Tiedje Lab - Projects
Biodegradation of polychlorinated biphenyls (PCBs) (Jorge Rodrigues)
Polychlorinated biphenyls (PCBs) have been of environmental concern since 1966 when unexplained peaks appeared in chromatographic analysis of water and soil samples. Although PCB production was banned in 1979, approximately 196 million kg of PCBs were released in the environment. Their high hydrophobic chemical characteristic contributes to persistence in the environment and accumulation in higher trophic level species like humans, in which they are known to cause liver damage and estrogen-like activity. Furthermore, known degradation mechanisms (e.g. photolysis and hydrolysis) play only a diminutive role on the removal of PCBs in nature. Microbiological degradation of PCBs has been regarded as the only effective, environmentally safe, and economical way for contamination removal.
Over the past 30 years, our laboratory has been actively searching (and researching) for microbiological mechanisms of PCB degradation. As major steps towards this goal, our laboratory was the first to shown that anaerobic microbial consortia can convert highly chlorinated PCBs into less chlorinated congeners, but leaving the biphenyl ring intact. This process, known as reductive dechlorination, is not important from the practical application for bioremediation, but also expands the range of anaerobic respiration processes, which occur in nature.
Secondly, we have isolated and characterized aerobic microorganisms with capability of growing on PCBs. Microorganisms with more versatile oxidative capabilities will cleave the biphenyl rings to yield chlorinated benzoates and pentanoic acid derivatives, later to be degraded by other bacteria. Alternatively, we engineered the best-known PCB degraders, Burholderia xenovorans strain LB400 and Rhodococcus sp. strain RHA1 with specific chlorobenzoate degradation genes, funneling products of PCB degradation into pre-existing pathways and preventing metabolic toxicity (1, 2, 3).
Lately, with advances in genomics and proteomics, our laboratory is continuing to study PCB degradation on three fronts:
- Transcriptome and proteome profiling of the B. xenovorans strain LB400. Recently, this microorganism had its entire genome completely sequenced, making it one of the largest bacterial genomes known. Its multiple catabolic pathways might explain the exceptional PCB degrading capacity of that strain. To understand how the metabolic network of biodegradative pathways is concatenated during PCB degradation (and how they are linked to the general cell metabolism) is primordial to devise new bioremediation methods and more favorable environmental conditions.
- Identification of active PCB degraders in soils using stable isotope probing (SIP). Although PCB-degrading microorganisms representing broad phylogenetic diversity have been cultivated and identified, it remains unknown which organisms are truly active in PCB degradation in soil. We are applying stable isotope probing techniques in a variety of soils and the rhizosphere to identify bacteria that derive carbon from 13C-biphenyl and to explore the functional genes they possess.
- Metagenomics of PCB contaminated soils. We seek to clone and study unknown PCB biodegradative pathways. Contaminated soils will very slowly degrade PCBs. If yet-to-be cultivated members of the indigenous soil microbial community are responsible for PCB degradation, we can shed light onto their general physiology and stimulate their activity, hence leading to higher rates of PCB degradation. In addition, new pathways for PCB degradation might be discovered, expanding the diversity of metabolic processes to be used for bioremediation.
- Rodrigues, J.L.M., O.V. Maltseva, T.V. Tsoi, R.R. Helton, J.F. Quensen, III, M. Fukuda and J.M. Tiedje. 2001. Development of a Rhodococcus recombinant strain for degradation of products from anaerobic dechlorination of PCBs. Environ. Sci. & Technol. 35:663-668.
- Rodrigues, J.L.M., M.R. Aiello, J.W. Urbance, T.V. Tsoi and J.M. Tiedje. 2002. Use of both 16S rRNA and engineered functional genes with real-time PCR to quantify an engineered, PCB-degrading Rhodococcus in soil. J. Microbiol. Methods 51:181-189.
- Rodrigues, J.L.M., C.A. Kachel, M.R. Aiello, J.F. Quensen, O.V. Maltseva, T.V. Tsoi and J.M. Tiedje. 2006. Degradation of Aroclor 1242 dechlorination products in sediments by Burkholderia xenovorans LB400 (ohb) and Rhodococcus sp. strain RHA1 (fcb). Appl. Environ. Microbiol. 72:2476-2482.
