IEG Research Interests
The Institute for Environmental Genomics has research interests in functional and comparative genomics, microbial ecology and community genomics, and development of metagenomic and bioinformatic tools. Our current projects include the experimental evolution of Desulfovibrio vulgaris Hildenborough under a variety of stress conditions, developing gene manipulation tools using the CRISPR-Cas9 system, examining the impact of climate change on microbial communities and the global carbon cycle, understanding the global water microbiome, examining the response of microbial communities to contaminants (U, nitrate, pH, Cr) important to the U.S. DOE, designing bioinformatics tools for ‘big data’ analysis, and developing and expanding high-throughput metagenomic tools and techniques such as microarrays, sequencing, and single cell genomics to examine microbial communities.
Functional and Comparative Genomics
This research area focuses on functional analysis of microorganisms important in environmental cleanup and bioenergy, particularly anaerobic microorganisms to understand gene function, regulation, networks and evolution.
A major challenge in microbiology is linking phenotype (functional traits) and genotypes (genes responsible for that trait) because phenotypes are often controlled by multiple genes and correlated to environmental conditions. Microbial experimental evolution can provide direct evidence of gene function in both essential and non-essential genes as well as non-coding regions. In this approach, microbial populations are propagated under controlled laboratory conditions and the underlying genomic changes are identified by sequencing the whole genomes of the evolved strain(s) or populations and aligning to the reference genome sequences of the ancestral strain.
Currently, the functional genomics studies are centered on the sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, and a denitrifier, Rhodanobacter denitrificans. Both species exist in contaminated field sites, but Rhodanobacter is dominant in ground water ecosystems with low pH and high heavy metal concentrations. We use genome sequencing, gene expression microarrays, qPCR, transposon libraries, and mutagenesis to analyze the function of genes involved in energy metabolism under different growth conditions, characterize stress response and global gene expression regulation, and examine their interactions with other species in artificial communities under various environmental conditions.
Targeted genome editing is critical for both fundamental molecular biology and applied genetic engineering, but genome editing is challenging in many microorganism due to the lack of efficient editing tools. The CRISPR-Cas9 system has been shown to be a powerful and versatile option for foreign gene knock-in and gene inactivation in eukaryotes, however, its application in microorganisms has been limited. Our lab has developed a single nick-triggered homologous recombination strategy using the Cas9 nickase and successfully edited specific genome loci in Clostridium cellulolyticum. Currently we are in the process of expanding the application of this powerful tool for use in other bacteria and as a high-throughput process.
Microbial Ecology and Community Genomics
Another key research direction at IEG is to use genomic technology to understand microbial community diversity, composition, structure, function and dynamics and determine the mechanisms controlling microbial community diversity. Microorganisms inhabit almost every imaginable environment on earth and play a large part in global geochemical cycles, yet the extent of microbial diversity and what mechanism control microbial communities are largely unknown. We use a variety of metagenomics approaches, such as functional gene arrays, high-throughput sequencing, and single cell genomics to address these areas. This work examines a variety of systems and environmental perturbations including global change, bioremediation, land use, bioenergy, and agricultural practices as well as across spatial and temporal differences.
We are involved in several projects involving the U. S. DOE Oak Ridge Integrated Field Research Challenge site. Oak Ridge was part of the Manhattan Project and has high levels of U, nitrate, and very low pH. Our focus is to understand the factors and mechanisms shaping microbial community structure at this contaminated site. Understanding the mechanisms controlling microbial community composition and function is extremely important because the knowledge gained may provide novel approaches to the manipulation of microbial communities for desired functions, and will be critical for successful bioaugmentation and biostimulation.
