Victor Kunin

Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite

From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis. Here we use a metagenomic analysis of the bacterial community resident in the hindgut paunch of a wood-feeding ‘higher’ Nasutitermes species (which do not contain cellulose-fermenting protozoa) to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H2 metabolism, CO2-reductive acetogenesis and N2 fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-μl environment can be.

Defining the core Arabidopsis thaliana root microbiome

Land plants associate with a root microbiota distinct from the complex microbial community present in surrounding soil. The microbiota colonizing the rhizosphere (immediately surrounding the root) and the endophytic compartment (within the root) contribute to plant growth, productivity, carbon sequestration and phytoremediation. Colonization of the root occurs despite a sophisticated plant immune system, suggesting finely tuned discrimination of mutualists and commensals from pathogens. Genetic principles governing the derivation of host-specific endophyte communities from soil communities are poorly understood. Here we report the pyrosequencing of the bacterial 16S ribosomal RNA gene of more than 600 Arabidopsis thaliana plants to test the hypotheses that the root rhizosphere and endophytic compartment microbiota of plants grown under controlled conditions in natural soils are sufficiently dependent on the host to remain consistent across different soil types and developmental stages, and sufficiently dependent on host genotype to vary between inbred Arabidopsis accessions. We describe different bacterial communities in two geochemically distinct bulk soils and in rhizosphere and endophytic compartments prepared from roots grown in these soils. The communities in each compartment are strongly influenced by soil type. Endophytic compartments from both soils feature overlapping, low-complexity communities that are markedly enriched in Actinobacteria and specific families from other phyla, notably Proteobacteria. Some bacteria vary quantitatively between plants of different developmental stage and genotype. Our rigorous definition of an endophytic compartment microbiome should facilitate controlled dissection of plant–microbe interactions derived from complex soil communities.

A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea.

Sequencing of bacterial and archaeal genomes has revolutionized our understanding of the many roles played by microorganisms. There are now nearly 1,000 completed bacterial and archaeal genomes available, most of which were chosen for sequencing on the basis of their physiology. As a result, the perspective provided by the currently available genomes is limited by a highly biased phylogenetic distribution. To explore the value added by choosing microbial genomes for sequencing on the basis of their evolutionary relationships, we have sequenced and analysed the genomes of 56 culturable species of Bacteria and Archaea selected to maximize phylogenetic coverage. Analysis of these genomes demonstrated pronounced benefits (compared to an equivalent set of genomes randomly selected from the existing database) in diverse areas including the reconstruction of phylogenetic history, the discovery of new protein families and biological properties, and the prediction of functions for known genes from other organisms. Our results strongly support the need for systematic ‘phylogenomic’ efforts to compile a phylogeny-driven ‘Genomic Encyclopedia of Bacteria and Archaea’ in order to derive maximum knowledge from existing microbial genome data as well as from genome sequences to come.

CRISPR — a widespread system that provides acquired resistance against phages in bacteria and archaea

Arrays of clustered, regularly interspaced short palindromic repeats (CRISPRs) are widespread in the genomes of many bacteria and almost all archaea. These arrays are composed of direct repeats that are separated by similarly sized non-repetitive spacers. CRISPR arrays, together with a group of associated proteins, confer resistance to phages, possibly by an RNA-interference-like mechanism. This Progress discusses the structure and function of this newly recognized antiviral mechanism.

Expansion of the BioCyc collection of pathway/genome databases to 160 genomes

The BioCyc database collection is a set of 160 pathway/genome databases (PGDBs) for most eukaryotic and prokaryotic species whose genomes have been completely sequenced to date. Each PGDB in the BioCyc collection describes the genome and predicted metabolic network of a single organism, inferred from the MetaCyc database, which is a reference source on metabolic pathways from multiple organisms. In addition, each bacterial PGDB includes predicted operons for the corresponding species. The BioCyc collection provides a unique resource for computational systems biology, namely global and comparative analyses of genomes and metabolic networks, and a supplement to the BioCyc resource of curated PGDBs. The Omics viewer available through the BioCyc website allows scientists to visualize combinations of gene expression, proteomics and metabolomics data on the metabolic maps of these organisms. This paper discusses the computational methodology by which the BioCyc collection has been expanded, and presents an aggregate analysis of the collection that includes the range of number of pathways present in these organisms, and the most frequently observed pathways. We seek scientists to adopt and curate individual PGDBs within the BioCyc collection. Only by harnessing the expertise of many scientists we can hope to produce biological databases, which accurately reflect the depth and breadth of knowledge that the biomedical research community is producing.

