Project 4: Application of comparative genomics, transcriptomics, and proteomics to optimize microbial reductive dehalogenation
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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Professor Alvarez-Cohen is a leader in understanding how microbes break down contaminants and what can be done to make bioremediation work for contaminants found at hazardous waste sites. She was elected to the National Academy of Engineering because of her discoveries of new organisms that can contribute to bioremediation. She has been involved for many years in providing advice through service on many committees at the National Academy of Sciences.
Investigators are working with microbes that can break down compounds that are common contaminants of groundwater at Superfund sites (and other hazardous sites). They are focusing on chlorinated compounds that are difficult to break down to non-toxic forms. Microbes that can break down these contaminants tend to act very slowly, so such bioremediation takes a long time. Based on their research on the biology and ecology of the micro-organisms, investigators are trying to determine how to speed-up this process. They are looking at adding other organisms to the mix and making changes in conditions. They hope to find better ways to remediate sites contaminated with TCE, the most common Superfund groundwater contaminant, which was formerly used as a solvent in everything from dry cleaning to white-out.
The micro-organisms they are using are bacteria of the genus Dehalococcoides (Deha). These bacteria degrade chloroethenes like TCE into a nontoxic form. Using these methods, the water stays within the contaminated aquifer during bioremediation, without pump and treat technologies. This is cost-effective and minimizes human and ecological exposure. The project uses cutting-edge molecular biological techniques to identify the bioprocesses responsible for efficient degradation of chloroethene compounds. The goal is to engineer the conditions to optimize dechlorination activities during bioremediation and to develop the tools needed to monitor conditions throughout this process.
Important discoveries so far
Investigators have developed molecular tools that allow them to study both the fundamental and applied aspects of Dehalococcoides, a bacterium capable of bioremediating chloroethenes. These tools have led to new discoveries about the growth of this organism, leading to optimization strategies that promote more successful bioremediation.
Accomplishments for the last year
One specific strategy of this research is to identify the enzymes of the Deha bacteria that contribute to robust growth and to degradation of TCE. Previous studies demonstrated that when Deha is grown with another kind of bacteria, the Deha grows to higher densities more rapidly, and TCE is broken down to ethene more quickly. (The other bacteria were a type that ferments lactate, known as Desulfovibrio vulgaris Hildenborough (DVH))
Over the past year, investigators compared the proteins produced when the two bacteria were grown together to the proteins produced when Deha was grown by itself. This was to find out what biological pathways were contributing to the greater growth of the bacteria and greater remediation of the TCE when the two bacteria were grown together.
When the two bacteria were grown together, proteins that were significantly regulated were involved in functions such as electron transfer and coenzyme acquisition for TCE degradation. This indicates that the greater growth for the two bacteria occurred because DVH produces compounds that help to meet metabolic requirements of Deha, known as syntrophy. This is an application of proteomics methods.
Work last year also dealt with the RNA responses of Deha to environmentally relevant parameters. We applied a custom-designed genus-wide microarray to a Dehalococcoides-containing microbial community (referred to as ANAS) that was enriched from a local Superfund site. Characterization of Deha DNA in ANAS revealed a unique collection of functional genes that differ from any currently sequenced Dehalococcoides strains. RNA was collected and analyzed at three time-points throughout the ANAS growth cycle in order to study Dehalococcoides under feast and famine growth conditions. Microarray analysis revealed that under feast conditions, the dominant functions represented by abundant RNA are associated with protein synthesis. On the other hand, under the famine condition, abundant RNA is dominantly related to stress response genes, such as chaperones, heat shock proteins, antioxidant genes and transcriptional regulation genes. These insights into the transcriptomes of Dehalococcoides under different environmental parameters will improve our understanding of the physiology of these bacteria in the environment. This is essential in the development of more effective strategies for in situ bioremediation of chloroethenes. On-going studies are also being conducted to compare the RNA profiles of unknown Dehalococcoides strains in other Superfund site enrichments grown under environmental conditions favoring methanogenesis and/or vitamin B12 availability.
What we plan to do next
This work has far-reaching significance because we have developed molecular tools that allow us to study both the fundamental and the applied aspects of Dehalococcoides, a bacterium capable of bioremediating chloroethenes. These tools have allowed us to make new discoveries about the growth of this organism, leading to optimization strategies that promote more successful in situ bioremediation.
