Large-scale replicated field study of maize rhizosphere identifies heritable microbes.

Soil microbes that colonize plant roots and are responsive to differences in plant genotype remain to be ascertained for agronomically important crops. From a very large-scale longitudinal field study of 27 maize inbred lines planted in three fields, with partial replication 5 y later, we identify root-associated microbiota exhibiting reproducible associations with plant genotype. Analysis of 4,866 samples identified 143 operational taxonomic units (OTUs) whose variation in relative abundances across the samples was significantly regulated by plant genotype, and included five of seven core OTUs present in all samples. Plant genetic effects were significant amid the large effects of plant age on the rhizosphere microbiome, regardless of the specific community of each field, and despite microbiome responses to climate events. Seasonal patterns showed that the plant root microbiome is locally seeded, changes with plant growth, and responds to weather events. However, against this background of variation, specific taxa responded to differences in host genotype. If shown to have beneficial functions, microbes may be considered candidate traits for selective breeding.

Trichodesmium genome maintains abundant, widespread noncoding DNA in situ, despite oligotrophic lifestyle.

Understanding the evolution of the free-living, cyanobacterial, diazotroph Trichodesmium is of great importance because of its critical role in oceanic biogeochemistry and primary production. Unlike the other >150 available genomes of free-living cyanobacteria, only 63.8% of the Trichodesmium erythraeum (strain IMS101) genome is predicted to encode protein, which is 20-25% less than the average for other cyanobacteria and nonpathogenic, free-living bacteria. We use distinctive isolates and metagenomic data to show that low coding density observed in IMS101 is a common feature of the Trichodesmium genus, both in culture and in situ. Transcriptome analysis indicates that 86% of the noncoding space is expressed, although the function of these transcripts is unclear. The density of noncoding, possible regulatory elements predicted in Trichodesmium, when normalized per intergenic kilobase, was comparable and twofold higher than that found in the gene-dense genomes of the sympatric cyanobacterial genera Synechococcus and Prochlorococcus, respectively. Conserved Trichodesmium noncoding RNA secondary structures were predicted between most culture and metagenomic sequences, lending support to the structural conservation. Conservation of these intergenic regions in spatiotemporally separated Trichodesmium populations suggests possible genus-wide selection for their maintenance. These large intergenic spacers may have developed during intervals of strong genetic drift caused by periodic blooms of a subset of genotypes, which may have reduced effective population size. Our data suggest that transposition of selfish DNA, low effective population size, and high-fidelity replication allowed the unusual "inflation" of noncoding sequence observed in Trichodesmium despite its oligotrophic lifestyle.

Environment-specific virocell metabolic reprogramming.

Viruses impact microbial systems through killing hosts, horizontal gene transfer, and altering cellular metabolism, consequently impacting nutrient cycles. A virus-infected cell, a "virocell," is distinct from its uninfected sister cell as the virus commandeers cellular machinery to produce viruses rather than replicate cells. Problematically, virocell responses to the nutrient-limited conditions that abound in nature are poorly understood. Here we used a systems biology approach to investigate virocell metabolic reprogramming under nutrient limitation. Using transcriptomics, proteomics, lipidomics, and endo- and exo-metabolomics, we assessed how low phosphate (low-P) conditions impacted virocells of a marine Pseudoalteromonas host when independently infected by two unrelated phages (HP1 and HS2). With the combined stresses of infection and nutrient limitation, a set of nested responses were observed. First, low-P imposed common cellular responses on all cells (virocells and uninfected cells), including activating the canonical P-stress response, and decreasing transcription, translation, and extracellular organic matter consumption. Second, low-P imposed infection-specific responses (for both virocells), including enhancing nitrogen assimilation and fatty acid degradation, and decreasing extracellular lipid relative abundance. Third, low-P suggested virocell-specific strategies. Specifically, HS2-virocells regulated gene expression by increasing transcription and ribosomal protein production, whereas HP1-virocells accumulated host proteins, decreased extracellular peptide relative abundance, and invested in broader energy and resource acquisition. These results suggest that although environmental conditions shape metabolism in common ways regardless of infection, virocell-specific strategies exist to support viral replication during nutrient limitation, and a framework now exists for identifying metabolic strategies of nutrient-limited virocells in nature.

