The exceptional form and function of the giant bacterium Ca. Epulopiscium viviparus revolves around its sodium motive force.

Epulopiscium spp. are the largest known heterotrophic bacteria; a large cigar-shaped individual is a million times the volume of Escherichia coli. To better understand the metabolic potential and relationship of Epulopiscium sp. type B with its host Naso tonganus, we generated a high-quality draft genome from a population of cells taken from a single fish. We propose the name Candidatus Epulopiscium viviparus to describe populations of this best-characterized Epulopiscium species. Metabolic reconstruction reveals more than 5% of the genome codes for carbohydrate active enzymes, which likely degrade recalcitrant host-diet algal polysaccharides into substrates that may be fermented to acetate, the most abundant short-chain fatty acid in the intestinal tract. Moreover, transcriptome analyses and the concentration of sodium ions in the host intestinal tract suggest that the use of a sodium motive force (SMF) to drive ATP synthesis and flagellar rotation is integral to symbiont metabolism and cellular biology. In natural populations, genes encoding both F-type and V-type ATPases and SMF generation via oxaloacetate decarboxylation are among the most highly expressed, suggesting that ATPases synthesize ATP and balance ion concentrations across the cell membrane. High expression of these and other integral membrane proteins may allow for the growth of its extensive intracellular membrane system. Further, complementary metabolism between microbe and host is implied with the potential provision of nitrogen and B vitamins to reinforce this nutritional symbiosis. The few features shared by all bacterial behemoths include extreme polyploidy, polyphosphate synthesis, and thus far, they have all resisted cultivation in the lab.

Competitive Exclusion and Metabolic Dependency among Microorganisms Structure the Cellulose Economy of an Agricultural Soil.

Microorganisms that degrade cellulose utilize extracellular reactions that yield free by-products which can promote interactions with noncellulolytic organisms. We hypothesized that these interactions determine the ecological and physiological traits governing the fate of cellulosic carbon (C) in soil. We performed comparative genomics with genome bins from a shotgun metagenomic-stable isotope probing experiment to characterize the attributes of cellulolytic and noncellulolytic taxa accessing 13C from cellulose. We hypothesized that cellulolytic taxa would exhibit competitive traits that limit access, while noncellulolytic taxa would display greater metabolic dependency, such as signatures of adaptive gene loss. We tested our hypotheses by evaluating genomic traits indicative of competitive exclusion or metabolic dependency, such as antibiotic production, growth rate, surface attachment, biomass degrading potential, and auxotrophy. The most 13C-enriched taxa were cellulolytic Cellvibrio (Gammaproteobacteria) and Chaetomium (Ascomycota), which exhibited a strategy of self-sufficiency (prototrophy), rapid growth, and competitive exclusion via antibiotic production. Auxotrophy was more prevalent in cellulolytic Actinobacteria than in cellulolytic Proteobacteria, demonstrating differences in dependency among cellulose degraders. Noncellulolytic taxa that accessed 13C from cellulose (Planctomycetales, Verrucomicrobia, and Vampirovibrionales) were also more dependent, as indicated by patterns of auxotrophy and 13C labeling (i.e., partial labeling or labeling at later stages). Major 13C-labeled cellulolytic microbes (e.g., Sorangium, Actinomycetales, Rhizobiales, and Caulobacteraceae) possessed adaptations for surface colonization (e.g., gliding motility, hyphae, attachment structures) signifying the importance of surface ecology in decomposing particulate organic matter. Our results demonstrated that access to cellulosic C was accompanied by ecological trade-offs characterized by differing degrees of metabolic dependency and competitive exclusion.IMPORTANCE Our study reveals the ecogenomic traits of microorganisms participating in the cellulose economy of soil. We identified three major categories of participants in this economy: (i) independent primary degraders, (ii) interdependent primary degraders, and (iii) secondary consumers (mutualists, opportunists, and parasites). Trade-offs between independent primary degraders, whose adaptations favor antagonism and competitive exclusion, and interdependent and secondary degraders, whose adaptations favor complex interspecies interactions, are expected to affect the fate of microbially processed carbon in soil. Our findings provide useful insights into the ecological relationships that govern one of the planet's most abundant resources of organic carbon. Furthermore, we demonstrate a novel gradient-resolved approach for stable isotope probing, which provides a cultivation-independent, genome-centric perspective into soil microbial processes.