- Mahendra S, Grostern A, Alvarez-Cohen L (2012) The impact of chlorinated solvent co-contaminants on the biodegradation kinetics of 1,4-dioxane. Chemosphere. Dec 10;http://dx.doi.org/10.1016/j.chemosphere.2012.10.104. PMID: 23237300 (PMC Journal- In Progress). [PDF]
- West KA, Lee PK, Johnson DR, Zinder SH, Alvarez-Cohen L (2012) Global gene expression of Dehalococcoides within a robust dynamic TCE-dechlorinating community under conditions of periodic substrate supply. Biotechnol Bioeng. Dec 27;doi: 10.1002/bit.24819. PMID: 23280440 (PMC Journal – In Process). [PDF]
- Yi S, Seth EC, Men YJ, Stabler SP, Allen RH, Alvarez-Cohen L, Taga ME (2012) Versatility in corrinoid salvaging and remodeling pathways supports corrinoid-dependent metabolism in Dehalococcoides mccartyi. Appl Environ Microbiol. Nov;78(21):7745-7752. PMCID: PMC3485714. [PDF]
- Grostern A, Sales CM, Zhuang WQ, Erbilgin O, Alvarez-Cohen L (2012) Glyoxylate metabolism is a key feature of the metabolic degradation of 1,4-dioxane by Pseudonocardia dioxanivorans strain CB1190. Appl. Environ. Microbiol. May;78(9):3298-3308. PMCID: PMC3346452. [PDF]
- Lee PK, Dill BD, Louie TS, Shah M, VerBerkmoes NC, Andersen GL, Zinder SH, Alvarez-Cohen L (2012) Global transcriptomic and proteomic responses of Dehalococcoides ethenogenes strain 195 to fixed nitrogen limitation. Appl. Environ. Microbiol. Mar;78(5):1424-36. PMCID: PMC3294477. [PDF]
- Men Y, Feil H, VerBerkmoes NC, Shah MB, Johnson DR, Lee PK, West KA, Zinder SH, Andersen GL, Alvarez-Cohen L (2012) Sustainable syntrophic growth of Dehalococcoides ethenogenes strain 195 with Desulfovibrio vulgaris Hildenborough and Methanobacterium congolense: global transcriptomic and proteomic analyses. ISME J. Feb 6;6(2):410-421. PMCID: PMC3260503. [PDF]
- Lee PK, Warnecke F, Brodie E, Macbeth T, Conrad ME, Andersen G, Alvarez-Cohen L (2012) Phylogenetic microarray analysis of a microbial community performing reductive dechlorination at a TCE-contaminated site. Environ. Sci. Technol. Jan 17;46(2):1044-1054. PMCID: PMC3461955. [PDF]
- Lee PK, Cheng D, Hu P, West KA, Dick GJ, Brodie EL, Andersen GL, Zinder SH, He J, Alvarez-Cohen L (2011) Comparative genomics of two newly isolated Dehalococcoides strains and an enrichment using a genus microarray. ISME J. Jun;5(6):1014-24. PMCID: PMC3131851. [PDF]
- Robrock KR, Mohn WW, Eltis LD, Alvarez-Cohen L (2010) Biphenyl and ethylbenzene dioxygenases of Rhodococcus jostii RHA1 transform PBDEs. Biotechnol Bioeng. Feb;108(2):313-21. PMID 20872819. [PDF]
- Lee PK, He J, Zinder SH, Alvarez-Cohen L (2009) Evidence for nitrogen fixation by “Dehalococcoides ethenogenes” strain 195. Appl Environ Microbiol. Dec; 75(23):7551-5. PMID: 19820162. PMCID: PMC2786412. [PDF]
- Tang YJ, Yi S, Zhuang WQ, Zinder SH, Keasling JD, Alvarez-Cohen L (2009) Investigation of carbon metabolism in “Dehalococcoides ethenogenes” strain 195 by use of isotopomer and transcriptomic analyses. J Bacteriol. Aug; 191(16):5224-31. PMID: 19525347. PMCID: PMC2725585. [PDF]
- Robrock KR, Coelhan M, Sedlak DL, Alvarez-Cohent L (2009) Aerobic biotransformation of polybrominated diphenyl ethers (PBDEs) by bacterial isolates. Environ Sci Technol. Aug 1; 43(15):5705-11. PMID: 19731666. [PDF]
- Johnson DR, Nemir A, Andersen GL, Zinder SH, Alvarez-Cohen L (2009) Transcriptomic microarray analysis of corrinoid responsive genes in Dehalococcoides ethenogenes strain 195. FEMS Microbiol Lett. May; 294(2):198-206. PMID: 19341394. [PDF]
- West KA, Johnson DR, Hu P, DeSantis TZ, Brodie EL, Lee PK, Feil H, Andersen GL, Zinder SH, Alvarez-Cohen L (2008) Comparative genomics of “Dehalococcoides ethenogenes” 195 and an enrichment culture containing unsequenced “Dehalococcoides” strains. Appl Environ Microbiol. Jun; 74(11):3533-40. PMID: 18359838. PMCID: PMC2423027. [PDF]