Genetic determinants of switchgrass-root-associated microbiota in field sites spanning its natural range.

A fundamental goal in plant microbiome research is to determine the relative impacts of host and environmental effects on root microbiota composition, particularly how host genotype impacts bacterial community composition. Most studies characterizing the effect of plant genotype on root microbiota undersample host genetic diversity and grow plants outside of their native ranges, making the associations between host and microbes difficult to interpret. Here, we characterized the root microbiota of a large diversity panel of switchgrass, a North American native C4 bioenergy crop, in three field locations spanning its native range. Our data, composed of 1,961 samples, suggest that field location is the primary determinant of microbiome composition; however, substantial heritable variation is widespread across bacterial taxa, especially those in the Sphingomonadaceae family. Despite diverse compositions, relatively few highly prevalent taxa make up the majority of the switchgrass root microbiota, a large fraction of which is shared across sites. Local genotypes preferentially recruit/filter for local microbes, supporting the idea of affinity between local plants and their microbiota. Using genome-wide association, we identified loci impacting the abundance of >400 microbial strains and found an enrichment of genes involved in immune responses, signaling pathways, and secondary metabolism. We found loci associated with over half of the core microbiota (i.e., microbes in >80% of samples), regardless of field location. Finally, we show a genetic relationship between a basal plant immunity pathway and relative abundances of root microbiota. This study brings us closer to harnessing and manipulating beneficial microbial associations via host genetics.

Taxonomic distribution of metabolic functions in bacteria associated with Trichodesmium consortia.

IMPORTANCE: Colonies of the cyanobacteria Trichodesmium act as a biological hotspot for the usage and recycling of key resources such as C, N, P, and Fe within an otherwise oligotrophic environment. While Trichodesmium colonies are known to interact and support a unique community of algae and particle-associated microbes, our understanding of the taxa that populate these colonies and the gene functions they encode is still limited. Characterizing the taxa and adaptive strategies that influence consortium physiology and its concomitant biogeochemistry is critical in a future ocean predicted to have increasingly resource-depleted regions.

Identification of beneficial and detrimental bacteria impacting sorghum responses to drought using multi-scale and multi-system microbiome comparisons.

Drought is a major abiotic stress limiting agricultural productivity. Previous field-level experiments have demonstrated that drought decreases microbiome diversity in the root and rhizosphere. How these changes ultimately affect plant health remains elusive. Toward this end, we combined reductionist, transitional and ecological approaches, applied to the staple cereal crop sorghum to identify key root-associated microbes that robustly affect drought-stressed plant phenotypes. Fifty-three Arabidopsis-associated bacteria were applied to sorghum seeds and their effect on root growth was monitored. Two Arthrobacter strains caused root growth inhibition (RGI) in Arabidopsis and sorghum. In the context of synthetic communities, Variovorax strains were able to protect plants from Arthrobacter-caused RGI. As a transitional system, high-throughput phenotyping was used to test the synthetic communities. During drought stress, plants colonized by Arthrobacter had reduced growth and leaf water content. Plants colonized by both Arthrobacter and Variovorax performed as well or better than control plants. In parallel, we performed a field trial wherein sorghum was evaluated across drought conditions. By incorporating data on soil properties into the microbiome analysis, we accounted for experimental noise with a novel method and were able to observe the negative correlation between the abundance of Arthrobacter and plant growth. Having validated this approach, we cross-referenced datasets from the high-throughput phenotyping and field experiments and report a list of bacteria with high confidence that positively associated with plant growth under drought stress. In conclusion, a three-tiered experimental system successfully spanned the lab-to-field gap and identified beneficial and deleterious bacterial strains for sorghum under drought.

Sodalis ligni Strain 159R Isolated from an Anaerobic Lignin-Degrading Consortium.

