Using Cartesian Doubt To Build a Sequencing-Based View of Microbiology.

The technological leap of DNA sequencing generated a tension between modern metagenomics and historical microbiology. We are forcibly harmonizing the output of a modern tool with centuries of experimental knowledge derived from culture-based microbiology. As a thought experiment, we borrow the notion of Cartesian doubt from philosopher Rene Descartes, who used doubt to build a philosophical framework from his incorrigible statement that "I think therefore I am." We aim to cast away preconceived notions and conceptualize microorganisms through the lens of metagenomic sequencing alone. Specifically, we propose funding and building analysis and engineering methods that neither search for nor rely on the assumption of independent genomes bound by lipid barriers containing discrete functional roles and taxonomies. We propose that a view of microbial communities based in sequencing will engender novel insights into metagenomic structure and may capture functional biology not reflected within the current paradigm.

CRISPRdisco: An Automated Pipeline for the Discovery and Analysis of CRISPR-Cas Systems.

CRISPR-Cas adaptive immune systems of bacteria and archaea have catapulted into the scientific spotlight as genome editing tools. To aid researchers in the field, we have developed an automated pipeline, named CRISPRdisco (CRISPR discovery), to identify CRISPR repeats and cas genes in genome assemblies, determine type and subtype, and describe system completeness. All six major types and 23 currently recognized subtypes and novel putative V-U types are detected. Here, we use the pipeline to identify and classify putative CRISPR-Cas systems in 2,777 complete genomes from the NCBI RefSeq database. This allows comparison to previous publications and investigation of the occurrence and size of CRISPR-Cas systems. Software available at http://github.com/crisprlab/CRISPRdisco provides reproducible, standardized, accessible, transparent, and high-throughput analysis methods available to all researchers in and beyond the CRISPR-Cas research community. This tool opens new avenues to enable classification within a complex nomenclature and provides analytical methods in a field that has evolved rapidly.

Genomic and phenotypic differentiation among Methanosarcina mazei populations from Columbia River sediment.

Methanogenic archaea are genotypically and phenotypically diverse organisms that are integral to carbon cycling in anaerobic environments. Owing to their genetic tractability and ability to be readily cultivated, Methanosarcina spp. have become a powerful model system for understanding methanogen biology at the cellular systems level. However, relatively little is known of how genotypic and phenotypic variation is partitioned in Methanosarcina populations inhabiting natural environments and the possible ecological and evolutionary implications of such variation. Here, we have identified how genomic and phenotypic diversity is partitioned within and between Methanosarcina mazei populations obtained from two different sediment environments in the Columbia River Estuary (Oregon, USA). Population genomic analysis of 56 M. mazei isolates averaging <1% nucleotide divergence revealed two distinct clades, which we refer to as 'mazei-T' and 'mazei-WC'. Genomic analyses showed that these clades differed in gene content and fixation of allelic variants, which point to potential differences in primary metabolism and also interactions with foreign genetic elements. This hypothesis of niche partitioning was supported by laboratory growth experiments that revealed significant differences in trimethylamine utilization. These findings improve our understanding of the ecologically relevant scales of genomic variation in natural systems and demonstrate interactions between genetic and ecological diversity in these easily cultivable and genetically tractable model methanogens.

ITEP: an integrated toolkit for exploration of microbial pan-genomes.

BACKGROUND: Comparative genomics is a powerful approach for studying variation in physiological traits as well as the evolution and ecology of microorganisms. Recent technological advances have enabled sequencing large numbers of related genomes in a single project, requiring computational tools for their integrated analysis. In particular, accurate annotations and identification of gene presence and absence are critical for understanding and modeling the cellular physiology of newly sequenced genomes. Although many tools are available to compare the gene contents of related genomes, new tools are necessary to enable close examination and curation of protein families from large numbers of closely related organisms, to integrate curation with the analysis of gain and loss, and to generate metabolic networks linking the annotations to observed phenotypes. RESULTS: We have developed ITEP, an Integrated Toolkit for Exploration of microbial Pan-genomes, to curate protein families, compute similarities to externally-defined domains, analyze gene gain and loss, and generate draft metabolic networks from one or more curated reference network reconstructions in groups of related microbial species among which the combination of core and variable genes constitute the their "pan-genomes". The ITEP toolkit consists of: (1) a series of modular command-line scripts for identification, comparison, curation, and analysis of protein families and their distribution across many genomes; (2) a set of Python libraries for programmatic access to the same data; and (3) pre-packaged scripts to perform common analysis workflows on a collection of genomes. ITEP's capabilities include de novo protein family prediction, ortholog detection, analysis of functional domains, identification of core and variable genes and gene regions, sequence alignments and tree generation, annotation curation, and the integration of cross-genome analysis and metabolic networks for study of metabolic network evolution. CONCLUSIONS: ITEP is a powerful, flexible toolkit for generation and curation of protein families. ITEP's modular design allows for straightforward extension as analysis methods and tools evolve. By integrating comparative genomics with the development of draft metabolic networks, ITEP harnesses the power of comparative genomics to build confidence in links between genotype and phenotype and helps disambiguate gene annotations when they are evaluated in both evolutionary and metabolic network contexts.

