Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Genomics:GTL Program Projects
University of Massachusetts, Amherst
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Progress Toward Genome-Scale Monitoring of In Situ Gene Expression During Uranium Bioremediation and Electricity Harvesting
Dawn Holmes* (dholmes@microbio.umass.edu), Kelly Nevin, Regina O’ Neil, Zhenya Shelbolina, Martin Lanthier, Jonathan Kaye, Brad Postier, and Derek Lovley
University of Massachusetts, Amherst, MA
The first two goals of our Genomics:GTL project are: 1) to determine the genome sequences of the Geobacteraceae that predominate during in situ bioremediation of uranium-contaminated waters and on the surface of electrodes harvesting electricity from waste organic matter and 2) to comprehensively determine genome-wide patterns of the expression of these Geobacteraceae genes in the environments of interest. This data will be used to modify in silico models of Geobacteraceae, that are initially being developed with data from well-studied pure cultures of Geobacteraceae so that these models will be able to more accurately predict the growth and metabolism under a variety of conditions in subsurface environments or on electrodes.
Substantial progress was made in 2004 in field-scale genomic studies of in situ uranium bioremediation and harvesting electricity from aquatic sediments. The in situ uranium bioremediation studies were conducted at the DOE-NABIR UMTRA field study site in Rifle, Colorado. Acetate was added to the groundwater in order to stimulate the activity of dissimilatory metal-reducing microorganisms. As previously observed, uranium was rapidly removed from the groundwater as soluble U(VI) was reduced to insoluble U(IV). This is important because it demonstrates the ability to conduct reproducible field experiments at the site.
The composition of the microbial community and groundwater chemistry were monitored daily during the 27 day experiment. The expression of a suite of key genes was monitored every other day. Furthermore, about every 5 days large quantities of groundwater (> 500 liters) were collected for analysis of the genome sequences of the predominant microorganisms. This level of molecular analysis of a subsurface environment is unprecedented.
Analysis of 16S rRNA gene sequences demonstrated that within 13 days Geobacteraceae increased from less than 5% of the microbial community when acetate was first injected to over 99% of the community. Quantitative analysis of multiple highly conserved Geobacteraceae genes indicated that the number of Geobacteraceae increased more than 4 orders of magnitude in this short time. Furthermore, the diversity of Geobacteraceae was extremely low. For example, during the height of uranium removal, 82% of the Geobacteraceae had 16S rRNA gene sequences that were 97.5-100% identical with 35% having an identical sequence within sequencing error. This is an incredible enrichment of closely related microorganisms in a natural environment. The finding that field experiments can be reproducibly conducted at the Rifle site and that the environment is highly enriched in a small cluster of highly related organisms demonstrates that this environment is ideal for the genome-enabled in silico environmental modeling we have proposed.
Furthermore, as the result of our detailed investigations of pure culture Geobacteraceae genomes in the first two years of our Genomics:GTL project, it was possible to use newly discovered Geobacteraceae-specific gene sequences to conduct detailed studies on levels of in situ gene expression in the subsurface. For example, from the analysis of the six available Geobacteraceae genomes it was possible to identify Geobacteraceae-specific sequences for unique genes such as OmpB, a unique Geobacteraceae-specific outer-membrane protein and GltA, a eukaryotic-like citrate synthase unique to Geobacteraceae, as well as for genes involved in nutrient uptake and a diversity of housekeeping genes.
Studies on expression of gltA in chemostat cultures demonstrated a positive correlation between rates of acetate metabolism and levels of gltA transcripts, suggesting that levels of gltA transcripts might provide an indication of rates of metabolism. In the field experiment there was a remarkable correspondence between acetate levels in the groundwater and levels of gltA transcripts. As acetate rose gltA transcript levels increased. Both acetate and gltA transcript levels dropped during a rain event that diluted the acetate with rainwater recharge, and then gltA levels increased concurrent with a renewed increase in acetate over time. This contrasted with the constant expression, relative to total RNA, of Geobacteraceae housekeeping genes such as recA, rpoD, and proC. The pattern of expression of ompB was similar to that of the housekeeping genes, consistent with pure culture results indicating that this gene is constitutively expressed and its transcript levels are not correlated with rates of metabolism. Expression of Geobacteraceae nifD, which encodes for a portion of the nitrogenase complex, followed a pattern similar to that of gltA. This provided further evidence that the metabolism of the Geobacteraceae was controlled by the availability of acetate and also demonstrated that the growth of Geobacteraceae during bioremediation was limited by the availability of fixed nitrogen.
These results demonstrate that high quality mRNA can be recovered from the subsurface and used to monitor the activity and metabolic state of Geobacteraceae during in situ uranium bioremediation. In order to monitor gene expression on a genome-wide basis a microarray approach is required. In order to develop this technique, pure cultures of Geobacter metallireducens were inoculated into sterilized sediments from the Rifle site which were amended with acetate in order to simulate growth of Geobacter species during in situ uranium bioremediation. High quality mRNA in sufficient quantities for microarray analysis could be extracted from the sediments and are currently being analyzed with a whole-genome microarray.
In order to construct microarrays that represent the genomes of the Geobacteraceae that predominate in the subsurface the genome sequences of these organisms are being determined with three approaches. Isolates with 16S rRNA gene sequences that are identical to those that predominate during in situ uranium bioremediation have been recovered and their genomes are being sequenced. High quality genomic DNA was extracted from the subsurface during the 2004 field experiment and is being used to construct both BAC and small insert libraries in order to obtain additional sequence from any Geobacteraceae that might not be isolated. Furthermore, samples have been preserved for single-cell genome sequencing. Sequence data from all three approaches should be available at the time of the meeting. This sequence data will be used to construct arrays for genome-scale analysis of gene expression using mRNA extracted for this purpose in the 2004 field experiment.
Field experiments on electricity harvesting were carried out in freshwater and marine sediments at the UMASS field station on Nantucket Island as well as with a swine waste digestor. Electrodes harvesting energy were highly enriched with Geobacteraceae of low diversity and representatives of the predominant Geobacteraceae were recovered in culture. Studies on gene expression and sequencing genomic DNA similar to those described above for the uranium bioremediation field experiment were carried out and will be presented.
* Presenting author
