Cultivation methods have contributed to our present knowledge about the presence and diversity of microbes in naturally occurring communities. However, it is well established that only a small fraction of prokaryotes have been cultivated by standard methods and, therefore, the prokaryotes that are cultivated may not reflect the composition and diversity within those communities. Of the two prokaryotic phylogenetic domains, Bacteria and Archaea, members of the former have been shown to be ubiquitous in nature, with ample evidence of vast assemblages of uncultured organisms. There is also now increasingly compelling evidence that the Archaea, which were once thought to occupy a limited number of environments, are also globally widespread. Here we report the use of molecular phylogenetic techniques, which are independent of microbial cultivation, to conduct an assessment of Archaea in a soil microbial community. Small subunit ribosomal RNA genes of Archaea were amplified from soil and cloned. Phylogenetic and nucleotide signature analyses of these cloned small subunit ribosomal RNA gene sequences revealed a cluster of Archaea from a soil microbial community that diverge deeply from the crenarchaeotal line of descent and has the closest affiliation to the lineage of planktonic Archaea. The identification and phylogenetic classification of this archaeal lineage from soil contributes to our understanding of the ecological significance of Archaea as a component of microbial communities in non-extreme environments.

Herbivores can gain indirect access to recalcitrant carbon present in plant cell walls through symbiotic associations with lignocellulolytic microbes. A paradigmatic example is the leaf-cutter ant (Tribe: Attini), which uses fresh leaves to cultivate a fungus for food in specialized gardens. Using a combination of sugar composition analyses, metagenomics, and whole-genome sequencing, we reveal that the fungus garden microbiome of leaf-cutter ants is composed of a diverse community of bacteria with high plant biomass-degrading capacity. Comparison of this microbiome's predicted carbohydrate-degrading enzyme profile with other metagenomes shows closest similarity to the bovine rumen, indicating evolutionary convergence of plant biomass degrading potential between two important herbivorous animals. Genomic and physiological characterization of two dominant bacteria in the fungus garden microbiome provides evidence of their capacity to degrade cellulose. Given the recent interest in cellulosic biofuels, understanding how large-scale and rapid plant biomass degradation occurs in a highly evolved insect herbivore is of particular relevance for bioenergy. Leaf-cutter ants form massive subterranean colonies containing millions of workers that harvest hundreds of kilograms of leaves each year. They use these leaves to grow a mutualistic fungus that serves as the colony's primary food source. By farming fungus in specialized garden chambers, these dominant Neotropical herbivores facilitate rapid large-scale plant biomass conversion. Our understanding of this degradation process, and the responsible microbial community, is limited. In this study, we track the degradation of plant polymers in leaf-cutter ant fungus gardens and characterize the microbial community potentially mediating this process. We show that cellulose and hemicelluloses are degraded in the fungus gardens and that a previously unknown microbial community containing a diversity of bacteria is present. Metagenomic analysis of this community's genetic content revealed many genes predicted to encode enzymes capable of degrading plant cell walls. The ability of leaf-cutter ants to maintain an external microbial community with high plant biomass-degrading capacity likely represents a key step in the establishment of these ants as widespread, dominant insect herbivores in the Neotropics. This system is an important model for understanding how microbial communities degrade plant biomass in natural systems and has direct relevancy for bioenergy, given recent interest in cellulosic biofuels.

The ability of phototrophs to convert light into biological energy is critical for life on Earth. However, there can be deleterious consequences associated with this bioenergetic conversion, including the production of toxic byproducts. For example, singlet oxygen (¹O₂) can be formed during photosynthesis by energy transfer from excited triplet-state chlorophyll pigments to O₂. By monitoring gene expression and growth in the presence of¹O₂, we show that the phototrophic bacteriumRhodobacter sphaeroidesmounts a transcriptional response to this reactive oxygen species (ROS) that requires the alternative σ factor, σ^E. An increase in σ^Eactivity is seen when cells are exposed to¹O₂generated either by photochemistry within the photosynthetic apparatus or the photosensitizer, methylene blue. Wavelengths of light responsible for the generating triplet-state chlorophyll pigments in the photosynthetic apparatus are sufficient for a sustained increase in σ^Eactivity. Continued exposure to¹O₂is required to maintain this transcriptional response, and other ROS do not cause a similar increase in σ^E-dependent gene expression. When a σ^Emutant produces low levels of carotenoids,¹O₂is bacteriocidal, suggesting that this response is essential for protecting cells from this ROS. In addition, global gene expression analysis identified ≈180 genes (≈60 operons) whose RNA levels increase ≥3-fold in cells with increased σ^Eactivity. Gene products encoded by four newly identified σ^E-dependent operons are predicted to be involved in stress response, protecting cells from¹O₂damage, or the conservation of energy.

A Puf- strain of Rhodobacter sphaeroides (PUFB1) was constructed by deleting a portion of the proximal region of the puf operon and inserting a kanamycin resistance gene cartridge. Southern blot analysis demonstrated that in PUFB1, the defective copy of the puf operon had replaced, through homologous recombination, the normal chromosomal copy. The Puf- phenotype was characterized by the inability of PUFB1 to grow photoheterotrophically (PS-), the lack of detectable puf-specific transcripts, the absence of the light-harvesting I complex and, by inference, the reaction center spectral complex, and greatly reduced levels of the light-harvesting II complex. The PS+ phenotype was restored to PUFB1 when a 13-kilobase BamHI restriction endonuclease fragment containing the entire puf operon and flanking regions was cloned into the broad-host-range plasmid vector RK2 derivative pRK404 and introduced by conjugation into PUFB1. In these complemented strains, there was an increased number of copies of the puf operon (four to six copies per defective chromosomal copy) as the result of plasmid copy number. However, there was no concomitant increase in either the specific bacteriochlorophyll content or the level of puf-specific transcripts when these strains were grown photoheterotrophically.

Microorganisms have shaped our planet and its inhabitants for over 3.5 billion years. Humankind has had a profound influence on the biosphere, manifested as global climate and land use changes, and extensive urbanization in response to a growing population. The challenges we face to supply food, energy, and clean water while maintaining and improving the health of our population and ecosystems are significant. Given the extensive influence of microorganisms across our biosphere, we propose that a coordinated, cross-disciplinary effort is required to understand, predict, and harness microbiome function. From the parallelization of gene function testing to precision manipulation of genes, communities, and model ecosystems and development of novel analytical and simulation approaches, we outline strategies to move microbiome research into an era of causality. These efforts will improve prediction of ecosystem response and enable the development of new, responsible, microbiome-based solutions to significant challenges of our time.