Diagnostic capabilities of nanopore long-read sequencing in muscular dystrophy.

Many individuals with muscular dystrophies remain genetically undiagnosed despite clinical diagnostic testing, including exome sequencing. Some may harbor previously undetected structural variants (SVs) or cryptic splice sites. We enrolled 10 unrelated families: nine had muscular dystrophy but lacked complete genetic diagnoses and one had an asymptomatic DMD duplication. Nanopore genomic long-read sequencing identified previously undetected pathogenic variants in four individuals: an SV in DMD, an SV in LAMA2, and two single nucleotide variants in DMD that alter splicing. The DMD duplication in the asymptomatic individual was in tandem. Nanopore sequencing may help streamline genetic diagnostic approaches for muscular dystrophy.

The potential for bacteria from carbon-limited deep terrestrial environments to participate in chlorine cycling.

Bacteria capable of dehalogenation via reductive or hydrolytic pathways are ubiquitous. Little is known, however, about the prevalence of bacterial dechlorination in deep terrestrial environments with a limited carbon supply. In this study we analyzed published genomes from three deep terrestrial subsurface sites: a deep aquifer in Western Siberia, the Sanford Underground Research Facility in South Dakota, USA, and the Soudan Underground Iron Mine (SUIM) in Minnesota, USA to determine if there was evidence to suggest that microbial dehalogenation was possible in these environments. Diverse dehalogenase genes were present in all analyzed metagenomes, with reductive dehalogenase and haloalkane dehalogenase genes the most common. Taxonomic analysis of both hydrolytic and reductive dehalogenase genes was performed to explore their affiliation; this analysis indicated that at the SUIM site, hydrolytic dehalogenase genes were taxonomically affiliated with Marinobacter species. Because of this affiliation, experiments were also performed with Marinobacter subterrani strain JG233 ('JG233'), an organism containing three predicted hydrolytic dehalogenase genes and isolated from the SUIM site, to determine whether hydrolytic dehalogenation was an active process and involved in growth on a chlorocarboxylic acid. Presence of these genes in genome appears to be functional, as JG233 was capable of chloroacetate dechlorination with simultaneous chloride release. Stable isotope experiments combined with confocal Raman microspectroscopy demonstrated that JG233 incorporated carbon from 13C-chloroacetate into its biomass. These experiments suggest that organisms present in these extreme and often low-carbon environments are capable of reductive and hydrolytic dechlorination and, based on laboratory experiments, may use this capability as a competitive advantage by utilizing chlorinated organic compounds for growth, either directly or after dechlorination.

The genome of the zebra mussel, Dreissena polymorpha: a resource for comparative genomics, invasion genetics, and biocontrol.

The zebra mussel, Dreissena polymorpha, continues to spread from its native range in Eurasia to Europe and North America, causing billions of dollars in damage and dramatically altering invaded aquatic ecosystems. Despite these impacts, there are few genomic resources for Dreissena or related bivalves. Although the D. polymorpha genome is highly repetitive, we have used a combination of long-read sequencing and Hi-C-based scaffolding to generate a high-quality chromosome-scale genome assembly. Through comparative analysis and transcriptomics experiments, we have gained insights into processes that likely control the invasive success of zebra mussels, including shell formation, synthesis of byssal threads, and thermal tolerance. We identified multiple intact steamer-like elements, a retrotransposon that has been linked to transmissible cancer in marine clams. We also found that D. polymorpha have an unusual 67 kb mitochondrial genome containing numerous tandem repeats, making it the largest observed in Eumetazoa. Together these findings create a rich resource for invasive species research and control efforts.

Development of a User-Friendly Pipeline for Mutational Analyses of HIV Using Ultra-Accurate Maximum-Depth Sequencing.

Human immunodeficiency virus type 2 (HIV-2) accumulates fewer mutations during replication than HIV type 1 (HIV-1). Advanced studies of HIV-2 mutagenesis, however, have historically been confounded by high background error rates in traditional next-generation sequencing techniques. In this study, we describe the adaptation of the previously described maximum-depth sequencing (MDS) technique to studies of both HIV-1 and HIV-2 for the ultra-accurate characterization of viral mutagenesis. We also present the development of a user-friendly Galaxy workflow for the bioinformatic analyses of sequencing data generated using the MDS technique, designed to improve replicability and accessibility to molecular virologists. This adapted MDS technique and analysis pipeline were validated by comparisons with previously published analyses of the frequency and spectra of mutations in HIV-1 and HIV-2 and is readily expandable to studies of viral mutation across the genomes of both viruses. Using this novel sequencing pipeline, we observed that the background error rate was reduced 100-fold over standard Illumina error rates, and 10-fold over traditional unique molecular identifier (UMI)-based sequencing. This technical advancement will allow for the exploration of novel and previously unrecognized sources of viral mutagenesis in both HIV-1 and HIV-2, which will expand our understanding of retroviral diversity and evolution.