NCBI's Virus Discovery Codeathon: Building "FIVE" -The Federated Index of Viral Experiments API Index.

Viruses represent important test cases for data federation due to their genome size and the rapid increase in sequence data in publicly available databases. However, some consequences of previously decentralized (unfederated) data are lack of consensus or comparisons between feature annotations. Unifying or displaying alternative annotations should be a priority both for communities with robust entry representation and for nascent communities with burgeoning data sources. To this end, during this three-day continuation of the Virus Hunting Toolkit codeathon series (VHT-2), a new integrated and federated viral index was elaborated. This Federated Index of Viral Experiments (FIVE) integrates pre-existing and novel functional and taxonomy annotations and virus-host pairings. Variability in the context of viral genomic diversity is often overlooked in virus databases. As a proof-of-concept, FIVE was the first attempt to include viral genome variation for HIV, the most well-studied human pathogen, through viral genome diversity graphs. As per the publication of this manuscript, FIVE is the first implementation of a virus-specific federated index of such scope. FIVE is coded in BigQuery for optimal access of large quantities of data and is publicly accessible. Many projects of database or index federation fail to provide easier alternatives to access or query information. To this end, a Python API query system was developed to enhance the accessibility of FIVE.

A Highly Prevalent and Pervasive Densovirus Discovered among Sea Stars from the North American Atlantic Coast.

The etiology of sea star wasting syndrome is hypothesized to be caused by a densovirus, sea star-associated densovirus (SSaDV), that has previously been reported on the Pacific and Atlantic Coasts of the United States. In this study, we reevaluated the presence of SSaDV among sea stars from the North American Atlantic Coast and in doing so discovered a novel densovirus that we have named Asterias forbesi-associated densovirus (AfaDV), which shares 78% nucleotide pairwise identity with SSaDV. In contrast to previous studies, SSaDV was not detected in sea stars from the North American Atlantic Coast. Using a variety of PCR-based techniques, we investigated the tissue tropism, host specificity, and prevalence of AfaDV among populations of sea stars at five locations along the Atlantic Coast. AfaDV was detected in three sea star species (Asterias forbesi, Asterias rubens, and Henricia sp.) found in this region and was highly prevalent (>80% of individuals tested; n = 134), among sampled populations. AfaDV was detected in the body wall, gonads, and pyloric caeca (digestive gland) of specimens but was not detected in their coelomic fluid. A significant difference in viral load (copies mg-1) was found between tissue types, with the pyloric caeca having the highest viral loads. Further investigation of Asterias forbesi gonad tissue found germ line cells (oocytes) to be virus positive, suggesting a potential route of vertical transmission. Taken together, these observations show that the presence of AfaDV is not an indicator of sea star wasting syndrome because AfaDV is a common constituent of these animals' microbiome, regardless of health.IMPORTANCE Sea star wasting syndrome is a disease primarily observed on the Pacific and Atlantic Coasts of North America that has significantly impacted sea star populations. The etiology of this disease is unknown, although it is hypothesized to be caused by a densovirus, SSaDV. However, previous studies have not found a correlation between SSaDV and sea star wasting syndrome on the North American Atlantic Coast. This study suggests that this observation may be explained by the presence of a genetically similar densovirus, AfaDV, that may have confounded previous studies. SSaDV was not present in sea stars screened in this study, and instead, AfaDV was commonly found in sea star populations across the New England region, with no apparent signs of disease. These results suggest that sea star densoviruses may be common constituents of the animals' microbiome, and the diversity and extent of these viruses among wild populations may be greater than previously recognized.

