Laurie A. Achenbach
Laurie A. Achenbach
Research Interests
Genetic Regulation of Perchlorate Reduction
In the last several years, perchlorate contamination of the environment has been recognized as an important health risk and perchlorate was recently added to the U.S. EPA's Office of Water Contaminant Candidate List. Microbial reduction of perchlorate has been identified as the most readily applicable form of remediation of this pollutant. In order to track (per)chlorate-reducing bacteria (DPRB) in the environment, we have designed a number of 16S rDNA primer sets specific to the most predominant groups of perchlorate reducers in the environment. However, due to the extreme phylogenetic diversity of perchlorate-reducing bacteria, 16S ribosomal RNA primer sets can only be designed to detect a few specific genera of (per)chlorate-reducing bacteria in the environment (e.g. Dechloromonas, Dechloromarinus, Dechlorospirillum, and Azospira species). A more inclusive approach for the detection of bacteria capable of (per)chlorate reduction is to develop a metabolic gene probe. We recently sequenced and genetically characterized two novel genes involved in (per)chlorate reduction, the cld gene encoding chlorite dismutase and the pcrABCD genes encoding perchlorate reductase.
Chlorite dismutase is now known to be a central enzyme involved in microbial perchlorate reduction. Our current research describes the identification and characterization of the cld gene encoding chlorite dismutase in a perchlorate-reducing bacterium, Dechloromonas agitata. As such, this is the first description of a functional gene associated with microbial perchlorate respiration, a ubiquitous metabolism in the environment. Analyses of the transcriptional regulation of the D. agitata cld gene demonstrated that transcription of the chlorite dismutase gene significantly increased when the organism was grown on perchlorate as opposed to aerobic growth, supporting previous observations on the environmental factors that influence microbial perchlorate reduction. The chlorite dismutase gene probe developed in that study hybridized to all DPRB tested regardless of their phylogenetic affiliation.
To detect DPRB in the environment, we developed and optimized two degenerate PCR primer sets targeting the chlorite dismutase (cld) gene. A nested PCR approach was used in conjunction with these primer sets to increase the sensitivity of the molecular detection method. Screening of environmental samples via amplification from genomic DNA and PCR product sequencing indicated that all products amplified by this method were cld gene sequences. These sequences were obtained from pristine (uncontaminated) sites as well as contaminated sites from which DPRB were isolated. More than one cld phylotype was also identified from some samples, indicating the presence of more than one DPRB strain at those sites. Using the nested primer sets in a reverse transcription-PCR (RT-PCR) approach, we have also demonstrated the functional activity of the cld gene in situ, enabling us to track the metabolic activity of DPRB throughout a bioremediative process. The use of these primer sets represents a direct and sensitive molecular method for the qualitative detection of (per)chlorate-reducing bacteria in the environment, thus offering another tool for monitoring natural attenuation.
Sequences of cld genes isolated in the course of this project were also generated from various DPRB and provided the first opportunity for a phylogenetic treatment of this metabolic gene. Comparisons of the cld and 16S rDNA gene trees indicated that the cld gene does not track 16S rDNA phylogeny, further implicating the possible role of horizontal transfer in the evolution of (per)chlorate respiration.
Another key enzyme involved in the metabolism of perchlorate is (per)chlorate reductase (also known as perchlorate reductase), an enzyme capable of reducing either chlorate or perchlorate. We have identified and characterized the (per)chlorate reductase genes pcrABCD from Dechloromonas aromatica and D. agitata. Sequence analysis of the pcrAB gene products exhibited similarity to alpha and beta subunits of archaeal nitrate reductases and bacterial selenate reductase, dimethyl sulphide dehydrogenase, ethylbenzene dehydrogenase, and chlorate reductase, all members of the dimethylsulfoxide reductase (DMSO) family. The pcrC gene product was similar to a c-type cytochrome, while the pcrD gene product exhibited similarity to molybdenum chaperone proteins of the aforementioned DMSO family members. Expression analysis of the pcrA gene from Dechloromonas agitata indicated transcription only under anaerobic (per)chlorate reducing conditions. The presence of oxygen completely inhibited pcrA expression regardless of the presence of perchlorate, chlorate, or nitrate. Deletion of the pcrA gene in Dechloromonas aromatica abolished growth in both perchlorate and chlorate but not nitrate, indicating a functional role of the pcrABCD genes in perchlorate reduction separate from nitrate reduction.
