2) to generate methane (26). PLoS Comput. 9, 157 (2018). One way to probe the lineage-resolved ecophysiology of uncultivated microbes is to assess if an environmental change elicits differential responses among subpopulations. Collectively, these data demonstrate that perturbation of iron metabolism-related genetic pathways in the root can impact rhizosphere composition, and that the observed shifts include changes in Actinobacterial abundance similar to those observed under drought. already built in. In contrast, bacteria with smaller genomes have high nutrient use efficiency but reduced nutrient demand for genome replication and therefore competitive advantages to survive in nutrient-limited environments [12, 13]. PDF | Plant growth-promoting microbes (PGPMs) have attracted increasing attention because they may be useful in increasing crop yield in a low-input and. FOIA This result is consistent with a model in which iron limitation in the root promotes Streptomyces growth and hinders the growth of Pseudomonas. Nat. Pyrosequencing-based assessment of the bacteria diversity in surface and subsurface peat layers of a northern wetland, with focus on poorly studied phyla and candidate divisions. The MAG analysis further suggests fermentative H2 production by the Holophaga, which potentially contributes to the elevated methane production by the hydrogenotrophic methanogen Methanobacterium. These data suggests that even within the phylum Actinobacteria distinct drought response phenotypes may exist. EMBnet. Ellipses show the parametric smallest area around the mean that contains 95% of the probability mass for each genotype. The ecology of Acidobacteria: moving beyond genes and genomes. Here, the shotgun data also revealed enrichment of two of these categories under drought stress at the peak of drought, specifically carbohydrate transport and metabolism and secondary metabolite transport and metabolism (Fig. Hierarchical clustering analysis demonstrates two broad patterns of gene expression: a set of genes exhibiting strong downregulation under drought stress (indicated with purple tree branches), and a set of genes exhibiting strong upregulation under drought stress (indicated with pink tree branches). On average, the 79 GCD-MAGs (ranging from 1.73 to 6.75 Mbp) had significantly (P<0.01) larger genomes (4.10 Mbp) than did the 393 non-GCD-MAGs (3.62 Mbp) (Figs. 2020 Society for Applied Microbiology and John Wiley & Sons Ltd. processed the metagenomes, assembled the MAGs, and analyzed the assembled genomes. Relative expression levels were calculated according to the 22CT (cycle threshold) method103, and the standard deviation was calculated among the three biological replicates. Plant Sci. However, these approaches have certain limitations. RNA was extracted using the QIAGEN miRNeasy Mini Kit (Cat. Schmidt MWI, Torn MS, Abiven S, Dittmar T, Guggenberger G, Janssens IA, Kleber M, Kgel-Knabner I, Lehmann J, Manning DAC, Nannipieri P, Rasse DP, Weiner S, Trumbore SE. Root total RNA was isolated using Trizol. This is an open-access article distributed under the terms of the, Acidobacteria, metagenomics, peatland biogeochemical cycling, peatland microbiome, soil redox dynamics, Metagenome-assembled genome statistics and taxonomic classifications, (A) Maximum likelihood phylogenomic tree inferred using a concatenated alignment of select ribosomal proteins. 30, 923930 (2014). At least in surface peat, diazotrophy appears to be mediated by Alphaproteobacteria (38, 57). However, the Anf and Vnf gene clusters in these genomes appear to be sufficient to produce a functional nitrogenase enzyme, as indicated by the presence of various genes required for cofactor biosynthesis (Fig. Nature 2007;449:80410. Here, we reconstructed 472 metagenome-assembled genomes (MAGs) covering agricultural soils from six long-term field trials across China. USA 112, E911E920 (2015). Lau, C. K. Y., Krewulak, K. D. & Vogel, H. J. Bacterial ferrous iron transport: the Feo system. Biotechnol. Recovery of nearly 8,000 metagenome-assembled genomes substantially expands the tree of life. Sieber, C. M. K. et al. Epub 2018 May 2. This culture-free approach, termed genome-resolved metagenomics 48, has been successfully applied to probe microbiome function in multiple environments, including hospital rooms, the human gut, and oceans 17, 49 - 51, and has improved our understanding of the microbial world and its response to perturbation. A drip system was utilized to apply all irrigation water during the growing season, consisting of drip lines placed on the surface of each furrow (0.76-m row spacing), with 0.3-m emitter spacing and 2L/h emitter output. Use the Previous and Next buttons to navigate three slides at a time, or the slide dot buttons at the end to jump three slides at a time. Molybdenum-based diazotrophy in a sphagnum peatland in northern Minnesota. Google Scholar. . Microbiol. 