Novel Microbial Groups Drive Productivity in an Archean Iron Formation.

Deep subsurface environments are decoupled from Earth's surface processes yet diverse, active, and abundant microbial communities thrive in these isolated environments. Microbes inhabiting the deep biosphere face unique challenges such as electron donor/acceptor limitations, pore space/fracture network limitations, and isolation from other microbes within the formation. Of the few systems that have been characterized, it is apparent that nutrient limitations likely facilitate diverse microbe-microbe interactions (i.e., syntrophic, symbiotic, or parasitic) and that these interactions drive biogeochemical cycling of major elements. Here we describe microbial communities living in low temperature, chemically reduced brines at the Soudan Underground Mine State Park, United States. The Soudan Iron mine intersects a massive hematite formation at the southern extent of the Canadian Shield. Fractured rock aquifer brines continuously flow from exploratory boreholes drilled circa 1960 and are enriched in deuterium compared to the global meteoric values, indicating brines have had little contact with surface derived waters, and continually degas low molecular weight hydrocarbons C1-C4. Microbial enrichments suggest that once brines exit the boreholes, oxidation of the hydrocarbons occur. Amplicon sequencing show these borehole communities are low in diversity and dominated by Firmicute and Proteobacteria phyla. From the metagenome assemblies, we recovered approximately thirty genomes with estimated completion over 50%. Analysis of genome taxonomy generally followed the amplicon data, and highlights that several of the genomes represent novel families and genera. Metabolic reconstruction shows two carbon-fixation pathways were dominant, the Wood-Ljungdahl (acetogenesis) and Calvin-Benson-Bassham (via RuBisCo), indicating that inorganic carbon likely enters into the microbial foodweb with differing carbon fractionation potentials. Interestingly, methanogenesis is likely driven by Methanolobus and suggests cycling of methylated compounds and not H2/CO2 or acetate. Furthermore, the abundance of sulfate in brines suggests cryptic sulfur cycling may occur, as we detect possible sulfate reducing and thiosulfate oxidizing microorganisms. Finally, a majority of the microorganisms identified contain genes that would allow them to participate in several element cycles, highlighting that in these deep isolated systems metabolic flexibility may be an important life history trait.

Chromosome rearrangements shape the diversification of secondary metabolism in the cyclosporin producing fungus Tolypocladium inflatum.

BACKGROUND: Genes involved in production of secondary metabolites (SMs) in fungi are exceptionally diverse. Even strains of the same species may exhibit differences in metabolite production, a finding that has important implications for drug discovery. Unlike in other eukaryotes, genes producing SMs are often clustered and co-expressed in fungal genomes, but the genetic mechanisms involved in the creation and maintenance of these secondary metabolite biosynthetic gene clusters (SMBGCs) remains poorly understood. RESULTS: In order to address the role of genome architecture and chromosome scale structural variation in generating diversity of SMBGCs, we generated chromosome scale assemblies of six geographically diverse isolates of the insect pathogenic fungus Tolypocladium inflatum, producer of the multi-billion dollar lifesaving immunosuppressant drug cyclosporin, and utilized a Hi-C chromosome conformation capture approach to address the role of genome architecture and structural variation in generating intraspecific diversity in SMBGCs. Our results demonstrate that the exchange of DNA between heterologous chromosomes plays an important role in generating novelty in SMBGCs in fungi. In particular, we demonstrate movement of a polyketide synthase (PKS) and several adjacent genes by translocation to a new chromosome and genomic context, potentially generating a novel PKS cluster. We also provide evidence for inter-chromosomal recombination between nonribosomal peptide synthetases located within subtelomeres and uncover a polymorphic cluster present in only two strains that is closely related to the cluster responsible for biosynthesis of the mycotoxin aflatoxin (AF), a highly carcinogenic compound that is a major public health concern worldwide. In contrast, the cyclosporin cluster, located internally on chromosomes, was conserved across strains, suggesting selective maintenance of this important virulence factor for infection of insects. CONCLUSIONS: This research places the evolution of SMBGCs within the context of whole genome evolution and suggests a role for recombination between chromosomes in generating novel SMBGCs in the medicinal fungus Tolypocladium inflatum.

Microbial biodegradation of biuret: defining biuret hydrolases within the isochorismatase superfamily.

