Paul M. Richardson

Community structure and metabolism through reconstruction of microbial genomes from the environment

Microbial communities are vital in the functioning of all ecosystems; however, most microorganisms are uncultivated, and their roles in natural systems are unclear. Here, using random shotgun sequencing of DNA from a natural acidophilic biofilm, we report reconstruction of near-complete genomes of Leptospirillum group II and Ferroplasma type II, and partial recovery of three other genomes. This was possible because the biofilm was dominated by a small number of species populations and the frequency of genomic rearrangements and gene insertions or deletions was relatively low. Because each sequence read came from a different individual, we could determine that single-nucleotide polymorphisms are the predominant form of heterogeneity at the strain level. The Leptospirillum group II genome had remarkably few nucleotide polymorphisms, despite the existence of low-abundance variants. The Ferroplasma type II genome seems to be a composite from three ancestral strains that have undergone homologous recombination to form a large population of mosaic genomes. Analysis of the gene complement for each organism revealed the pathways for carbon and nitrogen fixation and energy generation, and provided insights into survival strategies in an extreme environment.

The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis

Mycorrhizal symbioses—the union of roots and soil fungi—are universal in terrestrial ecosystems and may have been fundamental to land colonization by plants. Boreal, temperate and montane forests all depend on ectomycorrhizae. Identification of the primary factors that regulate symbiotic development and metabolic activity will therefore open the door to understanding the role of ectomycorrhizae in plant development and physiology, allowing the full ecological significance of this symbiosis to be explored. Here we report the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolor (Fig. 1) and highlight gene sets involved in rhizosphere colonization and symbiosis. This 65-megabase genome assembly contains ∼20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences. We detected unexpected genomic features, most notably a battery of effector-type small secreted proteins (SSPs) with unknown function, several of which are only expressed in symbiotic tissues. The most highly expressed SSP accumulates in the proliferating hyphae colonizing the host root. The ectomycorrhizae-specific SSPs probably have a decisive role in the establishment of the symbiosis. The unexpected observation that the genome of L. bicolor lacks carbohydrate-active enzymes involved in degradation of plant cell walls, but maintains the ability to degrade non-plant cell wall polysaccharides, reveals the dual saprotrophic and biotrophic lifestyle of the mycorrhizal fungus that enables it to grow within both soil and living plant roots. The predicted gene inventory of the L. bicolor genome, therefore, points to previously unknown mechanisms of symbiosis operating in biotrophic mycorrhizal fungi. The availability of this genome provides an unparalleled opportunity to develop a deeper understanding of the processes by which symbionts interact with plants within their ecosystem to perform vital functions in the carbon and nitrogen cycles that are fundamental to sustainable plant productivity.

The Genome of the Western Clawed Frog Xenopus tropicalis

Frog Genome The African clawed frog Xenopus tropicalis is the first amphibian to have its genome sequenced. Hellsten et al. (p. 633, see the cover) present an analysis of a draft assembly of the genome. The genome of the frog, which is an important model system for developmental biology, encodes over 20,000 protein-coding genes, of which more than 1700 genes have identified human disease associations. Detailed comparison of the content of protein-coding genes with other tetrapods—human and chicken—reveals extensive shared synteny, occasionally spanning entire chromosomes. Assembly, annotation, and analysis of the frog genome compares gene content and synteny with the human and chicken genomes. The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes more than 20,000 protein-coding genes, including orthologs of at least 1700 human disease genes. Over 1 million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like that of other tetrapods, the genome of X. tropicalis contains gene deserts enriched for conserved noncoding elements. The genome exhibits substantial shared synteny with human and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.

The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome

Following birth, the breast-fed infant gastrointestinal tract is rapidly colonized by a microbial consortium often dominated by bifidobacteria. Accordingly, the complete genome sequence of Bifidobacterium longum subsp. infantis ATCC15697 reflects a competitive nutrient-utilization strategy targeting milk-borne molecules which lack a nutritive value to the neonate. Several chromosomal loci reflect potential adaptation to the infant host including a 43 kbp cluster encoding catabolic genes, extracellular solute binding proteins and permeases predicted to be active on milk oligosaccharides. An examination of in vivo metabolism has detected the hallmarks of milk oligosaccharide utilization via the central fermentative pathway using metabolomic and proteomic approaches. Finally, conservation of gene clusters in multiple isolates corroborates the genomic mechanism underlying milk utilization for this infant-associated phylotype.