Phylogenetic analysis of the PcrA and other alpha subunits of the DMSO family indicated that perchlorate reductase forms a monophyletic group separate from chlorate reductase of Ideonella dechloratans. The separation of perchlorate reductase as distinct from chlorate reductase was further supported by DNA hybridization analysis of (per)chlorate- and chlorate-reducing strains using the pcrA gene as a probe. We have recently developed primer sets that specifically amplify the pcrA gene encoding the alpha subunit of perchlorate reductase from environmental samples using a nested PCR approach and are using these primer sets in RT-PCR analyses to assess functional activity of perchlorate reductase genes in situ.
Our analysis of Dechloromonas agitata, D. aromatica, and Azospira suillum indicates that not only are (per)chlorate- and chlorate-reducing bacteria phylogenetically diverse, but that they are diverse at the genetic level as well. One of the most interesting observations from the comparative analysis is the finding that the chlorite dismutase and (per)chlorate reductase genes are organized and oriented differently in different ClRB, possibly indicative of horizontal transfer of one or more of the genes involved in (per)chlorate reduction. A history of horizontal transfer would also explain the fact that many ClRB are phylogenetically very closely related to organisms not capable of (per)chlorate reduction with 16S rDNA sequence similarites of up to 99.8%.
Bacterial Diversity in Permanently-Frozen Antarctic Lakes
Lake Fryxell is a meromictic lake located 18 meters above sea level at the entrance of the Taylor Valley adjacent to McMurdo Sound, Antarctica. The lake is a closed basin with a maximum depth of 19 meters and has undergone several dry-down periods in the past resulting in very dense bottom waters overlaid with low solute water introduced from glacial melt water streams. These processes have resulted in a gradient of solutes in the water column that is further stabilized by a perennial ice cover of 3 to 5 meters. The water column is oxygen-saturated in the upper depths and anoxic in deeper waters due to limited gas exchange with the atmosphere imposed by the perennial ice cover that also serves as a barrier to light penetration allowing only a small amount of light to reach the water column.
There have been extensive studies on the geochemistry of Lake Fryxell, but to date only a few studies of the microbial communities inhabiting the lake have been conducted. These studies predominately focused on the presence of ammonia-oxidizing bacteria belonging to the gamma and beta subclasses of the Proteobacteria within the water column and the contributions of cyanobacterial populations inhabiting the lake's ice cover to the primary production of the Dry Valley ecosystem. Studies involving the bacterial diversity of microbial mat samples collected from moats that surround the lake during the austral summer season showed a large degree of microbial diversity including both gram -positive and gram-negative bacteria as well as Archaea.
Cultivation of bacteria from the environment is often limited and biased and, when successful, represents only a small portion of the bacterial diversity in complex microbial communities. The use of molecular techniques allows for the detection of bacteria in the environment in the absence of culturing, and the information gleaned from these molecular analyses can often aid in the subsequent isolation of organisms. The use of a PCR primer sets targeting 16S rRNA or metabolic genes allows the detection of specific bacteria in the environment. Coupling of PCR (polymerase chain reaction) and RT-PCR (reverse transcriptase PCR) with denaturing gradient gel electrophoresis (DGGE) to resolve unique metabolic gene sequences present in the environment allows one to assess both organismal diversity as well as metabolic activity. Given that the geochemical parameters present in Lake Fryxell are favorable for the growth of anoxygenic phototrophic bacteria, we investigated the presence of these organisms in the water column using a combination of PCR, RT-PCR, and denaturing gradient gel electrophoresis.
Photosynthetic Bacteria
Photosynthetic ability is widely distributed among microorganisms. Anoxygenic photosynthetic bacteria, or anoxyphototrophs, are prokaryotes capable of utilizing light as an energy source but, unlike cyanobacteria, do not evolve molecular oxygen. Anoxyphototrophs typically use sulfide or other reduced sulfur compounds as well as molecular hydrogen or a variety of small organic molecules as electron donors in photosynthesis. There are four known phylogenetic groups of anoxygenic phototrophs: the green sulfur bacteria, the green non-sulfur bacteria, the heliobacteria, and the purple bacteria. The presence of a year-round ice layer on Lake Fryxell creates stable gradients of oxygen, sulfide, and light throughout the water column suitable for the development of anoxygenic phototrophs. Anoxygenic phototrophs are often associated with highly stratified meromictic lakes, positioning themselves at depths where light and sulfide co-exist. Lake Fryxell has been shown to support photosynthetic activity with a deep chlorophyll maxima approximately two meters above the oxycline.