The Microbial Landscape of Sea Stars and the Anatomical and Interspecies Variability of Their Microbiome.

Sea stars are among the most important predators in benthic ecosystems worldwide which is partly attributed to their unique gastrointestinal features and feeding behaviors. Despite their ecological importance, the microbiome of these animals and its influence on adult host health and development largely remains unknown. To begin to understand such interactions we sought to understand what bacteria are associated with these animals, how the microbiome is partitioned across regions of the body and how seawater influences their microbiome. We analyzed the microbiome composition of a geographically and taxonomically diverse set of sea star taxa by using 16S rRNA gene amplicon sequencing and compared microorganisms associated with different regions of their body and to their local environment. In addition, we estimated the bacterial and coelomocyte abundance in the sea star coelomic fluid and bacterioplankton abundance in the surrounding seawater via epifluorescence microscopy. The average bacterial cell abundance observed in the coelomic fluid was one to two orders of magnitude lower than the bacterioplankton abundance in the surrounding seawater suggesting a selection against the presence of microorganisms in the coelomic fluid. The sea star microbiome was also significantly different from seawater with relatively few shared microbial taxa. Microbial communities were found to be significantly different between the pyloric caeca, gonads, coelomic fluid, and body wall of the animals. The most noticeable difference between anatomical sites was the greater relative abundance of Spirochaetae and Tenericutes found in hard tissues (gonads, pyloric caeca, and body wall) than in the coelomic fluid. The microbiome of sea stars thus appears to be anatomically partitioned, distinct from the microbial community of seawater and contains a relatively low abundance of bacteria within the coelomic cavity.

Diversification of Secondary Metabolite Biosynthetic Gene Clusters Coincides with Lineage Divergence in Streptomyces.

We have identified Streptomyces sister-taxa which share a recent common ancestor and nearly identical small subunit (SSU) rRNA gene sequences, but inhabit distinct geographic ranges demarcated by latitude and have sufficient genomic divergence to represent distinct species. Here, we explore the evolutionary dynamics of secondary metabolite biosynthetic gene clusters (SMGCs) following lineage divergence of these sister-taxa. These sister-taxa strains contained 310 distinct SMGCs belonging to 22 different gene cluster classes. While there was broad conservation of these 22 gene cluster classes among the genomes analyzed, each individual genome harbored a different number of gene clusters within each class. A total of nine SMGCs were conserved across nearly all strains, but the majority (57%) of SMGCs were strain-specific. We show that while each individual genome has a unique combination of SMGCs, this diversity displays lineage-level modularity. Overall, the northern-derived (NDR) clade had more SMGCs than the southern-derived (SDR) clade (40.7 ± 3.9 and 33.8 ± 3.9, mean and S.D., respectively). This difference in SMGC content corresponded with differences in the number of predicted open reading frames (ORFs) per genome (7775 ± 196 and 7093 ± 205, mean and S.D., respectively) such that the ratio of SMGC:ORF did not differ between sister-taxa genomes. We show that changes in SMGC diversity between the sister-taxa were driven primarily by gene acquisition and deletion events, and these changes were associated with an overall change in genome size which accompanied lineage divergence.

Unearthing the Ecology of Soil Microorganisms Using a High Resolution DNA-SIP Approach to Explore Cellulose and Xylose Metabolism in Soil.

We explored microbial contributions to decomposition using a sophisticated approach to DNA Stable Isotope Probing (SIP). Our experiment evaluated the dynamics and ecological characteristics of functionally defined microbial groups that metabolize labile and structural C in soils. We added to soil a complex amendment representing plant derived organic matter substituted with either (13)C-xylose or (13)C-cellulose to represent labile and structural C pools derived from abundant components of plant biomass. We found evidence for (13)C-incorporation into DNA from (13)C-xylose and (13)C-cellulose in 49 and 63 operational taxonomic units (OTUs), respectively. The types of microorganisms that assimilated (13)C in the (13)C-xylose treatment changed over time being predominantly Firmicutes at day 1 followed by Bacteroidetes at day 3 and then Actinobacteria at day 7. These (13)C-labeling dynamics suggest labile C traveled through different trophic levels. In contrast, microorganisms generally metabolized cellulose-C after 14 days and did not change to the same extent in phylogenetic composition over time. Microorganisms that metabolized cellulose-C belonged to poorly characterized but cosmopolitan soil lineages including Verrucomicrobia, Chloroflexi, and Planctomycetes.