Biuret is a minor component of urea fertilizer and an intermediate in s-triazine herbicide biodegradation. The microbial metabolism of biuret has never been comprehensively studied. Here, we enriched and isolated bacteria from a potato field that grew on biuret as a sole nitrogen source. We sequenced the genome of the fastest-growing isolate, Herbaspirillum sp. BH-1 and identified genes encoding putative biuret hydrolases (BHs). We purified and characterized a functional BH enzyme from Herbaspirillum sp. BH-1 and two other bacteria from divergent phyla. The BH enzymes reacted exclusively with biuret in the range of 2-11 µmol min-1 mg-1 protein. We then constructed a global protein superfamily network to map structure-function relationships in the BH subfamily and used this to mine > 7000 genomes. High-confidence BH sequences were detected in Actinobacteria, Alpha- and Beta-proteobacteria, and some fungi, archaea and green algae, but not animals or land plants. Unexpectedly, no cyanuric acid hydrolase homologs were detected in > 90% of genomes with BH homologs, suggesting BHs may have arisen independently of s-triazine ring metabolism. This work links genotype to phenotype by enabling accurate genome-mining to predict microbial utilization of biuret. Importantly, it advances understanding of the microbial capacity for biuret biodegradation in agricultural systems.

Complete Genome Sequence of Desulfovibrio desulfuricans Strain G11, a Model Sulfate-Reducing, Hydrogenotrophic, and Syntrophic Partner Organism.

Here, we report the draft genome of the Gram-negative, sulfate-reducing bacterium Desulfovibrio desulfuricans strain G11. Isolated from a rumen fluid enrichment, this culture has been a model syntrophic partner due to its metabolic flexibility. The assembly yielded a single circular chromosome of 3,414,943 bp and a 57% G+C content.

Isolation and Genomic Characterization of 'Desulfuromonas soudanensis WTL', a Metal- and Electrode-Respiring Bacterium from Anoxic Deep Subsurface Brine.

Reaching a depth of 713 m below the surface, the Soudan Underground Iron Mine (Soudan, MN, USA) transects a massive Archaean (2.7 Ga) banded iron formation, providing a remarkably accessible window into the terrestrial deep biosphere. Despite organic carbon limitation, metal-reducing microbial communities are present in potentially ancient anoxic brines continuously emanating from exploratory boreholes on Level 27. Using graphite electrodes deposited in situ as bait, we electrochemically enriched and isolated a novel halophilic iron-reducing Deltaproteobacterium, 'Desulfuromonas soudanensis' strain WTL, from an acetate-fed three-electrode bioreactor poised at +0.24 V (vs. standard hydrogen electrode). Cyclic voltammetry revealed that 'D. soudanensis' releases electrons at redox potentials approximately 100 mV more positive than the model freshwater surface isolate Geobacter sulfurreducens, suggesting that its extracellular respiration is tuned for higher potential electron acceptors. 'D. soudanensis' contains a 3,958,620-bp circular genome, assembled to completion using single-molecule real-time (SMRT) sequencing reads, which encodes a complete TCA cycle, 38 putative multiheme c-type cytochromes, one of which contains 69 heme-binding motifs, and a LuxI/LuxR quorum sensing cassette that produces an unidentified N-acyl homoserine lactone. Another cytochrome is predicted to lie within a putative prophage, suggesting that horizontal gene transfer plays a role in respiratory flexibility among metal reducers. Isolation of 'D. soudanensis' underscores the utility of electrode-based approaches for enriching rare metal reducers from a wide range of habitats.

Complete Genome Sequence of Streptomyces albus SM254, a Potent Antagonist of Bat White-Nose Syndrome Pathogen Pseudogymnoascus destructans.

We sequenced and annotated the complete 7,170,504-bp genome of a novel secondary metabolite-producingStreptomycesstrain,Streptomyces albusSM254, isolated from copper-rich subsurface fluids at ~220-m depth within the Soudan Iron Mine (Soudan, MN, USA).

Draft Genome Sequence of the Gram-Positive Thermophilic Iron Reducer Thermincola ferriacetica Strain Z-0001T.

A 3.19-Mbp draft genome of the Gram-positive thermophilic iron-reducing Firmicutes isolate from the Peptococcaceae family, Thermincola ferriacetica Z-0001, was assembled at ~100× coverage from 100-bp paired-end Illumina reads. The draft genome contains 3,274 predicted genes (3,187 protein coding genes) and putative multiheme c-type cytochromes.

Scarless Genome Editing and Stable Inducible Expression Vectors for Geobacter sulfurreducens.