Novel bacterial isolates with the capabilities of lignin depolymerization, catabolism, or both, could be pertinent to lignocellulosic biofuel applications. In this study, we aimed to identify anaerobic bacteria that could address the economic challenges faced with microbial-mediated biotechnologies, such as the need for aeration and mixing. Using a consortium seeded from temperate forest soil and enriched under anoxic conditions with organosolv lignin as the sole carbon source, we successfully isolated a novel bacterium, designated 159R. Based on the 16S rRNA gene, the isolate belongs to the genus Sodalis in the family Bruguierivoracaceae. Whole-genome sequencing revealed a genome size of 6.38 Mbp and a GC content of 55 mol%. To resolve the phylogenetic position of 159R, its phylogeny was reconstructed using (i) 16S rRNA genes of its closest relatives, (ii) multilocus sequence analysis (MLSA) of 100 genes, (iii) 49 clusters of orthologous groups (COG) domains, and (iv) 400 conserved proteins. Isolate 159R was closely related to the deadwood associated Sodalis guild rather than the tsetse fly and other insect endosymbiont guilds. Estimated genome-sequence-based digital DNA-DNA hybridization (dDDH), genome percentage of conserved proteins (POCP), and an alignment analysis between 159R and the Sodalis clade species further supported that isolate 159R was part of the Sodalis genus and a strain of Sodalis ligni. We proposed the name Sodalis ligni str. 159R (=DSM 110549 = ATCC TSD-177). IMPORTANCE Currently, in the paper industry, paper mill pulping relies on unsustainable and costly processes to remove lignin from lignocellulosic material. A greener approach is biopulping, which uses microbes and their enzymes to break down lignin. However, there are limitations to biopulping that prevent it from outcompeting other pulping processes, such as requiring constant aeration and mixing. Anaerobic bacteria are a promising alternative source for consolidated depolymerization of lignin and its conversion to valuable by-products. We presented Sodalis ligni str. 159R and its characteristics as another example of potential mechanisms that can be developed for lignocellulosic applications.

Genomes of three methylotrophs from a single niche reveal the genetic and metabolic divergence of the methylophilaceae.

The genomes of three representatives of the family Methylophilaceae, Methylotenera mobilis JLW8, Methylotenera versatilis 301, and Methylovorus glucosetrophus SIP3-4, all isolated from a single study site, Lake Washington in Seattle, WA, were completely sequenced. These were compared to each other and to the previously published genomes of Methylobacillus flagellatus KT and an unclassified Methylophilales strain, HTCC2181. Comparative analysis revealed that the core genome of Methylophilaceae may be as small as approximately 600 genes, while the pangenome may be as large as approximately 6,000 genes. Significant divergence between the genomes in terms of both gene content and gene and protein conservation was uncovered, including the varied presence of certain genes involved in methylotrophy. Overall, our data demonstrate that metabolic potentials can vary significantly between different species of Methylophilaceae, including organisms inhabiting the very same environment. These data suggest that genetic divergence among the members of this family may be responsible for their specialized and nonredundant functions in C1 cycling, which in turn suggests means for their successful coexistence in their specific ecological niches.

Genome sequence of Chthoniobacter flavus Ellin428, an aerobic heterotrophic soil bacterium.

Chthoniobacter flavus Ellin428 is the first isolate from the class Spartobacteria of the bacterial phylum Verrucomicrobia. C. flavus Ellin428 can metabolize many of the saccharide components of plant biomass but is incapable of growth on amino acids or organic acids other than pyruvate.

Genome sequence of the verrucomicrobium Opitutus terrae PB90-1, an abundant inhabitant of rice paddy soil ecosystems.

Bacteria of the deeply branching phylum Verrucomicrobia are rarely cultured yet commonly detected in metagenomic libraries from aquatic, terrestrial, and intestinal environments. We have sequenced the genome of Opitutus terrae PB90-1, a fermentative anaerobe within this phylum, isolated from rice paddy soil and capable of propionate production from plant-derived polysaccharides.

Genome sequence of "Pedosphaera parvula" Ellin514, an aerobic Verrucomicrobial isolate from pasture soil.

"Pedosphaera parvula" Ellin514 is an aerobically grown verrucomicrobial isolate from pasture soil. It is one of the few cultured representatives of subdivision 3 of the phylum Verrucomicrobia. Members of this group are widespread in terrestrial environments.