2019 Jul 19;20(4):1140-1150. doi: 10.1093/bib/bbx098. The analysis was performed at KEGG level 3 using DESeq2 and involved the 472 MAGs obtained in this study (e). J. Bot. Science (2020) https://doi.org/10.1126/science.abd0695. We wish to thank Dr. Jing Tian, Miss Qi Li, Mr. Tianxing Ma, and Mr. Zhibin He, who helped with soil sampling. Multiple nifDK clusters, including the longer nifKDBHX cluster that is illustrated here, were present in the Holophaga genomes. Article Three biological replicates were performed for each reverse transcription-PCR experiment. b Increased soil aeration leads to reduced iron availability for plants and microbes, as iron becomes increasingly stored as insoluble Fe3+ (shown in red text). While a redox-related differential enrichment of the Terracidiphilus and Holophaga was evident in each peat layer tested, there were notable differences in the population structures of the two groups between peat layers. Additionally, these new data help identifies new bacterial genetic traits associated with enrichment under drought stress, including the functional category inorganic ion transport. Evol. 49, 16 (2019). Shivlata, L. & Satyanarayana, T. Thermophilic and alkaliphilic actinobacteria: biology and potential applications. Agric. S5). Taken together, these results reiterate the potential importance of carbohydrate and amino acid transport, and implicate the new function of iron metabolism and transport, for microbial fitness in the drought-stressed rhizosphere. Download TableS1, PDF file, 0.2 MB. 5b). Target-enriched long-read sequencing (TELSeq) contextualizes antimicrobial resistance genes in metagenomes, Disentangling the genetic basis of rhizosphere microbiome assembly in tomato, Exploring rhizo-microbiome transplants as a tool for protective plant-microbiome manipulation, https://www.maizegdb.org/gene_center/gene/GRMZM2G063306, https://github.com/LingXu2018/EPICON_IRON, https://doi.org/10.1101/2020.12.21.419267, https://doi.org/10.1016/j.tplants.2020.03.014, https://doi.org/10.1038/s41579-020-0368-1, https://doi.org/10.1038/s41564-020-0719-8, https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, http://CRAN.R-project.org/package=pheatmap, http://creativecommons.org/licenses/by/4.0/. Proc. Mol. 2). A. et al. CAP analysis for all samples indicates that genotype explains 4.6% of the total variance, while under treatment, explains 53.4% (Supplementary Fig. Importantly, our data also suggest a previously unrecognized role of Acidobacteria in peat nitrogen cycling, as the potential for diazotrophy was found in both the Holophaga and the Terracidiphilus genomes. Ivanova AA, Zhelezova AD, Chernov TI, Dedysh SN. Our genome-centric approach enabled a detailed resolution of the mechanisms of niche differentiation within this abundant phylum across the O2 treatments. Neighborhood comparisons were also employed to improve confidence in annotations (e.g., nitrogenases). & McDowell, J. M. Iron homeostasis and plant immune responses: recent insights and translational implications. Are oxygen limitations under recognized regulators of organic carbon turnover in upland soils? Six of these MAGs represent a species-level cluster, sharing>97% average nucleotide identity (ANI) among them (Fig. 2005;165:899912. 2021. CAZyme annotations agreed upon by both HMMER and DIAMOND were retained for downstream analysis. Natl Acad. Rep. 9, 16144 (2019). 17, 14851494 (2019). In-depth knowledge and hands-on experience with several different types of datasets, including Whole-genome, RNA-Seq, and Metagenomics. PubMed Central The majority of the characterized members are within SD 1 (class Acidobacteriia, order Acidobacteriales) and SD 3 (class Acidobacteriia, order Bryobacterales) (4, 11, 12). 40, D1178D1186 (2012). See (26) for detailed descriptions of the layers. Diazotrophy has been attributed to Sphagnum-associated microorganisms as well as to free-living Cyanobacteria, methanotrophs, and heterotrophic bacteria across peat systems (38, 39, 41,47), although the relative contribution from each functional guild remains largely unknown. Microorganisms. 