Christopher S. Miller

Fermentation, Hydrogen, and Sulfur Metabolism in Multiple Uncultivated Bacterial Phyla

Bacterial PERegrinations Many branches of the bacterial domain of life are only known from sequences that turn up in metagenomic analyses and are still only named by acronym—for example, the phylum-level groups BD1-5, OP11, OD1, and the PERs. The parent organisms are probably widespread, but they have not been cultured, and very little is known about their metabolisms or their contributions and functions in the natural environment. Wrighton et al. (p. 1661) pumped acetate into an aquifer in Colorado to prompt the naturally occurring bacteria into action and then, from the runoff, filtered out the smaller microbial cells for further analysis. Mass-spectrometry–based proteomics was used to test for functional activity, and 49 distinct genomes were recovered, many with surprising functional attributes. All of the recovered organisms appeared to be strict anaerobes with a full glycolytic pathway that were capable of augmenting energy production by coupling proton-pumping activity to adenosine triphosphate synthase. Several hydrogenases were found that seemed to be able to switch between hydrogen production and polysulfide reduction, depending on the substrate available. Notably, carbon dioxide assimilation was a common feature, with many genes having similarity to those of archaea. Near-complete reconstruction of the genomes of 21 widespread uncultured environmental bacteria reveals metabolic novelties. BD1-5, OP11, and OD1 bacteria have been widely detected in anaerobic environments, but their metabolisms remain unclear owing to lack of cultivated representatives and minimal genomic sampling. We uncovered metabolic characteristics for members of these phyla, and a new lineage, PER, via cultivation-independent recovery of 49 partial to near-complete genomes from an acetate-amended aquifer. All organisms were nonrespiring anaerobes predicted to ferment. Three augment fermentation with archaeal-like hybrid type II/III ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) that couples adenosine monophosphate salvage with CO2 fixation, a pathway not previously described in Bacteria. Members of OD1 reduce sulfur and may pump protons using archaeal-type hydrogenases. For six organisms, the UGA stop codon is translated as tryptophan. All bacteria studied here may play previously unrecognized roles in hydrogen production, sulfur cycling, and fermentation of refractory sedimentary carbon.

EMIRGE: reconstruction of full-length ribosomal genes from microbial community short read sequencing data

Recovery of ribosomal small subunit genes by assembly of short read community DNA sequence data generally fails, making taxonomic characterization difficult. Here, we solve this problem with a novel iterative method, based on the expectation maximization algorithm, that reconstructs full-length small subunit gene sequences and provides estimates of relative taxon abundances. We apply the method to natural and simulated microbial communities, and correctly recover community structure from known and previously unreported rRNA gene sequences. An implementation of the method is freely available at https://github.com/csmiller/EMIRGE.

Microbes in the neonatal intensive care unit resemble those found in the gut of premature infants

BackgroundThe source inoculum of gastrointestinal tract (GIT) microbes is largely influenced by delivery mode in full-term infants, but these influences may be decoupled in very low birth weight (VLBW, <1,500 g) neonates via conventional broad-spectrum antibiotic treatment. We hypothesize the built environment (BE), specifically room surfaces frequently touched by humans, is a predominant source of colonizing microbes in the gut of premature VLBW infants. Here, we present the first matched fecal-BE time series analysis of two preterm VLBW neonates housed in a neonatal intensive care unit (NICU) over the first month of life.ResultsFresh fecal samples were collected every 3 days and metagenomes sequenced on an Illumina HiSeq2000 device. For each fecal sample, approximately 33 swabs were collected from each NICU room from 6 specified areas: sink, feeding and intubation tubing, hands of healthcare providers and parents, general surfaces, and nurse station electronics (keyboard, mouse, and cell phone). Swabs were processed using a recently developed ‘expectation maximization iterative reconstruction of genes from the environment’ (EMIRGE) amplicon pipeline in which full-length 16S rRNA amplicons were sheared and sequenced using an Illumina platform, and short reads reassembled into full-length genes. Over 24,000 full-length 16S rRNA sequences were produced, generating an average of approximately 12,000 operational taxonomic units (OTUs) (clustered at 97% nucleotide identity) per room-infant pair. Dominant gut taxa, including Staphylococcus epidermidis, Klebsiella pneumoniae, Bacteroides fragilis, and Escherichia coli, were widely distributed throughout the room environment with many gut colonizers detected in more than half of samples. Reconstructed genomes from infant gut colonizers revealed a suite of genes that confer resistance to antibiotics (for example, tetracycline, fluoroquinolone, and aminoglycoside) and sterilizing agents, which likely offer a competitive advantage in the NICU environment.ConclusionsWe have developed a high-throughput culture-independent approach that integrates room surveys based on full-length 16S rRNA gene sequences with metagenomic analysis of fecal samples collected from infants in the room. The approach enabled identification of discrete ICU reservoirs of microbes that also colonized the infant gut and provided evidence for the presence of certain organisms in the room prior to their detection in the gut.