Complete genome sequence of the Thermophilic Bacterium Exiguobacterium sp. AT1b.

Here we present the genome of strain Exiguobacterium sp. AT1b, a thermophilic member of the genus Exiguobacterium whose representatives were isolated from various environments along a thermal and physicochemical gradient. This genome was sequenced to be a comparative resource for the study of thermal adaptation with a psychroactive representative of the genus, Exiguobacterium sibiricum strain 255-15, that was previously sequenced by the U.S. Department of Energy's (DOE's) Joint Genome Institute (JGI) (http://genome.ornl.gov/microbial/exig/).

Genome sequence of Victivallis vadensis ATCC BAA-548, an anaerobic bacterium from the phylum Lentisphaerae, isolated from the human gastrointestinal tract.

Victivallis vadensis ATCC BAA-548 represents the first cultured representative from the novel phylum Lentisphaerae, a deep-branching bacterial lineage. Few cultured bacteria from this phylum are known, and V. vadensis therefore represents an important organism for evolutionary studies. V. vadensis is a strictly anaerobic sugar-fermenting isolate from the human gastrointestinal tract.

Sequencing of multiple clostridial genomes related to biomass conversion and biofuel production.

Modern methods to develop microbe-based biomass conversion processes require a system-level understanding of the microbes involved. Clostridium species have long been recognized as ideal candidates for processes involving biomass conversion and production of various biofuels and other industrial products. To expand the knowledge base for clostridial species relevant to current biofuel production efforts, we have sequenced the genomes of 20 species spanning multiple genera. The majority of species sequenced fall within the class III cellulosome-encoding Clostridium and the class V saccharolytic Thermoanaerobacteraceae. Species were chosen based on representation in the experimental literature as model organisms, ability to degrade cellulosic biomass either by free enzymes or by cellulosomes, ability to rapidly ferment hexose and pentose sugars to ethanol, and ability to ferment synthesis gas to ethanol. The sequenced strains significantly increase the number of noncommensal/nonpathogenic clostridial species and provide a key foundation for future studies of biomass conversion, cellulosome composition, and clostridial systems biology.

The genome sequence of Methanohalophilus mahii SLP(T) reveals differences in the energy metabolism among members of the Methanosarcinaceae inhabiting freshwater and saline environments.

Methanohalophilus mahii is the type species of the genus Methanohalophilus, which currently comprises three distinct species with validly published names. Mhp. mahii represents moderately halophilic methanogenic archaea with a strictly methylotrophic metabolism. The type strain SLP(T) was isolated from hypersaline sediments collected from the southern arm of Great Salt Lake, Utah. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,012,424 bp genome is a single replicon with 2032 protein-coding and 63 RNA genes and part of the Genomic Encyclopedia of Bacteria and Archaea project. A comparison of the reconstructed energy metabolism in the halophilic species Mhp. mahii with other representatives of the Methanosarcinaceae reveals some interesting differences to freshwater species.

Genome Sequences of 42 Bacteria Isolated from Sorghum bicolor Roots.

Forty-two bacterial strains were isolated from root samples of Sorghum bicolor The strains spanned 17 genera, including Dechloromonas, Duganella, Dyella, Flavobacterium, Herbaspirillum, Lutibacter, Mucilaginibacter, Novosphingobium, Paraburkholderia, Pedobacter, Pleomorphomonas, Rhizobacter, Rhizobium, Rhizomicrobium, Rugamonas, Variovorax, and Xanthobacter Their whole-genome sequences revealed diverse metabolic processes, including biological nitrogen fixation, in sorghum root microbiota.

Phototrophic Co-cultures From Extreme Environments: Community Structure and Potential Value for Fundamental and Applied Research.