Bacterial taxa that limit sulfur flux from the ocean.

Flux of dimethylsulfide (DMS) from ocean surface waters is the predominant natural source of sulfur to the atmosphere and influences climate by aerosol formation. Marine bacterioplankton regulate sulfur flux by converting the precursor dimethylsulfoniopropionate (DMSP) either to DMS or to sulfur compounds that are not climatically active. Through the discovery of a glycine cleavage T-family protein with DMSP methyltransferase activity, marine bacterioplankton in the Roseobacter and SAR11 taxa were identified as primary mediators of DMSP demethylation to methylmercaptopropionate. One-third of surface ocean bacteria harbor a DMSP demethylase homolog and thereby route a substantial fraction of global marine primary production away from DMS formation and into the marine microbial food web.

Genome sequence of Silicibacter pomeroyi reveals adaptations to the marine environment.

Since the recognition of prokaryotes as essential components of the oceanic food web, bacterioplankton have been acknowledged as catalysts of most major biogeochemical processes in the sea. Studying heterotrophic bacterioplankton has been challenging, however, as most major clades have never been cultured or have only been grown to low densities in sea water. Here we describe the genome sequence of Silicibacter pomeroyi, a member of the marine Roseobacter clade (Fig. 1), the relatives of which comprise approximately 10-20% of coastal and oceanic mixed-layer bacterioplankton. This first genome sequence from any major heterotrophic clade consists of a chromosome (4,109,442 base pairs) and megaplasmid (491,611 base pairs). Genome analysis indicates that this organism relies upon a lithoheterotrophic strategy that uses inorganic compounds (carbon monoxide and sulphide) to supplement heterotrophy. Silicibacter pomeroyi also has genes advantageous for associations with plankton and suspended particles, including genes for uptake of algal-derived compounds, use of metabolites from reducing microzones, rapid growth and cell-density-dependent regulation. This bacterium has a physiology distinct from that of marine oligotrophs, adding a new strategy to the recognized repertoire for coping with a nutrient-poor ocean.

Silicibacter pomeroyi sp. nov. and Roseovarius nubinhibens sp. nov., dimethylsulfoniopropionate-demethylating bacteria from marine environments.

Three Gram-negative, rod-shaped, aerobic bacteria that were capable of degrading dimethylsulfoniopropionate (DMSP) were isolated from marine waters. These isolates (DSS-3(T), DSS-10 and ISM(T)) exhibited the ability to demethylate and cleave DMSP, as well as to degrade other sulfur compounds related to DMSP that are cycled in marine environments. Intracellular poly-beta-hydroxybutyrate inclusions, surface blebs and one polar, complex flagellum that rotated exclusively in the clockwise direction were observed for DSS-3(T). The outer membrane of ISM(T) was separated from the cytoplasm at the poles in a toga-like morphology. The primary fatty acid in both strains was C(18 : 1)omega7c. DNA G+C contents for the isolates were 68.0+/-0.1, 68.1+/-0.1 and 66.0+/-0.2 mol% for DSS-3(T), DSS-10 and ISM(T), respectively. 16S rRNA gene sequence analyses placed these organisms within the Roseobacter lineage of the alpha-PROTEOBACTERIA: Closely related species were Silicibacter lacuscaerulensis and Ruegeria atlantica (DSS-3(T) and DSS-10) and Roseovarius tolerans (ISM(T)). Neither DSS-3(T) nor ISM(T) exhibited 16S rRNA similarity >97 % or DNA-DNA hybridization values >45 % to their nearest described relatives. Genotypic and phenotypic analyses support the creation of two novel species: Silicibacter pomeroyi sp. nov. with strain DSS-3(T) (=ATCC 700808(T)=DSM 15171(T)) as the type strain, and Roseovarius nubinhibens sp. nov. with strain ISM(T) (=ATCC BAA-591(T)=DSM 15170(T)) as the type strain.