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Reconstructing genomic segments from metagenomics data is a highly complex task. In addition to general challenges, such as repeats and sequencing errors, metagenomic assembly needs to tolerate the uneven depth of coverage among organisms in a community and differences between nearly identical strains. Previous methods have addressed these issues by smoothing genomic variants. We present a variant-aware metagenomic scaffolder called MetaCarvel, which combines new strategies for repeat detection with graph analytics for the discovery of variants. We show that MetaCarvel can accurately reconstruct genomic segments from complex microbial mixtures and correctly identify and characterize several classes of common genomic variants.

Bacteria that directly use electrodes as metabolic electron donors (biocathodes) have been proposed for applications ranging from microbial electrosynthesis to advanced bioelectronics for cellular communication with machines. However, just as we understand very little about oxidation of analogous natural insoluble electron donors, such as iron oxide, the organisms and extracellular electron transfer (EET) pathways underlying the electrode-cell direct electron transfer processes are almost completely unknown. Biocathodes are a stable biofilm cultivation platform to interrogate both the rate and mechanism of EET using electrochemistry and to study the electroautotrophic organisms that catalyze these reactions. Here we provide new evidence supporting the hypothesis that the uncultured bacterium “ Candidatus Tenderia electrophaga” directly couples extracellular electron transfer to CO 2 fixation. Our results provide insight into developing biocathode technology, such as microbial electrosynthesis, as well as advancing our understanding of chemolithoautotrophy. Biocathodes provide a stable electron source to drive reduction reactions in electrotrophic microbial electrochemical systems. Electroautotrophic biocathode communities may be more robust than monocultures in environmentally relevant settings, but some members are not easily cultivated outside the electrode environment. We previously used metagenomics and metaproteomics to propose a pathway for coupling extracellular electron transfer (EET) to carbon fixation in “ Candidatus Tenderia electrophaga,” an uncultivated but dominant member of an electroautotrophic biocathode community. Here we validate and refine this proposed pathway using metatranscriptomics of replicate aerobic biocathodes poised at the growth potential level of 310 mV and the suboptimal 470 mV (versus the standard hydrogen electrode). At both potentials, transcripts were more abundant from “ Ca. Tenderia electrophaga” than from any other constituent, and its relative activity was positively correlated with current. Several genes encoding key components of the proposed “ Ca. Tenderia electrophaga” EET pathway were more highly expressed at 470 mV, consistent with a need for cells to acquire more electrons to obtain the same amount of energy as at 310 mV. These included cyc2 , encoding a homolog of a protein known to be involved in iron oxidation. Mean expression of all CO 2 fixation-related genes is 0.27 log 2 -fold higher at 310 mV, indicating that reduced energy availability at 470 mV decreased CO 2 fixation. Our results substantiate the claim that “ Ca. Tenderia electrophaga” is the key electroautotroph, which will help guide further development of this community for microbial electrosynthesis. IMPORTANCE Bacteria that directly use electrodes as metabolic electron donors (biocathodes) have been proposed for applications ranging from microbial electrosynthesis to advanced bioelectronics for cellular communication with machines. However, just as we understand very little about oxidation of analogous natural insoluble electron donors, such as iron oxide, the organisms and extracellular electron transfer (EET) pathways underlying the electrode-cell direct electron transfer processes are almost completely unknown. Biocathodes are a stable biofilm cultivation platform to interrogate both the rate and mechanism of EET using electrochemistry and to study the electroautotrophic organisms that catalyze these reactions. Here we provide new evidence supporting the hypothesis that the uncultured bacterium “ Candidatus Tenderia electrophaga” directly couples extracellular electron transfer to CO 2 fixation. Our results provide insight into developing biocathode technology, such as microbial electrosynthesis, as well as advancing our understanding of chemolithoautotrophy.

The yeast Exophiala dermatitidis exhibits high resistance to γ-radiation in comparison to many other fungi. Several aspects of this phenotype have been characterized, including its dependence on homologous recombination for the repair of radiation-induced DNA damage, and the transcriptomic response invoked by acute γ-radiation exposure in this organism. However, these findings have yet to identify unique γ-radiation exposure survival strategies—many genes that are induced by γ-radiation exposure do not appear to be important for recovery, and the homologous recombination machinery of this organism is not unique compared to more sensitive species. To identify features associated with γ-radiation resistance, here we characterized the proteomes of two E. dermatitidis strains—the wild type and a hyper-resistant strain developed through adaptive laboratory evolution—before and after γ-radiation exposure. The results demonstrate that protein intensities do not change substantially in response to this stress. Rather, the increased resistance exhibited by the evolved strain may be due in part to increased basal levels of single-stranded binding proteins and a large increase in ribosomal content, possibly allowing for a more robust, induced response during recovery. This experiment provides evidence enabling us to focus on DNA replication, protein production, and ribosome levels for further studies into the mechanism of γ-radiation resistance in E. dermatitidis and other fungi.