Application of a High-Density Oligonucleotide Microarray Approach To Study Bacterial Population Dynamics during Uranium Reduction and Reoxidation

ABSTRACT Reduction of soluble uranium U(VI) to less-soluble uranium U(IV) is a promising approach to minimize migration from contaminated aquifers. It is generally assumed that, under constant reducing conditions, U(IV) is stable and immobile; however, in a previous study, we documented reoxidation of U(IV) under continuous reducing conditions (Wan et al., Environ. Sci. Technol. 2005, 39:6162-6169). To determine if changes in microbial community composition were a factor in U(IV) reoxidation, we employed a high-density phylogenetic DNA microarray (16S microarray) containing 500,000 probes to monitor changes in bacterial populations during this remediation process. Comparison of the 16S microarray with clone libraries demonstrated successful detection and classification of most clone groups. Analysis of the most dynamic groups of 16S rRNA gene amplicons detected by the 16S microarray identified five clusters of bacterial subfamilies responding in a similar manner. This approach demonstrated that amplicons of known metal-reducing bacteria such as Geothrix fermentans (confirmed by quantitative PCR) and those within the Geobacteraceae were abundant during U(VI) reduction and did not decline during the U(IV) reoxidation phase. Significantly, it appears that the observed reoxidation of uranium under reducing conditions occurred despite elevated microbial activity and the consistent presence of metal-reducing bacteria. High-density phylogenetic microarrays constitute a powerful tool, enabling the detection and monitoring of a substantial portion of the microbial population in a routine, accurate, and reproducible manner.

Genome sequence of the lignocellulose-bioconverting and xylose-fermenting yeast Pichia stipitis.

Xylose is a major constituent of plant lignocellulose, and its fermentation is important for the bioconversion of plant biomass to fuels and chemicals. Pichia stipitis is a well-studied, native xylose-fermenting yeast. The mechanism and regulation of xylose metabolism in P. stipitis have been characterized and genes from P. stipitis have been used to engineer xylose metabolism in Saccharomyces cerevisiae. We have sequenced and assembled the complete genome of P. stipitis. The sequence data have revealed unusual aspects of genome organization, numerous genes for bioconversion, a preliminary insight into regulation of central metabolic pathways and several examples of colocalized genes with related functions. The genome sequence provides insight into how P. stipitis regulates its redox balance while very efficiently fermenting xylose under microaerobic conditions.

Genomic analysis of the uncultivated marine crenarchaeote Cenarchaeum symbiosum

Crenarchaeota are ubiquitous and abundant microbial constituents of soils, sediments, lakes, and ocean waters. To further describe the cosmopolitan nonthermophilic Crenarchaeota, we analyzed the genome sequence of one representative, the uncultivated sponge symbiont Cenarchaeum symbiosum. C. symbiosum genotypes coinhabiting the same host partitioned into two dominant populations, corresponding to previously described a- and b-type ribosomal RNA variants. Although they were syntenic, overlapping a- and b-type ribotype genomes harbored significant variability. A single tiling path comprising the dominant a-type genotype was assembled and used to explore the genomic properties of C. symbiosum and its planktonic relatives. Of 2,066 ORFs, 55.6% matched genes with predicted function from previously sequenced genomes. The remaining genes partitioned between functional RNAs (2.4%) and hypotheticals (42%) with limited homology to known functional genes. The latter category included some genes likely involved in the archaeal–sponge symbiotic association. Conversely, 525 C. symbiosum ORFs were most highly similar to sequences from marine environmental genomic surveys, and they apparently represent orthologous genes from free-living planktonic Crenarchaeota. In total, the C. symbiosum genome was remarkably distinct from those of other known Archaea and shared many core metabolic features in common with its free-living planktonic relatives.