Metagenomic analysis of two enhanced biological phosphorus removal (EBPR) sludge communities

Enhanced biological phosphorus removal (EBPR) is one of the best-studied microbially mediated industrial processes because of its ecological and economic relevance. Despite this, it is not well understood at the metabolic level. Here we present a metagenomic analysis of two lab-scale EBPR sludges dominated by the uncultured bacterium, “Candidatus Accumulibacter phosphatis.” The analysis sheds light on several controversies in EBPR metabolic models and provides hypotheses explaining the dominance of A. phosphatis in this habitat, its lifestyle outside EBPR and probable cultivation requirements. Comparison of the same species from different EBPR sludges highlights recent evolutionary dynamics in the A. phosphatis genome that could be linked to mechanisms for environmental adaptation. In spite of an apparent lack of phylogenetic overlap in the flanking communities of the two sludges studied, common functional themes were found, at least one of them complementary to the inferred metabolism of the dominant organism. The present study provides a much needed blueprint for a systems-level understanding of EBPR and illustrates that metagenomics enables detailed, often novel, insights into even well-studied biological systems.

Experimental factors affecting PCR-based estimates of microbial species richness and evenness

Pyrosequencing of 16S rRNA gene amplicons for microbial community profiling can, for equivalent costs, yield more than two orders of magnitude more sensitivity than traditional PCR cloning and Sanger sequencing. With this increased sensitivity and the ability to analyze multiple samples in parallel, it has become possible to evaluate several technical aspects of PCR-based community structure profiling methods. We tested the effect of amplicon length and primer pair on estimates of species richness (number of species) and evenness (relative abundance of species) by assessing the potentially tractable microbial community residing in the termite hindgut. Two regions of the 16S rRNA gene were sequenced from one of two common priming sites, spanning the V1–V2 or V8 regions, using amplicons ranging in length from 352 to 1443 bp. Our results show that both amplicon length and primer pair markedly influence estimates of richness and evenness. However, estimates of species evenness are consistent among different primer pairs targeting the same region. These results highlight the importance of experimental methodology when comparing diversity estimates across communities.

Evolutionary conservation of sequence and secondary structures in CRISPR repeats

BackgroundClustered regularly interspaced short palindromic repeats (CRISPRs) are a novel class of direct repeats, separated by unique spacer sequences of similar length, that are present in approximately 40% of bacterial and most archaeal genomes analyzed to date. More than 40 gene families, called CRISPR-associated sequences (CASs), appear in conjunction with these repeats and are thought to be involved in the propagation and functioning of CRISPRs. It has been recently shown that CRISPR provides acquired resistance against viruses in prokaryotes.ResultsHere we analyze CRISPR repeats identified in 195 microbial genomes and show that they can be organized into multiple clusters based on sequence similarity. Some of the clusters present stable, highly conserved RNA secondary structures, while others lack detectable structures. Stable secondary structures exhibit multiple compensatory base changes in the stem region, indicating evolutionary and functional conservation.ConclusionWe show that the repeat-based classification corresponds to, and expands upon, a previously reported CAS gene-based classification, including specific relationships between CRISPR and CAS subtypes.

A Bioinformatician's Guide to Metagenomics

SUMMARY As random shotgun metagenomic projects proliferate and become the dominant source of publicly available sequence data, procedures for the best practices in their execution and analysis become increasingly important. Based on our experience at the Joint Genome Institute, we describe the chain of decisions accompanying a metagenomic project from the viewpoint of the bioinformatic analysis step by step. We guide the reader through a standard workflow for a metagenomic project beginning with presequencing considerations such as community composition and sequence data type that will greatly influence downstream analyses. We proceed with recommendations for sampling and data generation including sample and metadata collection, community profiling, construction of shotgun libraries, and sequencing strategies. We then discuss the application of generic sequence processing steps (read preprocessing, assembly, and gene prediction and annotation) to metagenomic data sets in contrast to genome projects. Different types of data analyses particular to metagenomes are then presented, including binning, dominant population analysis, and gene-centric analysis. Finally, data management issues are presented and discussed. We hope that this review will assist bioinformaticians and biologists in making better-informed decisions on their journey during a metagenomic project.

A korarchaeal genome reveals insights into the evolution of the Archaea

The candidate division Korarchaeota comprises a group of uncultivated microorganisms that, by their small subunit rRNA phylogeny, may have diverged early from the major archaeal phyla Crenarchaeota and Euryarchaeota. Here, we report the initial characterization of a member of the Korarchaeota with the proposed name, “Candidatus Korarchaeum cryptofilum,” which exhibits an ultrathin filamentous morphology. To investigate possible ancestral relationships between deep-branching Korarchaeota and other phyla, we used whole-genome shotgun sequencing to construct a complete composite korarchaeal genome from enriched cells. The genome was assembled into a single contig 1.59 Mb in length with a G + C content of 49%. Of the 1,617 predicted protein-coding genes, 1,382 (85%) could be assigned to a revised set of archaeal Clusters of Orthologous Groups (COGs). The predicted gene functions suggest that the organism relies on a simple mode of peptide fermentation for carbon and energy and lacks the ability to synthesize de novo purines, CoA, and several other cofactors. Phylogenetic analyses based on conserved single genes and concatenated protein sequences positioned the korarchaeote as a deep archaeal lineage with an apparent affinity to the Crenarchaeota. However, the predicted gene content revealed that several conserved cellular systems, such as cell division, DNA replication, and tRNA maturation, resemble the counterparts in the Euryarchaeota. In light of the known composition of archaeal genomes, the Korarchaeota might have retained a set of cellular features that represents the ancestral archaeal form.