Metaproteomics is an increasingly popular methodology that provides information regarding the metabolic functions of specific microbial taxa and has potential for contributing to ocean ecology and biogeochemical studies. A blinded multi-laboratory intercomparison was conducted to assess comparability and reproducibility of taxonomic and functional results and their sensitivity to methodological variables. Euphotic zone samples from the Bermuda Atlantic Time-series Study (BATS) in the North Atlantic Ocean collected by in situ pumps and the autonomous underwater vehicle (AUV) Clio were distributed with a paired metagenome, and one-dimensional (1D) liquid chromatographic data-dependent acquisition mass spectrometry analysis was stipulated. Analysis of mass spectra from seven laboratories through a common bioinformatic pipeline identified a shared set of 1056 proteins from 1395 shared peptide constituents. Quantitative analyses showed good reproducibility: pairwise regressions of spectral counts between laboratories yielded R2 values averaged 0.62±0.11, and a Sørensen similarity analysis of the top 1000 proteins revealed 70 %–80 % similarity between laboratory groups. Taxonomic and functional assignments showed good coherence between technical replicates and different laboratories. A bioinformatic intercomparison study, involving 10 laboratories using eight software packages, successfully identified thousands of peptides within the complex metaproteomic datasets, demonstrating the utility of these software tools for ocean metaproteomic research. Lessons learned and potential improvements in methods were described. Future efforts could examine reproducibility in deeper metaproteomes, examine accuracy in targeted absolute quantitation analyses, and develop standards for data output formats to improve data interoperability. Together, these results demonstrate the reproducibility of metaproteomic analyses and their suitability for microbial oceanography research, including integration into global-scale ocean surveys and ocean biogeochemical models.

The impact of microbial communities, also known as the microbiome, on human health and the environment is receiving increased attention. Studying translated gene products (proteins) and comparing metaproteomic profiles may elucidate how microbiomes respond to specific environmental stimuli, and interact with host organisms. Characterizing proteins expressed by a complex microbiome and interpreting their functional signature requires sophisticated informatics tools and workflows tailored to metaproteomics. Additionally, there is a need to disseminate these informatics resources to researchers undertaking metaproteomic studies, who could use them to make new and important discoveries in microbiome research. The Galaxy for proteomics platform (Galaxy-P) offers an open source, web-based bioinformatics platform for disseminating metaproteomics software and workflows. Within this platform, we have developed easily-accessible and documented metaproteomic software tools and workflows aimed at training researchers in their operation and disseminating the tools for more widespread use. The modular workflows encompass the core requirements of metaproteomic informatics: (a) database generation; (b) peptide spectral matching; (c) taxonomic analysis and (d) functional analysis. Much of the software available via the Galaxy-P platform was selected, packaged and deployed through an online metaproteomics “Contribution Fest“ undertaken by a unique consortium of expert software developers and users from the metaproteomics research community, who have co-authored this manuscript. These resources are documented on GitHub and freely available through the Galaxy Toolshed, as well as a publicly accessible metaproteomics gateway Galaxy instance. These documented workflows are well suited for the training of novice metaproteomics researchers, through online resources such as the Galaxy Training Network, as well as hands-on training workshops. Here, we describe the metaproteomics tools available within these Galaxy-based resources, as well as the process by which they were selected and implemented in our community-based work. We hope this description will increase access to and utilization of metaproteomics tools, as well as offer a framework for continued community-based development and dissemination of cutting edge metaproteomics software.

The black yeast Exophiala lecanii-corni of the order Chaetothyriales is notable for its ability to produce abundant quantities of DHN-melanin. While many other Exophiala species are frequent causal agents of human infection, E. lecanii-corni CBS 102400 lacks the thermotolerance requirements that enable pathogenicity, making it appealing for use in targeted functional studies and biotechnological applications. Here, we report the stress tolerance characteristics of E. lecanii-corni, with an emphasis on the influence of melanin on its resistance to various forms of stress. We find that E. lecanii-corni has a distinct stress tolerance profile that includes variation in resistance to temperature, osmotic, and oxidative stress relative to the extremophilic and pathogenic black yeast Exophiala dermatitidis. Notably, the presence of melanin substantially impacts stress resistance in E. lecanii-corni, while this was not found to be the case in E. dermatitidis. The cellular context, therefore, influences the role of melanin in stress protection. In addition, we present a detailed analysis of the E. lecanii-corni genome, revealing key differences in functional genetic content relative to other ascomycetous species, including a significant decrease in abundance of genes encoding ribosomal proteins. In all, this study provides insight into how genetics and physiology may underlie stress tolerance and enhances understanding of the genetic diversity of black yeasts.

Summary Biocathode microbial communities are proposed to catalyse a range of useful reactions. Unlike bioanodes, model biocathode organisms have not yet been successfully cultivated in isolation highlighting the need for culture‐independent approaches to characterization. Biocathode MCL (Marinobacter, Chromatiaceae, Labrenzia) is a microbial community proposed to couple CO2 fixation to extracellular electron transfer and O2 reduction. Previous metagenomic analysis of a single MCL bioelectrochemical system (BES) resulted in resolution of 16 bin genomes. To further resolve bin genomes and compare community composition across replicate MCL BES, we performed shotgun metagenomic and 16S rRNA gene (16S) sequencing at steady‐state current. Clustering pooled reads from replicate BES increased the number of resolved bin genomes to 20, over half of which were > 90% complete. Direct comparison of unassembled metagenomic reads and 16S operational taxonomic units (OTUs) predicted higher community diversity than the assembled/clustered metagenome and the predicted relative abundances did not match. However, when 16S OTUs were mapped to bin genomes and genome abundance was scaled by 16S gene copy number, estimated relative abundance was more similar to metagenomic analysis. The relative abundance of the bin genome representing ‘Ca. Tenderia electrophaga’ was correlated with increasing current, further supporting the hypothesis that this organism is the electroautotroph. Results presented here show a positive correlation between the relative abundance of “Candidatus Tenderia electrophaga” and the amount of current at the cathode, substantiating its role as an electroautotroph. This finding is original and significant because to date there are no reports of previously uncharacterized marine bacteria directly linked to autotrophic extracellular electron transfer (EET) in a mixed microbial population enriched from the environment.”

Here, we present the complete genome sequence of Labrenzia sp. strain CP4, isolated from an electricity-consuming marine biocathode biofilm. Labrenzia sp. strain CP4 consists of a circular 5.2 Mbp chromosome and an 88 Kbp plasmid.