Glycoside Hydrolase Activities of Thermophilic Bacterial Consortia Adapted to Switchgrass

ABSTRACT Industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. Thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. Compost-derived microbial consortia were adapted to switchgrass at 60°C to develop thermophilic biomass-degrading consortia for detailed studies. Microbial community analysis using small-subunit rRNA gene amplicon pyrosequencing and short-read metagenomic sequencing demonstrated that thermophilic adaptation to switchgrass resulted in low-diversity bacterial consortia with a high abundance of bacteria related to thermophilic paenibacilli, Rhodothermus marinus, and Thermus thermophilus. At lower abundance, thermophilic Chloroflexi and an uncultivated lineage of the Gemmatimonadetes phylum were observed. Supernatants isolated from these consortia had high levels of xylanase and endoglucanase activities. Compared to commercial enzyme preparations, the endoglucanase enzymes had a higher thermotolerance and were more stable in the presence of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid used for biomass pretreatment. The supernatants were used to saccharify [C2mim][OAc]-pretreated switchgrass at elevated temperatures (up to 80°C), demonstrating that these consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to more extreme reaction conditions.

Biostimulation induces syntrophic interactions that impact C, S and N cycling in a sediment microbial community

Stimulation of subsurface microorganisms to induce reductive immobilization of metals is a promising approach for bioremediation, yet the overall microbial community response is typically poorly understood. Here we used proteogenomics to test the hypothesis that excess input of acetate activates complex community functioning and syntrophic interactions among autotrophs and heterotrophs. A flow-through sediment column was incubated in a groundwater well of an acetate-amended aquifer and recovered during microbial sulfate reduction. De novo reconstruction of community sequences yielded near-complete genomes of Desulfobacter (Deltaproteobacteria), Sulfurovum- and Sulfurimonas-like Epsilonproteobacteria and Bacteroidetes. Partial genomes were obtained for Clostridiales (Firmicutes) and Desulfuromonadales-like Deltaproteobacteria. The majority of proteins identified by mass spectrometry corresponded to Desulfobacter-like species, and demonstrate the role of this organism in sulfate reduction (Dsr and APS), nitrogen fixation and acetate oxidation to CO2 during amendment. Results indicate less abundant Desulfuromonadales, and possibly Bacteroidetes, also actively contributed to CO2 production via the tricarboxylic acid (TCA) cycle. Proteomic data indicate that sulfide was partially re-oxidized by Epsilonproteobacteria through nitrate-dependent sulfide oxidation (using Nap, Nir, Nos, SQR and Sox), with CO2 fixed using the reverse TCA cycle. We infer that high acetate concentrations, aimed at stimulating anaerobic heterotrophy, led to the co-enrichment of, and carbon fixation in Epsilonproteobacteria. Results give an insight into ecosystem behavior following addition of simple organic carbon to the subsurface, and demonstrate a range of biological processes and community interactions were stimulated.