Identification of Methyl Coenzyme M Reductase A (mcrA) Genes Associated with Methane-Oxidizing Archaea

ABSTRACT Phylogenetic and stable-isotope analyses implicated two methanogen-like archaeal groups, ANME-1 and ANME-2, as key participants in the process of anaerobic methane oxidation. Although nothing is known about anaerobic methane oxidation at the molecular level, the evolutionary relationship between methane-oxidizing archaea (MOA) and methanogenic archaea raises the possibility that MOA have co-opted key elements of the methanogenic pathway, reversing many of its steps to oxidize methane anaerobically. In order to explore this hypothesis, the existence and genomic conservation of methyl coenzyme M reductase (MCR), the enzyme catalyzing the terminal step in methanogenesis, was studied in ANME-1 and ANME-2 archaea isolated from various marine environments. Clone libraries targeting a conserved region of the alpha subunit of MCR (mcrA) were generated and compared from environmental samples, laboratory-incubated microcosms, and fosmid libraries. Four out of five novel mcrA types identified from these sources were associated with ANME-1 or ANME-2 group members. Assignment of mcrA types to specific phylogenetic groups was based on environmental clone recoveries, selective enrichment of specific MOA and mcrA types in a microcosm, phylogenetic congruence between mcrA and small-subunit rRNA tree topologies, and genomic context derived from fosmid sequences. Analysis of the ANME-1 and ANME-2 mcrA sequences suggested the potential for catalytic activity based on conservation of active-site amino acids. These results provide a basis for identifying methanotrophic archaea with mcrA sequences and define a functional genomic link between methanogenic and methanotrophic archaea.

Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and climate

Significance We demonstrate that grape-associated microbial biogeography is nonrandomly associated with regional, varietal, and climatic factors across multiscale viticultural zones. This poses a paradigm shift in our understanding of food and agricultural systems beyond grape and wine production, wherein patterning of whole microbial communities associated with agricultural products may associate with downstream quality characteristics. Elucidating the relationship between production region, climate, and microbial patterns may enhance biological control within these systems, improving the supply, consumer acceptance, and economic value of important agricultural commodities. Wine grapes present a unique biogeography model, wherein microbial biodiversity patterns across viticultural zones not only answer questions of dispersal and community maintenance, they are also an inherent component of the quality, consumer acceptance, and economic appreciation of a culturally important food product. On their journey from the vineyard to the wine bottle, grapes are transformed to wine through microbial activity, with indisputable consequences for wine quality parameters. Wine grapes harbor a wide range of microbes originating from the surrounding environment, many of which are recognized for their role in grapevine health and wine quality. However, determinants of regional wine characteristics have not been identified, but are frequently assumed to stem from viticultural or geological factors alone. This study used a high-throughput, short-amplicon sequencing approach to demonstrate that regional, site-specific, and grape-variety factors shape the fungal and bacterial consortia inhabiting wine-grape surfaces. Furthermore, these microbial assemblages are correlated to specific climatic features, suggesting a link between vineyard environmental conditions and microbial inhabitation patterns. Taken together, these factors shape the unique microbial inputs to regional wine fermentations, posing the existence of nonrandom “microbial terroir” as a determining factor in regional variation among wine grapes.

Environmental genomics reveals a single-species ecosystem deep within earth

DNA from low-biodiversity fracture water collected at 2.8-kilometer depth in a South African gold mine was sequenced and assembled into a single, complete genome. This bacterium, Candidatus Desulforudis audaxviator, composes >99.9% of the microorganisms inhabiting the fluid phase of this particular fracture. Its genome indicates a motile, sporulating, sulfate-reducing, chemoautotrophic thermophile that can fix its own nitrogen and carbon by using machinery shared with archaea. Candidatus Desulforudis audaxviator is capable of an independent life-style well suited to long-term isolation from the photosphere deep within Earths crust and offers an example of a natural ecosystem that appears to have its biological component entirely encoded within a single genome.