Cyanobacteria are found in most illuminated environments and are key players in global carbon and nitrogen cycling. Although significant efforts have been made to advance our understanding of this important phylum, still little is known about how members of the cyanobacteria affect and respond to changes in complex biological systems. This lack of knowledge is in part due to our dependence on pure cultures when determining the metabolism and function of a microorganism. We took advantage of the Culture Collection of Microorganisms from Extreme Environments (CCMEE), a collection of more than 1,000 publicly available photosynthetic co-cultures maintained at the Pacific Northwest National Laboratory, and assessed via 16S rRNA amplicon sequencing if samples readily available from public culture collection could be used in the future to generate new insights into the role of microbial communities in global and local carbon and nitrogen cycling. Results from this work support the existing notion that culture depositories in general hold the potential to advance fundamental and applied research. Although it remains to be seen if co-cultures can be used at large scale to infer roles of individual organisms, samples that are publicly available from existing co-cultures depositories, such as the CCMEE, might be an economical starting point for such studies. Access to archived biological samples, without the need for costly field work, might in some circumstances be one of the few remaining ways to advance the field and to generate new insights into the biology of ecosystems that are not easily accessible. The current COVID-19 pandemic, which makes sampling expeditions almost impossible without putting the health of the participating scientists on the line, is a very timely example.

Diversity and Distribution of a Novel Genus of Hyperthermophilic Aquificae Viruses Encoding a Proof-Reading Family-A DNA Polymerase.

Despite the high abundance of Aquificae in many geothermal systems, these bacteria are difficult to culture and no viruses infecting members of this phylum have been isolated. Here, we describe the complete, circular dsDNA Uncultivated Virus Genome (UViG) of Thermocrinis Octopus Spring virus (TOSV), derived from metagenomic data, along with eight related UViGs representing three additional viral species. Despite low overall similarity among viruses from different hot springs, the genomes shared a high degree of synteny, and encoded numerous genes for nucleotide metabolism, including a PolA-type DNA polymerase polyprotein with likely accessory functions, a DNA Pol III sliding clamp, a thymidylate kinase, a DNA gyrase, a helicase, and a DNA methylase. Also present were conserved genes predicted to code for phage capsid, large and small subunits of terminase, portal protein, holin, and lytic transglycosylase, all consistent with a distant relatedness to cultivated Caudovirales. These viruses are predicted to infect Aquificae, as multiple CRISPR spacers matching the viral genomes were identified within the genomes and metagenomic contigs from these bacteria. Based on the predicted atypical bi-directional replication strategy, low sequence similarity to known viral genomes, and unique position in gene-sharing networks, we propose a new putative genus, "Pyrovirus," in the order Caudovirales.

Phage-specific metabolic reprogramming of virocells.

Ocean viruses are abundant and infect 20-40% of surface microbes. Infected cells, termed virocells, are thus a predominant microbial state. Yet, virocells and their ecosystem impacts are understudied, thus precluding their incorporation into ecosystem models. Here we investigated how unrelated bacterial viruses (phages) reprogram one host into contrasting virocells with different potential ecosystem footprints. We independently infected the marine Pseudoalteromonas bacterium with siphovirus PSA-HS2 and podovirus PSA-HP1. Time-resolved multi-omics unveiled drastically different metabolic reprogramming and resource requirements by each virocell, which were related to phage-host genomic complementarity and viral fitness. Namely, HS2 was more complementary to the host in nucleotides and amino acids, and fitter during infection than HP1. Functionally, HS2 virocells hardly differed from uninfected cells, with minimal host metabolism impacts. HS2 virocells repressed energy-consuming metabolisms, including motility and translation. Contrastingly, HP1 virocells substantially differed from uninfected cells. They repressed host transcription, responded to infection continuously, and drastically reprogrammed resource acquisition, central carbon and energy metabolisms. Ecologically, this work suggests that one cell, infected versus uninfected, can have immensely different metabolisms that affect the ecosystem differently. Finally, we relate phage-host genome complementarity, virocell metabolic reprogramming, and viral fitness in a conceptual model to guide incorporating viruses into ecosystem models.

Metagenomes from Experimental Hydrologic Manipulation of Restored Coastal Plain Wetland Soils (Tyrell County, North Carolina).

Hydrologic changes modify microbial community structure and ecosystem functions, especially in wetland systems. Here, we present 24 metagenomes from a coastal freshwater wetland experiment in which we manipulated hydrologic conditions and plant presence. These wetland soil metagenomes will deepen our understanding of how hydrology and vegetation influence microbial functional diversity.