Transcriptome and proteome profiling of Burkholderia xenovorans LB400 (Jacob Parnell, Vincent Denef, Joonhong Park, Tamara Tsoi)
Polychlorinated biphenyls (PCBs) are organic chemicals belonging to a class of hydrocarbons that are among the most important environmental contaminants as they persist in the environment and bioaccumulate. Over the past thirty years, Burkholderia xenovorans strain LB400 has become one of the model organisms in aerobic PCB degradation due to the wide range of congeners it can mineralize. Unfortunately, much of the research on PCB degradation to date has been reductionist, focusing primarily on the genes in the biphenyl pathway and not on the organism as a whole. We use a multi-phasic approach connecting whole cell physiology, genomics, proteomics and metabolomics to converge on mechanisms involved in PCB degradation.
In collaboration with our laboratory, the LB400 genome was sequenced by The Joint Genome Institute and annotated by The Oak Ridge National Laboratories. The LB400 genome is one of the largest prokaryotic genomes sequenced to date with a total genome size of ~9.7 Mbp. We have recently conducted research that provides insight into the general physiology, metabolism, biochemistry and management of the metabolic warehouse of this large genome. The presence of at least eleven "central aromatic" and nineteen "peripheral aromatic" pathways in LB400 are among the highest in any sequenced bacterial genome. Due to numerous and often redundant pathways as well as high genome plasticity, LB400 is versatile and able to adapt to complex or diverse niches.
To enhance our knowledge of LB400, and more specifically how its genomic context enables its success as a PCB degrader, a microarray was constructed to explore genes potentially important in the biodegradation of PCBs. Initial fundamental studies involving the expression of central metabolism pathways in LB400 required for PCB degradation (biphenyl, benzoate, and succinate) indicate functional redundancy of multiple benzoate-degrading systems. These pathways have since been confirmed via proteomic analysis and knockout studies. We further investigated the whole-genome differential expression patterns when LB400 was exposed to PCBs during growth on biphenyl, benzoate, and succinate relative to PCB(-) growth on the same carbon source. Through these analyses, we have found that LB400 is among the microorganisms most tolerant to PCBs. Additionally, we have found expression of several proteins in LB400 that may convey tolerance.
- Denef, V.J., J. Park, J.L.M. Rodrigues, T.V. Tsoi, S.A. Hashsham and J.M. Tiedje. 2003. Validation of a more sensitive method for using spotted oligonucleotide DNA microarrays for functional genomics studies on bacterial communities. Environ. Microbiol. 5:933-943.
- Denef, V.J., J. Park, T.V. Tsoi, J.-M. Rouillard, H. Zhang, J.A. Wibbenmeyer, W. Verstraete, E. Gulari, S.A. Hashsham and J.M. Tiedje. 2004. Biphenyl and benzoate metabolism in a genomic context: Outlining genome-wide metabolic networks in Burkholderia xenovorans LB400. Appl. Environ. Microbiol. 70:4961-4970.
- Denef V.J., M.A. Patrauchan, C. Florizone, J. Park, T.V. Tsoi, W. Verstraete, J.M. Tiedje, L.D. Eltis. 2005. Growth substrate and phase specific expression of biphenyl, benzoate and C1 metabolic pathways in Burkholderia xenovorans LB400. J. Bacteriol. 187:7996-8005.
- Denef, V.J., J.A. Klappenbach, M.A. Patrauchan, C. Florizone, J.L.M. Rodrigues, T.V. Tsoi, W. Verstraete, L.D. Eltis and J.M. Tiedje. 2006. Genetic and genomic insights into the role of benzoate-catabolic pathway redundancy in Burkholderia xenovorans LB400. Appl. Environ. Microbiol. 72:585-595.
Identification of active PCB-degrading bacteria in soil using stable isotope probing
Understanding the identity of microorganisms and functional genes capable of biodegrading pollutants like polychlorinated biphenyls (PCBs) is important to the development of successful bioremediation technologies. Culture-based methods are severely limited in their ability to detect many organisms in the environment, and are incapable of revealing which organisms are truly active in degradative processes. In order to identify organisms and genes active in PCB degradation, we are applying and expanding stable isotope probing (SIP) methods.