Because the Oak Ridge site has been the subject of so much study and there are decades worth of data related to the site geology, geochemistry, hydrology as well as microbial studies, it is an ideal site to study the stability, resilience, resistance, and evolution of microorganisms and mechanisms that influence their assembly and performance. Samples from over 100 sampling wells have been collected to better understand the response of microbial communities to environmental conditions. Ongoing studies include characterizing the diversity of protists and fungi in groundwater and examining the relationship between functional diversity and phylogenetic diversity. Shotgun sequencing is being employed to determine the diversity and metabolic capacity of the subsurface microbial communities. Mechanisms of community assembly for free-living and particle-associated bacteria are also being determined.
Climate change is an ongoing concern as the average temperature of Earth continues to rise and weather patterns become more extreme. Understanding the responses, adaptations and feedback mechanisms of biological communities to climate change is critical in order to predict the future state of Earth and climate systems. Although a great deal is known regarding the feedbacks of aboveground communities to climate change, the response of belowground microbial communities is still poorly understood. Thus, it is important to advance system-level predictive understanding of the feedbacks of belowground microbial communities to multiple climate change factors and their impacts on soil carbon (C) cycling. Microbial decomposition of permafrost C is one of the most likely potential positive feedbacks from terrestrial ecosystems to the atmosphere in a warmer world, so we have been studying microbial communities from a tundra warming experiment in Alaska. In in a tall grass prairie ecosystem, we have been characterizing community changes resulting from warming and clipping (harvest of plant tops), a practice commonly used in agriculture and for biofuels feedstock harvesting.
Another area of research is examining how microbial communities impact plant growth and what functions are responsible for improved plant growth. This could have important implications to biofuels feedstock growth and harvesting.
Development of Metagenomic and Bioinformatic Tools
IEG researchers have pioneered the development and application of functional gene arrays (e.g., GeoChip), and metagenomic sequencing (e.g., MiSeq sequencing of phylogenetic and functional gene amplicons) for microbial community and quantitative analysis. Current research directions include the establishment of a Raman-based single cell genomics facility and methods for analyzing ‘big data’.
GeoChip microarrays are the most comprehensive FGAs available to date. The GeoChip arrays for probes for functional genes involved in C, N, S, and P cycles, degradation genes environmental contaminants (organic solvents, pesticides, BTEX, etc.), metal homeostasis, antibiotic resistance, stress response, energy processing, as well as several other categories (viral and protist genes, etc). We continue to expand the number and coverage functional gene groups, develop and improve on software and algorithms used for sequence selection, probe design, and data processing, improve on sample preparation and hybridization protocols, and develop new uses for the microarray technology.
High-throughput sequencing has enabled microbiologists to address many previously unanswerable research questions and sequencing amplified gene markers to determine phylogenetic and functional diversity is becoming a common approach. The Illumina platform, particularly the MiSeq, has become an attractive sequencing option due to lower cost, rapid analysis, and higher accuracy. IEG is equipped with a MiSeq benchtop sequencer. A major challenge for MiSeq amplicon sequencing is the so-called low sequence diversity or unbalanced base composition in template DNA sequences. We have optimized protocols for de novo sequencing of 16S rRNA gene for bacteria and archaea and developed a novel phasing amplicon sequencing approach to overcome this issue by shifting sequencing phases among different community samples by adding spacers consisting of 0-7 bases. We continue to improve our sequencing protocols to increase quality and reduce cost and to increase available primer sets to allow sequencing of more genes.
In addition to the above, IEG is expanding into single cell genomics using Raman sorting. A great challenge in single cell genomics is identifying the isolated cells for subsequent sequencing. Raman sorting allows for the identification of specific bacterial species based on their spectral pattern. The spectra can also provide information on physiological differences, growth phase of particular cells, and incorporation of stable isotopes. We are currently working on setting up protocols and methods for this new project.
Although the rapidly expanding genomic technologies provide powerful analysis tools, as data sets become larger and larger, it is increasingly more difficult to analyze this ‘big data’. IEG has been working on development and implementing bioinformatics tools for processing this data. Using random matrix theory, we have been identified cellular interaction networks consisting of many individual modules and automatic methods for identifying these functional modules. Data processing and analysis pipelines have been developed for microarray and sequencing data.