Dynamics of microbial community composition and soil organic carbon mineralization in soil following addition of pyrogenic and fresh organic matter.

Pyrogenic organic matter (PyOM) additions to soils can have large impacts on soil organic carbon (SOC) cycling. As the soil microbial community drives SOC fluxes, understanding how PyOM additions affect soil microbes is essential to understanding how PyOM affects SOC. We studied SOC dynamics and surveyed soil bacterial communities after OM additions in a field experiment. We produced and mixed in either 350 °C corn stover PyOM or an equivalent initial amount of dried corn stover to a Typic Fragiudept soil. Stover increased SOC-derived and total CO2 fluxes (up to 6x), and caused rapid and persistent changes in bacterial community composition over 82 days. In contrast, PyOM only temporarily increased total soil CO2 fluxes (up to 2x) and caused fewer changes in bacterial community composition. Of the operational taxonomic units (OTUs) that increased in response to PyOM additions, 70% also responded to stover additions. These OTUs likely thrive on easily mineralizable carbon (C) that is found both in stover and, to a lesser extent, in PyOM. In contrast, we also identified unique PyOM responders, which may respond to substrates such as polyaromatic C. In particular, members of Gemmatimonadetes tended to increase in relative abundance in response to PyOM but not to fresh organic matter. We identify taxa to target for future investigations of the mechanistic underpinnings of ecological phenomena associated with PyOM additions to soil.

Non-cyanobacterial diazotrophs mediate dinitrogen fixation in biological soil crusts during early crust formation.

Biological soil crusts (BSCs) are key components of ecosystem productivity in arid lands and they cover a substantial fraction of the terrestrial surface. In particular, BSC N2-fixation contributes significantly to the nitrogen (N) budget of arid land ecosystems. In mature crusts, N2-fixation is largely attributed to heterocystous cyanobacteria; however, early successional crusts possess few N2-fixing cyanobacteria and this suggests that microorganisms other than cyanobacteria mediate N2-fixation during the critical early stages of BSC development. DNA stable isotope probing with (15)N2 revealed that Clostridiaceae and Proteobacteria are the most common microorganisms that assimilate (15)N2 in early successional crusts. The Clostridiaceae identified are divergent from previously characterized isolates, though N2-fixation has previously been observed in this family. The Proteobacteria identified share >98.5% small subunit rRNA gene sequence identity with isolates from genera known to possess diazotrophs (for example, Pseudomonas, Klebsiella, Shigella and Ideonella). The low abundance of these heterotrophic diazotrophs in BSCs may explain why they have not been characterized previously. Diazotrophs have a critical role in BSC formation and characterization of these organisms represents a crucial step towards understanding how anthropogenic change will affect the formation and ecological function of BSCs in arid ecosystems.

The khmer software package: enabling efficient nucleotide sequence analysis.

The khmer package is a freely available software library for working efficiently with fixed length DNA words, or k-mers. khmer provides implementations of a probabilistic k-mer counting data structure, a compressible De Bruijn graph representation, De Bruijn graph partitioning, and digital normalization. khmer is implemented in C++ and Python, and is freely available under the BSD license at  https://github.com/dib-lab/khmer/.

Structure and Inhibition of Microbiome ß-Glucuronidases Essential to the Alleviation of Cancer Drug Toxicity.