Our lab designed primer sets to target specific 16S rDNA sequences of photosynthetic bacteria including the green sulfur bacteria, the green non-sulfur bacteria, and the Heliobacteriaceae. Due to the phylogenetic diversity of purple sulfur and purple non-sulfur phototrophs, the 16S rDNA gene was not an appropriate target for phylogenetic rDNA primers. Thus, we designed a primer set that targets the pufM gene encoding the M subunit of the photosynthetic reaction center, universally distributed among purple phototrophic bacteria. The pufM primer set amplified DNA from not only purple sulfur and purple non-sulfur phototrophs, but from Chloroflexus species, which also produce a purple bacterial-like reaction center. Although the purple bacterial reaction center structurally resembles green plant photosystem II, the pufM primers did not amplify cyanobacterial DNA, further indicating their specificity for purple anoxyphototrophs. This combination of phylogenetic and photosynthesis-specific primers covers all groups of known anoxygenic phototrophs, and as such shows promise as a molecular tool for the rapid assessment of natural samples in ecological studies of these organisms
Water samples collected from Lake Fryxell were filtered and total RNA or genomic DNA was isolated. PCR amplification, DGGE and sequencing of the pufM gene from environmental gDNA yielded several distinct phylotypes of purple non-sulfur bacteria throughout the water column. pufM phylotypes showing similarity to aerobic anoxygenic phototrophs were also detected. Only one group of clones showed high pufM sequence similarity to the pufM gene of a cultured anoxygenic phototroph. The pufM clone sequences obtained were representative of the alpha and beta groups of the Proteobacteria. However, detailed analysis of the sequences in a phylogenetic framework could not be performed as the pufM gene is known to be subject to lateral transfer. The pufM phylotypes showed distinct patterns of distribution throughout the water column with respect to light, oxygen, and sulfide concentrations. In addition, RT-PCR of pufM mRNA indicated photosynthetic activity of anoxygenic phototrophs was greatest at 9 and 11 meter depths, decreased activity at 14 meters, and no detectable activity at 17 meters depth. The limited amount of light available to anoxyphototrophs within the water column would suggested that those phylotypes that are actively photosynthesizing likely represent low light-adapted phototrophs. Because the purple non-sulfur bacteria are also able to grow chemoheterotrophically in the presence of oxygen as well as by anaerobic respiration or fermentation, this metabolic adaptability provides these organisms alternate means for survival in the water column, thus potentially allowing them a means of survival during the extended darkness of the Antarctic winter.
Sulfur-Cycling Prokaryotes
Sulfate-reducers are not uncommon in permanently cold environments and several psychrophilic representatives of sulfate-reducing bacteria (SRB) have been isolated in recent years. Sulfate reduction appears to be a major process in oceanic sediments, an environment dominated by low temperatures, where SRB play an important role in the sulfur cycle as well as the remineralization of carbon. With members residing in the d subdivision of the Proteobacteria, gram-positive subdivision of Bacteria and the Archaeal domain, this group of organisms reduces sulfate to sulfide using a variety of electron donors including H2, fatty acids, and alcohols.
We are also investigating inorganic sulfur-cycling bacteria in these Dry Valley Antarctic lakes. Using group-specific metabolic gene probes, this research will identify and track sulfate-reducing prokaryotes through the water column of Lake Fryxell. To investigate the population of sulfate-reducing bacteria in Lake Fryxell, both 16S rDNA and metabolic primer sets targeting the dsrA gene for the dissimilatory sulfite reductase alpha subunit were employed to analyze environmental DNA obtained from the water column and sediments of Lake Fryxell. In addition, enrichment cultures of sulfate-reducing bacteria established at 4°C from Lake Fryxell water were also screened using the dsrA primer set. The sequence information obtained showed that a diverse group of sulfate-reducing prokaryotes of the domain Bacteria inhabit Lake Fryxell. With one exception, the enrichment culture sequences were not represented within the environmental sequences. Sequence data were compared with the geochemical profile of Lake Fryxell to identify possible connections between the diversity of sulfate-reducing bacteria and limnological conditions. Several clone groups were highly localized with respect to lake depth and, therefore, experienced specific physiochemical conditions. However, all sulfate-reducing bacteria inhabiting Lake Fryxell must function under the constantly cold conditions characteristic of this extreme environment.