Stable isotope probing (SIP) is an elegant technique enabling the direct identification of organisms that degrade a specific substrate within the context of a complex microbial community (Manefield et al., 2002, Radajewski et al., 2003). In SIP experiments, a 13C-labelled compound (i.e. biphenyl) is provided to a microbial community and then organisms that utilize the substrate incorporate 13C into their nucleic acids. Nucleic acids are extracted and subjected to density gradient centrifugation to separate labeled from unlabeled material. Community profiling and sequence analyses of 16S rRNA genes in the 13C-labeled nucleic acids then reveal the phylogenetic identity of the active organisms. Through a cooperation with the laboratory of Dr. Mark Bailey at the NERC Center for Ecology and Hydrology (CEH), Oxford, U. K. [http://www.ceh.ac.uk/sites/oxford.html] we have adapted and optimized both DNA and RNA-based SIP to investigate biphenyl-degrading populations in soil. Currently, we are integrating advanced metagenomic and functional gene microarray analyses with SIP to investigate not only the identity but also the genetic capabilities of active organisms in soils and the rhizosphere of plants from a variety of geographic locations.
- Manefield, M., A. S. Whiteley, R. I. Griffiths, and M. J. Bailey. 2002. RNA stable isotope probing, a novel means of linking microbial community function to phylogeny. Applied and Environmental Microbiology 68:5367-5373.
- Radajewski, S., I. McDonald and J. C. Murrell. 2003. Stable-isotope probing of nucleic acids: a window to the function of uncultured microorganisms. Current Opinion in Biotechnology 14:296-302.
PCB-degrading bacteria from diverse PCB contaminated sites (Woo Jun Sul, Joonhong Park and Mary Beth Leigh)
Organisms active in PCB degradation have been identified in several different contaminated soils and sediments from locations in Michigan, New Jersey and the Czech Republic. Both RNA- and DNA-based SIP methods have been applied to identify organisms that derive carbon from the PCB analogue, biphenyl, in soil and sediment microcosms. Analyses of 13C-nucleic acids are being conducted at a series of incubation times to explore how carbon flows through the soil microbial community as a result of biphenyl/PCB degradation.
PCB-degrading bacteria important to rhizosphere bioremediation in the root zone of pine
Organisms potentially important to rhizosphere bioremediation of PCBs are being identified in soil from the root zone of Austrian pine (Pinus nigra), which was previously found to be associated with increased populations of PCB-degraders at a contaminated site in the Czech Republic (1). The identity of active degraders identified with SIP are being compared with those detected using culture-based methods to provide insight into culture biases. In addition to phylogenetically identifying active biphenyl-degraders, the functional genes possessed by the active populations are being explored using a comprehensive functional gene microarray (FGA) developed by collaborator Jizhong Zhou at Oak Ridge National Laboratory. The FGA includes over 23,000 probes targeting functional genes important to environmental microbial processes including nearly all known to be involved in the degradation of biphenyl, PCBs, mono- and polyaromatic compounds and many associated with plant aromatic compound degradation. The results will provide new insights into the organisms and degradative pathways associated with plants that may be important to the rhizoremediation of aromatic pollutants.
Leigh, Mary Beth, Petra Prouzova, Martina Mackova, Thomas Macek, David P. Nagle and John S. Fletcher. 2006. Influence of mature trees on populations of polychlorinated biphenyl-degrading bacteria in a PCB-contaminated site. Applied and Environmental Microbiology 72:2331-2342.
Influence of global climate changes on microbial rhizosphere populations of grass land plants (Stephan Gantner)
Biphenyl and benzoate degrading bacteria in the rhizosphere of plants are being identified using stable isotope probing in the context of a field experiment examining the influence of climate change on specific bacterial functional groups. [see project summary in Rhizosphere]
Metagenomics of PCB-degrading bacteria in soil (Woo Jun Sul)
The microbial world is diverse owing its 3.7 billion years of evolution, which provides for both the opportunity of undiscovered metabolic capacity, including that for pollutant degradation, and the challenge of detecting and recovering this activity. Its well known that more than 99% of the microbial world has not been cultured and hence remains undiscovered. Our purpose of this project is to explore and recover genes for two key biodegradative steps in the detoxication of chlorinated polyaromatic compounds from the DNA of this uncultured microbial diversity, and then to use the molecular markers from this study to aid in site assessment and quantitative predictions of biodegradation at contaminated sites. Stable isotope probing (SIP) is an elegant technique for separating and concentrating nucleic acids from organisms actively engaged in the biodegradation of a substrate. SIP is achieved by providing microbial communities with a specific 13C-labeled compound, resulting in the incorporation of 13C into the nucleic acids of microorganisms that grow on the labeled compound. 13C-labeled DNA is then separated from the unlabeled background DNA by density gradient centrifugation, producing concentrated genomic DNA derived from an entire functional group of organisms active in biodegradation. Metagenomics is the culture-independent genomic analysis or the functional and sequence-based analysis of the collective microbial genomes contained in an environmental sample. When combined with metagenomics, SIP would provide sequence data of DNA of greater relevance and would not be limited by previous sequence knowledge.