Perturbation and restoration of the fathead minnow gut microbiome after low-level triclosan exposure.

BackgroundTriclosan is a widely used antimicrobial compound and emerging environmental contaminant. Although the role of the gut microbiome in health and disease is increasingly well established, the interaction between environmental contaminants and host microbiome is largely unexplored, with unknown consequences for host health. This study examined the effects of low, environmentally relevant levels of triclosan exposure on the fish gut microbiome. Developing fathead minnows (Pimephales promelas) were exposed to two low levels of triclosan over a 7-day exposure. Fish gastrointestinal tracts from exposed and control fish were harvested at four time points: immediately preceding and following the 7-day exposure and after 1 and 2 weeks of depuration.ResultsA total of 103 fish gut bacterial communities were characterized by high-throughput sequencing and analysis of the V3-V4 region of the 16S rRNA gene. By measures of both alpha and beta diversity, gut microbial communities were significantly differentiated by exposure history immediately following triclosan exposure. After 2 weeks of depuration, these differences disappear. Independent of exposure history, communities were also significantly structured by time. This first detailed census of the fathead minnow gut microbiome shows a bacterial community that is similar in composition to those of zebrafish and other freshwater fish. Among the triclosan-resilient members of this host-associated community are taxa associated with denitrification in wastewater treatment, taxa potentially able to degrade triclosan, and taxa from an unstudied host-associated candidate division.ConclusionsThe fathead minnow gut microbiome is rapidly and significantly altered by exposure to low, environmentally relevant levels of triclosan, yet largely recovers from this short-term perturbation over an equivalently brief time span. These results suggest that even low-level environmental exposure to a common antimicrobial compound can induce significant short-term changes to the gut microbiome, followed by restoration, demonstrating both the sensitivity and resilience of the gut flora to challenges by environmental toxicants. This short-term disruption in a developing organism may have important long-term consequences for host health. The identification of multiple taxa not often reported in the fish gut suggests that microbial nitrogen metabolism in the fish gut may be more complex than previously appreciated.

Short-read assembly of full-length 16S amplicons reveals bacterial diversity in subsurface sediments.

In microbial ecology, a fundamental question relates to how community diversity and composition change in response to perturbation. Most studies have had limited ability to deeply sample community structure (e.g. Sanger-sequenced 16S rRNA libraries), or have had limited taxonomic resolution (e.g. studies based on 16S rRNA hypervariable region sequencing). Here, we combine the higher taxonomic resolution of near-full-length 16S rRNA gene amplicons with the economics and sensitivity of short-read sequencing to assay the abundance and identity of organisms that represent as little as 0.01% of sediment bacterial communities. We used a new version of EMIRGE optimized for large data size to reconstruct near-full-length 16S rRNA genes from amplicons sheared and sequenced with Illumina technology. The approach allowed us to differentiate the community composition among samples acquired before perturbation, after acetate amendment shifted the predominant metabolism to iron reduction, and once sulfate reduction began. Results were highly reproducible across technical replicates, and identified specific taxa that responded to the perturbation. All samples contain very high alpha diversity and abundant organisms from phyla without cultivated representatives. Surprisingly, at the time points measured, there was no strong loss of evenness, despite the selective pressure of acetate amendment and change in the terminal electron accepting process. However, community membership was altered significantly. The method allows for sensitive, accurate profiling of the “long tail” of low abundance organisms that exist in many microbial communities, and can resolve population dynamics in response to environmental change.