The selective inhibition of bacterial ß-glucuronidases was recently shown to alleviate drug-induced gastrointestinal toxicity in mice, including the damage caused by the widely used anticancer drug irinotecan. Here, we report crystal structures of representative ß-glucuronidases from the Firmicutes Streptococcus agalactiae and Clostridium perfringens and the Proteobacterium Escherichia coli, and the characterization of a ß-glucuronidase from the Bacteroidetes Bacteroides fragilis. While largely similar in structure, these enzymes exhibit marked differences in catalytic properties and propensities for inhibition, indicating that the microbiome maintains functional diversity in orthologous enzymes. Small changes in the structure of designed inhibitors can induce significant conformational changes in the ß-glucuronidase active site. Finally, we establish that ß-glucuronidase inhibition does not alter the serum pharmacokinetics of irinotecan or its metabolites in mice. Together, the data presented advance our in vitro and in vivo understanding of the microbial ß-glucuronidases, a promising new set of targets for controlling drug-induced gastrointestinal toxicity.

The effect of carbon subsidies on marine planktonic niche partitioning and recruitment during biofilm assembly.

The influence of resource availability on planktonic and biofilm microbial community membership is poorly understood. Heterotrophic bacteria derive some to all of their organic carbon (C) from photoautotrophs while simultaneously competing with photoautotrophs for inorganic nutrients such as phosphorus (P) or nitrogen (N). Therefore, C inputs have the potential to shift the competitive balance of aquatic microbial communities by increasing the resource space available to heterotrophs (more C) while decreasing the resource space available to photoautotrophs (less mineral nutrients due to increased competition from heterotrophs). To test how resource dynamics affect membership of planktonic communities and assembly of biofilm communities we amended a series of flow-through mesocosms with C to alter the availability of C among treatments. Each mesocosm was fed with unfiltered seawater and incubated with sterilized microscope slides as surfaces for biofilm formation. The highest C treatment had the highest planktonic heterotroph abundance, lowest planktonic photoautotroph abundance, and highest biofilm biomass. We surveyed bacterial 16S rRNA genes and plastid 23S rRNA genes to characterize biofilm and planktonic community membership and structure. Regardless of resource additions, biofilm communities had higher alpha diversity than planktonic communities in all mesocosms. Heterotrophic plankton communities were distinct from heterotrophic biofilm communities in all but the highest C treatment where heterotrophic plankton and biofilm communities resembled each other after 17 days. Unlike the heterotrophs, photoautotrophic plankton communities were different than photoautotrophic biofilm communities in composition in all treatments including the highest C treatment. Our results suggest that although resource amendments affect community membership and structure, microbial lifestyle (biofilm vs. planktonic) has a stronger influence on community composition.

Cyanobacterial construction of hot spring siliceous stromatolites in Yellowstone National Park.

Living stromatolites growing in a hot spring in Yellowstone National Park are composed of silica-encrusted cyanobacterial mats. Two cyanobacterial mat types grow on the stromatolite surfaces and are preserved as two distinct lithofacies. One mat is present when the stromatolites are submerged or at the water-atmosphere interface and the other when stromatolites protrude from the hot spring. The lithofacies created by the encrustation of submerged mats constitutes the bulk of the stromatolites, is comprised of silica-encrusted filaments, and is distinctly laminated. To better understand the cyanobacterial membership and community structure differences between the mats, we collected mat samples from each type. Molecular methods revealed that submerged mat cyanobacteria were predominantly one novel phylotype while the exposed mats were predominantly heterocystous phylotypes (Chlorogloeopsis HTF and Fischerella). The cyanobacterium dominating the submerged mat type does not belong in any of the subphylum groups of cyanobacteria recognized by the Ribosomal Database Project and has also been found in association with travertine stromatolites in a Southwest Japan hot spring. Cyanobacterial membership profiles indicate that the heterocystous phylotypes are 'rare biosphere' members of the submerged mats. The heterocystous phylotypes likely emerge when the water level of the hot spring drops. Environmental pressures tied to water level such as sulfide exposure and possibly oxygen tension may inhibit the heterocystous types in submerged mats. These living stromatolites are finely laminated and therefore, in texture, may better represent similarly laminated ancient forms compared with more coarsely laminated living marine examples.