(Meta) Genomic And Functional Analyses Of Microbial Populations Involved In Polychlorinated Biphenyl Degradation In Contaminated Environmental Matrices
Polychlorinated biphenyls (PCBs) are among most toxic and persistent man-made chemical pollutants that threaten both the natural ecosystem and human health. Various PCB-degrading microorganisms have been isolated and biodegradative pathways responsible for partial PCB biological oxidation have been extensively studied, However, there is currently limited knowledge concerning the active microbial community involved in the biotransformation processes of PCBs in the environment. This lead us to seek more information about the active functional genes involved in PCB degradation and the metabolic networks essential to degradation in contaminated environmental matrices.
Bioremediation of Metals and Nitrate
Stable isotope probing for biostimulated metal, uranium and nitrate reducing bacteria [Mary Beth Leigh and Erick Cardenas, with Terence Marsh (http://www.mmg.msu.edu/114.html) and Nathaniel Ostrom (http://www.msu.edu/user/ostrom/ostrom%20lab/index.htm)]
Stable isotope probing (SIP) techniques are being applied to understand the ecology and physiology of microorganisms and communities important for the reduction of U, Cr, Tc and nitrate at the U. S. Department of Energy's NABIR-Field Research Center (http://www.esd.ornl.gov/nabirfrc/) at Oak Ridge National Laboratory (ORNL) and to identify those that respond under field implementation of bioremediation. An experimental groundwater treatment system (http://public.ornl.gov/nabirfrc/FactSheet-Criddle.pdf) developed by Craig Criddle (Stanford University) (http://www.stanford.edu/group/evpilot/) and Phil Jardine (ORNL) (http://www.esd.ornl.gov/people/jardine/) has been implemented, which involves aboveground conditioning of contaminated groundwater, including the removal of nitrate and pH adjustment, followed by reinjection into the aquifer and subsequent biostimulation of metal and uranium reduction. The aboveground treatment system includes a fluidized-bed denitrifying reactor that effectively removes nitrate when biostimulated with ethanol. In the Tiedje Lab, stable-isotope probing (SIP) methods are being applied to identify denitrifying microorganisms important to reactor function that incorporate the biostimulatory substrate. SIP is currently being combined with functional gene analyses to detect a variety of denitrification genes in biostimulated organisms using a functional gene array in cooperation with Jizhong Zhou (ORNL) (http://www.esd.ornl.gov/people/zhou/zhou.html) and with targeted PCR primers.
To investigate and optimize the belowground treatment process, SIP experiments will also be conducted on FRC sediment samples to identify bacteria important to metal and radionuclide reduction that respond to biostimulation by the provision of various electron donors.
Exploring the Genome and Proteome of Desulfitobacterium hafniense DCB-2 for its Protein Complexes Involved in Metal Reduction and Dechlorination [Sang-Hoon Kim and Christina Harzman, with Terence L. Marsh, David DeWitt, Joan Broderick (Montana State University) and John Davis (Columbus State University, Columbus, GA)]
Desulfitobacteria are Gram-positive, spore-forming, nitrogen fixing anaerobes with the ability to reduce many electron acceptors including Fe(III), U(VI), Cr(VI), As(V), Mn(IV), Se(VI), NO3-, CO2, sulfite, fumarate and humates. They can grow by halorespiration on chlorinated aromatic and aliphatic pollutants and use a variety of electron donors. Using Desulfitobacterium hafniense DCB-2 as a model microbe, the main goals of our project are: (i) to gain insight into the genetic and metabolic pathways involved in dissimilatory metal reduction, reductive dechlorination and N2 fixation, (ii) to discern the commonalities among these electron-accepting processes, (iii) to identify multi-protein complexes catalyzing these functions and (iv) to elucidate the coordination in expression of these pathways and processes.
We use high-throughput technologies to reveal the transcriptome and proteome of D. hafniense DCB-2 grown under different reducing conditions to identify and classify the genes and proteins involved in the targeted pathways. We also use selective expression and mutagenesis of the genes of interest to help understand their functions, to aid the structural and functional study of the expressed proteins and to study the tertiary structures of the multi-protein complexes involved in the metabolic pathways.