Project 4: Application of comparative genomics, transcriptomics, and proteomics to optimize microbial reductive dehalogenation
Summary
This project applies advanced molecular tools to understand and optimize the microbial detoxification of common Superfund pollutants, perchloroethene (PCE) and trichloroethene (TCE), which pose a significant threat to human and ecological health. By studying the fundamental processes responsible for anaerobic microbial degradation of PCE and TCE, this work promotes improved situ bioremediation processes. Previous NIEHS-funded research sought to identify and understand the microbes within complex communities responsible for dechlorination. This work takes the next logical step by focusing on the only genus of bacteria, Dehalococcoides, known to completely reduce PCE and TCE to ethene. Whole-genome microarrays, proteomic analyses, and quantitative PCR are being used to characterize the genomic differences between a variety of Dehalococcoides strains and to evaluate gene expression and proteomic changes caused by reductive dechlorination of a variety of substrates, growth in simple and complex microbial communities, and other physiological perturbations. Genomic and transcriptomic comparison of Dehalococcoides strains with different degradation abilities identifies the pathways responsible for specific and general metabolism as well as reveals the evolutionary relationship between the various isolated strains. Transcriptomic comparison of Dehalococcoides strains in pure and mixed cultures identifies pathways involved in inter-species interactions, reveals the nutritional needs and metabolic roles of Dehalococcoides in consortia, and addresses the limitation in bioremediation applications presented by the poor growth of isolated Dehalococcoides strains. Data from strain identification, gene expression, and protein production is being complied into kinetic models that can be used to predict rates of reductive dechlorination by poorly characterized microbial communities. This research meets the SBRP goal of limiting the human exposure and toxicity of chemicals commonly found at Superfund sites by advancing the development of in situ bioremediation of PCE and TCE, a technology that destroys contaminants in their subsurface location without extraction to the surface, avoiding potential human and ecological exposure.
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Project Update
This project seeks to develop new tools to optimize the application of microorganisms to biodegrade common Superfund pollutants such as trichloroethene (TCE) within contaminated groundwater aquifers, a process called in situ bioremediation. In situ bioremediation is a promising and cost effective method for cleaning these contaminants up without bringing them to the surface and risking human exposure. The work of Drs. Lisa Alvarez-Cohen and Gary Anderson focuses on Dehalococcoides, the only known bacteria that can completely convert TCE to non-toxic ethane gas. A better understanding of the DNA, RNA and proteins of Dehalococcoides will greatly improve the understanding of how to effectively grow these finicky organisms, so that their abundance and activity at bioremediation sites can be maximized.
Onc of the research group’s objectives is to study the growth of Dehalococcoides in complex microbial communities such as one that they enriched from a local Superfund site (ANAS) that is capable of rapid and complete conversion of TCE to ethene. The researchers compared the DNA and RNA of strains of Dehalococcoides in the ANAS microbial community with that of a well-studied isolate in the laboratory, and confirmed that the genes associated with central metabolism, including an apparently incomplete carbon fixation pathway, vitamin B12 salvaging system, nitrogen fixation pathway, and central respiratory enzymes are present and active in both. Also, although one gene known to be responsible for TCE conversion was detected, 13 of the 19 other potential genes that could carry out this reaction were not detected, suggesting the occurrence of horizontal gene transfer. Application of a high-density phylogenetic microarray to study the microbial population in the ANAS culture identified the dominant and active members, including fermentors, sulfate reducers and methane generators. Understanding the microbial community that composes this stable and efficient bioremediating culture furthers understanding of microbial community dynamics. Extensive examination of this model community is planned.
Some of the researchers’ work this year dealt with exploring mechanisms to grow Dehalococcoides more rapidly and to a higher density. Current studies are underway that evaluate the effects of degradation products vinyl chloride and ethene on cell growth. They also tested the effects of nitrogen stress on the culture and showed that the isolate can actually obtain its nitrogen from atmospheric nitrogen gas. However, this reaction causes stress to the cells, resulting in slower growth and biodegradation. In contrast, when the isolate is grown in co-culture with a sulfate-reducing bacterium, it grows more rapidly and robustly to higher densities. Analysis of the RNA and proteins generated by the co-culture confirm that there are important interrelationships between the organisms, allowing them to grow more effectively together than alone.
The significance of this work is that the research group has developed molecular tools that allow them to study both the fundamental and the applied aspects of Dehalococcoides, a bacterium capable of bioremediating chlorinated solvents. These tools have allowed the researchers to make new discoveries about the growth of this organism, so that engineering processes may be designed for more successful in situ bioremediation.
Publications
- Johnson, David R., E.L. Brodie, Alan E. Hubbard, Gary L. Andersen, Steven H. Zinder, and Lisa Alvarez-Cohen. 2008. Temporal transcriptomic microarray analysis of “Dehalococcoides ethenogenes” strain 195 during the transition into stationary phase. Applied and Environmental Microbiology. (http://aem.asm.org/)
74(9):2864-72. - Lee, Patrick K H, Tamzen W. Macbeth, K.S. Sorenson, and Lisa Alvarez-Cohen. 2008. Quantifying Genes and Transcripts to Assess the In Situ Physiology of Dehalococcoides spp. in a Trichloroethene-Contamlnated Field Site. Applied and Environmental Microbiology. (http://aem.asm.org/) 74:2728-2739.
- Lee, Patrick K H, Tamzen W. Macbeth, Kent S. Sorenson, Rula A. Deeb, and Lisa Alvarez-Cohen. 2008. Quantifying genes and transcripts to assess the in situ physiology of “Dehalococcoides” spp. in a trichloroethene-contaminated groundwater site. Applied and Environmental Microbiology. (http://aem.asm.org/) 74(9):2728-39. doi:10.1128/AEM.02199-07 (http://dx.doi.org/10.1128/AEM.02199-07)
- Robrock, Kristin R., P. Korytar, and Lisa Alvarez-Cohen. 2008. Pathways for the Anaerobic Microbial Debromination of Polybrominated Diphenyl Ethers. Environmental Science & Technology. (http://pubs.acs.org/journals/esthag/) 42(8):2845-52. doi:10.1021/es0720917 (http://dx.doi.org/10.1021/es0720917)