Metal reduction by members of the Geobacteraceae is encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent of Geobacter biofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacterium Geobacter sulfurreducens. A motile strain of G. sulfurreducens was constructed by precise removal of a transposon interrupting the fgrM flagellar regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochrome imcH. We also show that RK2-based plasmids were maintained in G. sulfurreducens for over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutive Geobacter promoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented ΔimcH mutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerous G. sulfurreducens strain constructs simultaneously and shows the sensitivity of imcH expression by the vanillate system. These tools will enable more sophisticated genetic studies in G. sulfurreducens without polar insertion effects or need for multiple antibiotics.

Complete Genome Sequence of Achromobacter xylosoxidans MN001, a Cystic Fibrosis Airway Isolate.

The genome of Achromobacter xylosoxidans MN001, a strain isolated from sputum derived from an adult cystic fibrosis patient, was sequenced using combined single-molecule real-time and Illumina sequencing. Assembly of the complete genome resulted in a 5,876,039-bp chromosome, representing the smallest A. xylosoxidans genome sequenced to date.

Complete Genome of Geobacter pickeringii G13T, a Metal-Reducing Isolate from Sedimentary Kaolin Deposits.

We used PacBio sequencing to assemble the genome of the pristine freshwater isolate Geobacter pickeringii G13(T) into a single 3,618,700-bp circular chromosome polished to 99.999% accuracy (quality value [QV], 50). This isolate shares several features with other Geobacter spp., including genes for degradation of aromatics and an abundance of multiheme c-type cytochromes.

Genomes of Geoalkalibacter ferrihydriticus Z-0531T and Geoalkalibacter subterraneus Red1T, Two Haloalkaliphilic Metal-Reducing Deltaproteobacteria.

We sequenced and annotated genomes of two haloalkaliphilic Deltaproteobacteria, Geoalkalibacter ferrihydriticus Z-0531(T) (DSM 17813) and Geoalkalibacter subterraneus Red1(T) (DSM 23483). During assembly, we discovered that the DSMZ stock culture of G. subterraneus was contaminated. We reisolated G. subterraneus in axenic culture and redeposited it in DSMZ and JCM.

Coupling dark metabolism to electricity generation using photosynthetic cocultures.

We investigated the role of green sulfur bacteria inlight-responsive electricity generation in microbial electrochemical cells (MXCs). We operated MXCs containing either monocultures or defined cocultures of previously enriched phototrophic Chlorobium and anode-respiring Geobacter under anaerobic conditions in the absence of electron donor. Monoculture control MXCs containing Geobacter or Chlorobium neither responded to light nor produced current, respectively. Instead, light-responsive current generation occurred only in coculture MXCs. Current increased above background levels only in the dark and declined slowly over 96 h. This pattern suggested that Chlorobium exhausted intracellular glycogen reserves via dark fermentation to supply an electron donor, presumably acetate, to Geobacter. With medium containing sulfide as the sole photosynthetic electron donor, current generation had a similar and reproducible negative light response. To investigate whether this metabolic interaction also occurred without an electrode, we performed coculture experiments in batch serum bottles. In this setup, sulfide served as the sole electron donor, whose oxidation by Chlorobium was required to provide S(0) as the electron acceptor to Geobacter. Copies of Geobacter 16S rDNA increased approximately 14-fold in batch bottle cocultures containing sulfide compared to those lacking sulfide, and did not decline after termination of sulfide feeding. These results suggest that products of both photosynthesis and dark fermentation by Chlorobium were sufficient both to yield an electrochemical response by Geobacter biofilms, and to promote Geobacter growthin batch cocultures. Our work expands upon the fusion of MXCs with coculture techniques and reinforces the utility of microbial electrochemistry for sensitive, real-time monitoring of microbial interactions in which a metabolic intermediate can be converted to electrical current.

Generation of high current densities by pure cultures of anode-respiring Geoalkalibacter spp. under alkaline and saline conditions in microbial electrochemical cells.