- Johnson DR, Brodie EL, Hubbard AE, Andersen GL, Zinder SH, Alvarez-Cohen L (2008) Temporal transcriptomic microarray analysis of “Dehalococcoides ethenogenes” strain 195 during the transition into stationary phase. Appl Environ Microbiol. May; 74(9):2864-72. PMID: 18310438. PMCID: PMC2394897. [PDF]
- Lee PK, Macbeth TW, Sorenson KS, Jr., Deeb RA, Alvarez-Cohen L (2008) Quantifying genes and transcripts to assess the in situ physiology of “Dehalococcoides” spp. in a trichloroethene-contaminated groundwater site. Appl Environ Microbiol. May; 74(9):2728-39. PMID: 18326677. PMCID: PMC2394903. [PDF]
- Robrock KR, Korytar P, Alvarez-Cohen L (2008) Pathways for the anaerobic microbial debromination of polybrominated diphenyl ethers. Environ Sci Technol. Apr 15; 42(8):2845-52. PMID: 18497133. [PDF]
- Sharp JO, Sales CM, LeBlanc JC, Liu J, Wood TK, Eltis LD, Mohn WW, Alvarez-Cohen L (2007) An inducible propane monooxygenase is responsible for N-nitrosodimethylamine degradation by Rhodococcus sp. strain RHA1. Appl Environ Microbiol. Nov; 73(21):6930-8. PMID: 17873074. PMCID: PMC2074979. [PDF]
- Lee PK, Conrad ME, Alvarez-Cohen L (2007) Stable carbon isotope fractionation of chloroethenes by dehalorespiring isolates. Environ Sci Technol. Jun 15; 41(12):4277-85. PMID: 17626425. [PDF]
- He J, Holmes VF, Lee PK, Alvarez-Cohen L (2007) Influence of vitamin B12 and cocultures on the growth of Dehalococcoides isolates in defined medium. Appl Environ Microbiol. May; 73(9):2847-53. PMID: 17337553. PMC1892872. [PDF]
- Rahm BG, Chauhan S, Holmes VF, Macbeth TW, Sorenson KS, Jr., Alvarez-Cohen L (2006) Molecular characterization of microbial populations at two sites with differing reductive dechlorination abilities. Biodegradation. Dec; 17(6):523-34. PMID: 16477354. [PDF]
- Holmes VF, He J, Lee PK, Alvarez-Cohen L (2006) Discrimination of multiple Dehalococcoides strains in a trichloroethene enrichment by quantification of their reductive dehalogenase genes. Appl Environ Microbiol. Sep; 72(9):5877-83. PMID: 16957207. PMC1563660. [PDF]
- Lee PK, Johnson DR, Holmes VF, He J, Alvarez-Cohen L (2006) Reductive dehalogenase gene expression as a biomarker for physiological activity of Dehalococcoides spp. Appl Environ Microbiol. Sep; 72(9):6161-8. PMID: 16957242. PMCID: PMC1563655. [PDF]
- Mahendra S, Alvarez-Cohen L (2006) Kinetics of 1,4-dioxane biodegradation by monooxygenase-expressing bacteria. Environ Sci Technol. Sep 1; 40(17):5435-42. PMID: 16999122. [PDF]
- He J, Robrock KR, Alvarez-Cohen L (2006) Microbial reductive debromination of polybrominated diphenyl ethers (PBDEs). Environ Sci Technol. Jul 15; 40(14):4429-34. PMID: 16903281. [PDF]
- Johnson DR, Lee PK, Holmes VF, Fortin AC, Alvarez-Cohen L (2005) Transcriptional expression of the tceA gene in a Dehalococcoides-containing microbial enrichment. Appl Environ Microbiol. Nov; 71(11):7145-51. PMID: 16269753. PMCID: PMC1287711. [PDF]
- Freeborn RA, West KA, Bhupathiraju VK, Chauhan S, Rahm BG, Richardson RE, Alvarez-Cohen L (2005) Phylogenetic analysis of TCE-dechlorinating consortia enriched on a variety of electron donors. Environ Sci Technol. Nov 1; 39(21):8358-68. PMID: 16294874. [PDF]
IN PRESS AT LAST SUBMISSION
- Johnson DR, Lee PK, Holmes VF, Alvarez-Cohen L (2005) An internal reference technique for accurately quantifying specific mRNAs by real-time PCR with application to the tceA reductive dehalogenase gene. Appl Environ Microbiol. Jul; 71(7):3866-71. PMID: 16000799. PMC1169012. [PDF]
- Mahendra S, Alvarez-Cohen L (2005) Pseudonocardia dioxanivorans sp. nov., a novel actinomycete that grows on 1,4-dioxane. Int J Syst Evol Microbiol. Mar; 55(Pt 2):593-8. PMID: 15774630. [PDF]
- Sharp JO, Wood TK, Alvarez-Cohen L (2005) Aerobic biodegradation of N-nitrosodimethylamine (NDMA) by axenic bacterial strains. Biotechnol Bioeng. Mar 5; 89(5):608-18. PMID: PMC15672376. [PDF]