Heterotrophic Archaea Contribute to Carbon Cycling in Low-pH, Suboxic Biofilm Communities

ABSTRACT Archaea are widely distributed and yet are most often not the most abundant members of microbial communities. Here, we document a transition from Bacteria- to Archaea-dominated communities in microbial biofilms sampled from the Richmond Mine acid mine drainage (AMD) system (∼pH 1.0, ∼38°C) and in laboratory-cultivated biofilms. This transition occurs when chemoautotrophic microbial communities that develop at the air-solution interface sink to the sediment-solution interface and degrade under microaerobic and anaerobic conditions. The archaea identified in these sunken biofilms are from the class Thermoplasmata, and in some cases, the highly divergent ARMAN nanoarchaeal lineage. In several of the sunken biofilms, nanoarchaea comprise 10 to 25% of the community, based on fluorescent in situ hybridization and metagenomic analyses. Comparative community proteomic analyses show a persistence of bacterial proteins in sunken biofilms, but there is clear evidence for amino acid modifications due to acid hydrolysis. Given the low representation of bacterial cells in sunken biofilms based on microscopy, we infer that hydrolysis reflects proteins derived from lysed cells. For archaea, we detected ∼2,400 distinct proteins, including a subset involved in proteolysis and peptide uptake. Laboratory cultivation experiments using complex carbon substrates demonstrated anaerobic enrichment of Ferroplasma and Aplasma coupled to the reduction of ferric iron. These findings indicate dominance of acidophilic archaea in degrading biofilms and suggest that they play roles in anaerobic nutrient cycling at low pH.

Disturbed subsurface microbial communities follow equivalent trajectories despite different structural starting points.

Microbial community structure, and niche and neutral processes can all influence response to disturbance. Here, we provide experimental evidence for niche versus neutral and founding community effects during a bioremediation-related organic carbon disturbance. Subsurface sediment, partitioned into 22 flow-through columns, was stimulated in situ by the addition of acetate as a carbon and electron donor source. This drove the system into a new transient biogeochemical state characterized by iron reduction and enriched Desulfuromonadales, Comamonadaceae and Bacteroidetes lineages. After approximately 1 month conditions favoured sulfate reduction, and were accompanied by a substantial increase in the relative abundance of Desulfobulbus, Desulfosporosinus, Desulfitobacterium and Desulfotomaculum. Two subsets of four to five columns each were switched from acetate to lactate amendment during either iron (earlier) or sulfate (later) reduction. Hence, subsets had significantly different founding communities. All lactate treatments exhibited lower relative abundances of Desulfotomaculum and Bacteroidetes, enrichments of Clostridiales and Psychrosinus species, and a temporal succession from highly abundant Clostridium sensu stricto to Psychrosinus. Regardless of starting point, lactate-switch communities followed comparable structural trajectories, whereby convergence was evident 9 to 16 days after each switch, and significant after 29 to 34 days of lactate addition. Results imply that neither the founding community nor neutral processes influenced succession following perturbation.

A novel three-unit tRNA splicing endonuclease found in ultrasmall Archaea possesses broad substrate specificity

tRNA splicing endonucleases, essential enzymes found in Archaea and Eukaryotes, are involved in the processing of pre-tRNA molecules. In Archaea, three types of splicing endonuclease [homotetrameric: α4, homodimeric: α2, and heterotetrameric: (αβ)2] have been identified, each representing different substrate specificity during the tRNA intron cleavage. Here, we discovered a fourth type of archaeal tRNA splicing endonuclease (ε2) in the genome of the acidophilic archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2 and its closely related species, ARMAN-1. The enzyme consists of two duplicated catalytic units and one structural unit encoded on a single gene, representing a novel three-unit architecture. Homodimeric formation was confirmed by cross-linking assay, and site-directed mutagenesis determined that the conserved L10-pocket interaction between catalytic and structural unit is necessary for the assembly. A tRNA splicing assay reveal that ε2 endonuclease cleaves both canonical and non-canonical bulge–helix–bulge motifs, similar to that of (αβ)2 endonuclease. Unlike other ARMAN and Euryarchaeota, tRNAs found in ARMAN-2 are highly disrupted by introns at various positions, which again resemble the properties of archaeal species with (αβ)2 endonuclease. Thus, the discovery of ε2 endonuclease in an archaeon deeply branched within Euryarchaeota represents a new example of the coevolution of tRNA and their processing enzymes.