UNLABELLED: Anode-respiring bacteria (ARB) generate electric current in microbial electrochemical cells (MXCs) by channeling electrons from the oxidation of organic substrates to an electrode. Production of high current densities by monocultures in MXCs has resulted almost exclusively from the activity of Geobacter sulfurreducens, a neutrophilic freshwater Fe(III)-reducing bacterium and the highest-current-producing member documented for the Geobacteraceae family of the Deltaproteobacteria. Here we report high current densities generated by haloalkaliphilic Geoalkalibacter spp., thus broadening the capability for high anode respiration rates by including other genera within the Geobacteraceae. In this study, acetate-fed pure cultures of two related Geoalkalibacter spp. produced current densities of 5.0 to 8.3 and 2.4 to 3.3 A m(-2) under alkaline (pH 9.3) and saline (1.7% NaCl) conditions, respectively. Chronoamperometric studies of halophilic Glk. subterraneus DSM 23483 and alkaliphilic Glk. ferrihydriticus DSM 17813 suggested that cells performed long-range electron transfer through electrode-attached biofilms and not through soluble electron shuttles. Glk. ferrihydriticus also oxidized ethanol directly to produce current, with maximum current densities of 5.7 to 7.1 A m(-2) and coulombic efficiencies of 84 to 95%. Cyclic voltammetry (CV) elicited a sigmoidal response with characteristic onset, midpoint, and saturation potentials, while CV performed in the absence of an electron donor suggested the involvement of redox molecules in the biofilm that were limited by diffusion. These results matched those previously reported for actively respiring Gb. sulfurreducens biofilms producing similar current densities (~5 to 9 A m(-2)). IMPORTANCE: This study establishes the highest current densities ever achieved by pure cultures of anode-respiring bacteria (ARB) under alkaline and saline conditions in microbial electrochemical cells (MXCs) and provides the first electrochemical characterization of the genus Geoalkalibacter. Production of high current densities among the Geobacteraceae is no longer exclusive to Geobacter sulfurreducens, suggesting greater versatility for this family in fundamental and applied microbial electrochemical cell (MXC) research than previously considered. Additionally, this work raises the possibility that different members of the Geobacteraceae have conserved molecular mechanisms governing respiratory extracellular electron transfer to electrodes. Thus, the capacity for high current generation may exist in other uncultivated members of this family. Advancement of MXC technology for practical uses must rely on an expanded suite of ARB capable of using different electron donors and producing high current densities under various conditions. Geoalkalibacter spp. can potentially broaden the practical capabilities of MXCs to include energy generation and waste treatment under expanded ranges of salinity and pH.

Light-responsive current generation by phototrophically enriched anode biofilms dominated by green sulfur bacteria.

The objective of this study was to employ microbial electrochemical cells (MXCs) to selectively enrich and examine anoxygenic photosynthetic bacteria for potential anaerobic respiration capabilities using electrodes. In the process, we designed a novel enrichment strategy that manipulated the poised anode potential, light, nitrogen availability, and media supply to promote growth of phototrophic bacteria while minimizing co-enrichment of non-phototrophic anode-respiring bacteria (ARB). This approach resulted in light-responsive electricity generation from fresh- and saltwater inocula. Under anoxic conditions, current showed a negative light response, suggesting that the enriched phototrophic consortia shifted between phototrophic and anaerobic respiratory metabolism. Molecular, physical, and electrochemical analyses elucidated that anode biofilms were dominated by green sulfur bacteria, and biofilms exhibited anode respiration kinetics indicative of non-mediated electron transfer, but kinetic parameters differed from values previously reported for non-phototrophic ARB. These results invite the utilization of MXCs as microbiological tools for exploring anaerobic respiratory capabilities among anoxygenic photosynthetic bacteria.

Quantitative PCR for tracking the megaplasmid-borne biodegradation potential of a model sphingomonad.

We developed a quantitative PCR method for tracking the dxnA1 gene, the initial, megaplasmid-borne gene in Sphingomonas wittichii RW1's dibenzo-p-dioxin degradation pathway. We used this method on complex environmental samples and report on growth of S. wittichii RW1 in landfill leachate, thus furnishing a novel tool for monitoring megaplasmid-borne, dioxygenase-encoding genes.

Effects of temperature shifts on growth rate and lipid characteristics of Synechocystis sp. PCC6803 in a bench-top photobioreactor.

Synechocystis sp. PCC6803 exhibited a high degree of variation in biomass and lipid production rates in response to temperature changes in a photobioreactor. Compared with an optimal temperature of 30-33°C, a higher temperature of 44°C and lower temperatures of 22°C and 18°C severely inhibited the specific growth rate (up to a 66% decrease), biomass production rate (up to a 71% decrease), nutrient utilization rates (up to a 77% decrease), and lipid production rate (up to a 80% decrease). Temperature stress triggered changes in the relative percentage of individual fatty acids (mainly for C16:0 and C18:3), and degree of unsaturation significantly changed: 0.87 at 30°C, 0.62 at 44°C, and 1.29 at 18°C. Although PCC6803 survived temperature stress and maintained its predominate position in the culture, it could not fully recover from long-term temperature stress. Thus, avoiding prolonged exposure to extreme temperature is crucial for using PCC6803 as feedstock for biofuel production.