Archaea
We have also detected Archaea in permanently frozen Lake Fryxell, Antarctica, using 16S rRNA diversity analyses. Two clusters of methanogens were detected in the sediments and another cluster of possibly methanotrophic Euryarchaeota in the anoxic water column just above the sediments. One crenarchaeote was detected from water just below the oxycline. Although the presence of Archaea in constantly cold marine waters is well known, our discovery of a diverse group of Archaea in Lake Fryxell is the first such report from the water column and sediments of a permanently frozen continental Antarctic lake. The Archaea present in Lake Fryxell are likely involved in the major biogeochemical cycles that occur there. Continued study of the Archaea in Lake Fryxell, the only lake in the Taylor Valley that supports extensive methanogenesis and sulfidogenesis, may reveal new connections between the sulfur and methane cycles in extremely cold environments.
Recent Publications
Thrash, J.C., J.I. Van Trump, K.A. Weber, E. Miller, L.A. Achenbach, and J.D. Coates. 2007. Electrochemical stimulation of microbial perchlorate reduction. Environ. Sci. Technol. 41:1740-1746.
Achenbach, L.A., K.S. Bender, Y. Sun, and J.D. Coates. 2006. The biochemistry and genetics of perchlorate reduction. In: B. Gu and J.D. Coates (eds.) Perchlorate: Environmental Occurrence, Interactions, and Treatment. Springer. pp. 297-310.
Coates, J.D., and L.A. Achenbach. 2006. The microbiology of perchlorate reduction and its bioremediative application. In: B. Gu and J.D. Coates (eds.) Perchlorate: Environmental Occurrence, Interactions, and Treatment. Springer. pp. 279-295.
Weber, K.A., J. Pollock, K.A. Cole, S.M. O'Connor, L.A. Achenbach, and J.D. Coates. 2006. Anaerobic nitrate-dependent iron(II) bio-oxidation by a novel lithoautotrophic betaproteobacterium, Strain 2002. Appl. Environ. Microbiol. 72:686-694.
Karr, E.A., J.M. Ng, S.M. Belchik, W.M. Sattley, M.T. Madigan, and L.A. Achenbach. 2006. Biodiversity of methanogenic and other Archaea in the permanently frozen Lake Fryxell, Antarctica. Appl. Environ. Microbiol. 72:1663-1666.
Weber, K.A., L.A. Achenbach, and J.D. Coates. 2006. Microbes pumping iron: anaerobic microbial iron oxidation and reduction. Nature Rev. Microbiol. 4:752-764.
Bender, K.S., C. Shang, R. Chakraborty, S.M. Belchik, J.D. Coates, and L.A. Achenbach. 2005. Identification, characterization, and classification of genes encoding perchlorate reductase. J. Bacteriol. 187:5090-5096.
Karr, E.A., W.M. Sattley, M.R. Rice, D.O. Jung, M.T. Madigan, and L.A. Achenbach. 2005. Diversity and distribution of sulfate-reducing bacteria in the permanently frozen Lake Fryxell, McMurdo Dry Valleys, Antarctica. Appl. Environ. Micro. 71:6353-6359.
Coates, J.D., K.A. Cole, U. Michaelidou, J. Patrick, M.J. McInerney, and L.A. Achenbach. 2005. Biological odor control in hog waste through stimulated microbial Fe(III) reduction. Appl. Environ. Micro. 71:4728-4735.
Madigan, M.T., D.O. Jung, E.A. Karr, W.M. Sattley, L.A. Achenbach, and M.T.J. van der Meer. 2005. Diversity of anoxygenic phototrophs in contrasting extreme environments. In: Geothermal Biology and Geochemistry in Yellowstone National Park. W. Inskeep (ed). Thermal Biology Institute, Montana State University.
Zhang, H., B.E. Logan, J. M. Regan, L. A. Achenbach, and M.A. Bruns. 2005. Molecular assessment of inoculated and indigenous bacteria in biofilms from a pilot-scale perchlorate-reducing bioreactor. Micro. Ecol. 49:388-398.