2) and may instead benefit from the breakdown products of polysaccharide hydrolysis that can be acquired using the diverse repertoire of transporters found in these genomes (Fig. S1). Next, we used the Ribo-Zero rRNA Removal Kit (Bacteria, Illumina, Catalog No. ISME J. 1A). PubMed C.G. Large-scale differences in microbial biodiversity discovery between 16S amplicon and shotgun sequencing. The resulting amplicon libraries produced on average approximately 45259, 41582, and 34649 reads per sample for soils, rhizospheres, and roots, respectively. While Acidobacteria, particularly Holophaga that were enriched in the O2-pretreated incubations, appeared to be key members of the microbial community following oxygenation, their specific roles in the biogeochemical response to transient oxygenation remained unclear. ISME J. Santos-Medelln, C., Edwards, J., Liechty, Z., Nguyen, B. Nat Ecol Evol. While these dynamics of iron competition and exchange have long been conjectured to be critical for shaping microbemicrobe and microbehost dynamics78, it remains challenging to measure and assess them in the context of complex communities in and on host tissues, such as roots and rhizospheres. Front. The tree was re-rooted using an Archaeal outgroup clade. 1C) appears to result largely from the O2 stimulating the degradation of polyphenolic and other complex plant compounds (26), which provides additional carbon and energy flux for anaerobic carbon conversions, possibly supported by the removal of the phenolic inhibition of hydrolase activity (27, 55). The PCR conditions for the amplification were 95C for 2min, and 50 two-step cycles (95C for 10s, 60Cs for 25s) followed by a plate read; a melt curve was generated by heating from 72 to 95C with 0.2C increments. Among these genes, biomarker genes are involved in carbon utilization (K00114 alcohol dehydrogenase, K05349 beta-glucosidase, K01785 aldose 1-epimerase), cell motility (K02556 chemotaxis protein MotA) and biosynthesis of enediyne antibiotics (K21162 enediyne biosynthesis protein E4). The concentration was assessed using a Qubit 3 Fluorometer (Invitrogen, Carlsbad, CA, USA) and quality was assessed using an Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). 290, 2768827699 (2015). Indeed, the Holophaga isolate H. foetida harbors a low-affinity terminal oxidase, despite being described as obligately anaerobic (5). Plant Stress 5, 3241 (2011). ISME J. For all plots analyzed in this study, one of two irrigation treatments (drought or control) was applied. MaizeGDB 2018: the maize multi-genome genetics and genomics database. 1A). mBio. 2008. Front. Despite the importance of PSB for mediating phosphorus limitation in soils, genome-centric metagenomics research assessing phosphate solubilization potential in terrestrial microbiomes is scarce. Spohn M, Kuzyakov Y. Phosphorus mineralization can be driven by microbial need for carbon. 1B). Nat. Peter Daszak's EcoHealth Alliance repurposed grants to Wuhan Insistitue of Virology for the Gain-of-Threat experiments on bat viruses causing Covid-19 Pandemic. 1C). Genome-resolved metagenomics bioinformatics. 2010;3:55761. Dots to the right of a bar indicate non-significantly enriched MAGs, p-value > 0.05; asterisks to the right of the bar indicate significantly enriched MAGs, p-value < 0.05. Our results improve the knowledge of both the diversity and the genetic composition of phosphate-solubilizing bacteria. PLoS One 9, e95445 (2014). Drought treatment was imposed on one-half of the blocks and consisted of a complete lack of irrigation until rewetting at the ninth week (TP9) after planting (WAP). Knorr, K.-H. & Blodau, C. Impact of experimental drought and rewetting on redox transformations and methanogenesis in mesocosms of a northern fen soil. In addition to providing insight into community-wide shifts in functional capacity, metagenomic sequencing also enables the reassembly of bacterial genomes from environmental samples for within-community comparative genomic analyses47. 2014. The typical genome-resolved analysis of metagenomes, obtained through any source, typically involves four steps i.e. Black, unhighlighted leaves represent publicly available Acidobacteria MAGs downloaded from GenBank.
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