Coates, J.D., and L.A. Achenbach. 2004. Microbial perchlorate reduction: rocket-fuelled metabolism. Nature Rev. Microbiol. 2:569-580.
Bender, K.S., M.R. Rice, W.H. Fugate, J.D. Coates, and L.A. Achenbach. 2004. Metabolic primers for the detection of (per)chlorate-reducing bacteria in the environment and phylogenetic analysis of cld gene sequences. Appl. Env. Micro. 70:5651-5658.
Jung, D.O., L.A. Achenbach, E.A. Karr, S. Takaichi, and M.T. Madigan. 2004. A gas vesiculate planktonic strain of the purple nonsulfur bacterium Rhodoferax antarcticus isolated from Lake Fryxell, Dry Valleys, Antarctica. Arch. Microbiol. 182:236-243.
Karr, E.A., W.M. Sattley, D.O. Jung, M.T. Madigan, and L.A. Achenbach. 2003. Remarkable diversity of phototrophic purple bacteria from a permanently frozen Antarctic lake. Appl. Environ. Microbiol. 69:4910-4914.
Cummings, D.E., O.L. Snoeyenbos-West, D.T. Newby, A.M. Niggemyer, D.R. Lovley, L.A. Achenbach, and R.F. Rosenzweig. 2003. Diversity of Geobacteraceae species inhabiting metal-polluted freshwater lake sediments ascertained by 16S rDNA analyses. Micro. Ecol. 46:257-269.
Bender, K.S., S.M. O'Connor, R. Chakraborty, J.D. Coates, and L.A. Achenbach. 2002. Sequencing and transcriptional analysis of the chlorite dismutase gene of Dechloromonas agitata and its use as a metabolic probe. Appl. Environ. Microbiol. 68:4820-4826.
Chaudhuri, S.K., S.M. O'Connor, R. Gustavson, L.A. Achenbach, and J.D. Coates. 2002. Environmental factors that control microbial perchlorate reduction. Appl. Environ. Microbiol. 68:4425-4430.
Lack, J.G., S.K. Chaudhuri, R. Chakrabortey, L.A. Achenbach, and J.D. Coates. 2002. Anaerobic biooxidation of Fe(II) by Dechlorosoma suillum. Micro. Ecol. 43:424-431.
Coates, J.D., K.A. Cole, R. Chakraborty, S.M. O'Connor, and L.A. Achenbach. 2002. Diversity and ubiquity of bacteria capable of utilizing humic substances as electron donors for anaerobic respiration. Appl. Environ. Microbiol. 68:2445-2452.
Coates, J.D., and L.A. Achenbach. 2001. The biogeochemistry of aquifer systems. In: Manual of Environmental Microbiology, 2nd edition. C.J. Hurst, R.L. Crawford, G.R. Knudsen, M.J. McInerney, and L.D. Stetzenbach (eds.). ASM Press, Washington DC. pp. 719-727.
Achenbach, L.A., J. Carey, and M.T. Madigan. 2001. Phylogenetic and photosynthetic primers for the detection of anoxygenic phototrophs in natural environments. Appl. Environ. Microbiol. 67:2922-2926.
Coates, J.D., R. Chakraborty, J.G. Lack, S.M. O'Connor, K.A. Cole, K.S. Bender, and L.A. Achenbach. 2001. Anaerobic benzene oxidation coupled to nitrate reduction in pure culture by two strains of Dechloromonas. Nature 411:1039-1043.
Achenbach, L.A., U. Michaelidou, R.A. Bruce, J. Fryman, and J.D. Coates. 2001. Dechloromonas agitata gen. nov., sp. nov. and Dechlorosoma suillum gen. nov., sp. nov., two novel environmentally dominant (per)chlorate-reducing bacteria and their phylogenetic position. Int. J. Syst. Evol. Microbiol. 51:527-533.
Coates, J.D., V. Bhupathiraju, L.A. Achenbach, M.J. McInerney, and D.R. Lovley. 2001. Geobacter hydrogenophilus, Geobacter chapelleii, and Geobacter grbicium, three new, strictly anaerobic, dissimilatory Fe(III)-reducers. Int. J. Syst. Evol. Microbiol. 51:581-588.
Department of Microbiology e-mail: microbiology@micro.siu.edu
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SIUC / Microbiology / Laurie Achenbach / 01-May-06 / laa