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7 MONITORING THE FATE AND ACTIVITY OF DIARYL ETHER-DEGRADING BACTERIA IN SOIL R.U. Halden, G.W. Mundfrom, E.G. Peters, and D.F. Dwyer Department of Civil Engineering University of Minnesota Minneapolis, Minnesota 55455-0220 INTRODUCTION Bioaugmentation of natural microbial communities with microorganisms that are adapted or engineered to degrade target pollutants is one approach to in situ bioremediation. In order to assess the efficacy of this approach, one must be able to monitor the environmental fate of applied bioagents. Molecular-genetic methods that couple the polymerase chain reaction (PCR) to DNA hybridization allow one to unambiguously identify introduced microorganisms in complex environmental samples via detection of specific target-DNA sequences.1'3 These methods are also suitable for determining whether genes encoding degradative enzymes are retained by bioagents and/or transferred to indigenous microorganisms. Pseudomonas pseudoalcaligenes strain POB310 (POB310) uses 3- and 4-phenoxybenzoic acid (3-POB and 4-POB) as sole sources of carbon and energy.4 This ability suggests that POB310 may potentially be used as a bioremediation agent for the cleanup of sites containing diaryl ether compounds. The degradative pathway for 3-POB and 4-POB in POB310 relies on an enzyme, 4- POB-dioxygenase, that is encoded on a self-transmissible, unstable plasmid (pPOB). Previously, genes encoding the strain's 4-POB-dioxygenase as well as its 16S rRNA were cloned and the corresponding nucleic acid sequences derived.5 In this study, we have used the sequence data to construct PCR primers and oligonucleotide probes that facilitate the detection and identification of POB310 and the genes encoding 4-POB-dioxygenase in environmental samples. During our research, it became obvious that POB310 rapidly loses its ability to degrade phe- noxybenzoate compounds in non-selective growth conditions.5 Thus, we created a novel phe- noxybenzoate-degrading bacterium, P. sp. strain B13 (pD30.9), by transfer of the genes encoding 4-POB-dioxygenase into P. sp. strain B13. We hypothesized that the new strain might retain and express the genes more effectively than POB310 during environmental applications, since strain B13 has previously been observed to survive and degrade target pollutants in river, lake, and aquifer sediments as well as in activated sludge.6"9 This hypothesis was tested in soil microcosm experiments involving both POB310 and B13 (pD30.9), the genetically engineered microorganism (GEM). In these experiments, B13 (pD30.9) was able to persist at higher densities and degrade the target compound, 3-POB, more rapidly than POB310. MATERIALS AND METHODS Development and Testing of PCR Primers and Probes Sequence Analyses of the 16S rDNA ofPOBHO A partial sequence of the 16S rDNA of POB310 was provided courtesy of Dr. Edward Moore. GBF, Braunschweig, Germany. Analysis of the DNA sequence was performed with computer programs available through the GCG-Wisconsin Package (Genetics Computer Group, Madison, WI) and the Ribosomal Database Project server.10 PCR primers and internal probes were synthesized by National Biosciences, Inc., Minneapolis, MN. 50th Purdue Industrial Waste Conference Proceedings. 199.S, Ann Arbor Press, inc., Chelsea. Michigan 48118. Printed in U.S.A. 57
Object Description
Purdue Identification Number | ETRIWC199507 |
Title | Monitoring the fate and activity of diaryl ether-degrading bacteria in soil |
Author |
Halden, R. U. Mundfrom, G. W. Peters, E. G. Dwyer, D. F. |
Date of Original | 1995 |
Conference Title | Proceedings of the 50th Industrial Waste Conference |
Conference Front Matter (copy and paste) | http://e-archives.lib.purdue.edu/u?/engext,45474 |
Extent of Original | p. 57-64 |
Collection Title | Engineering Technical Reports Collection, Purdue University |
Repository | Purdue University Libraries |
Rights Statement | Digital object copyright Purdue University. All rights reserved. |
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Description
Title | page 57 |
Collection Title | Engineering Technical Reports Collection, Purdue University |
Repository | Purdue University Libraries |
Rights Statement | Digital copyright Purdue University. All rights reserved. |
Language | eng |
Type (DCMI) | text |
Format | JP2 |
Capture Device | Fujitsu fi-5650C |
Capture Details | ScandAll 21 |
Transcript | 7 MONITORING THE FATE AND ACTIVITY OF DIARYL ETHER-DEGRADING BACTERIA IN SOIL R.U. Halden, G.W. Mundfrom, E.G. Peters, and D.F. Dwyer Department of Civil Engineering University of Minnesota Minneapolis, Minnesota 55455-0220 INTRODUCTION Bioaugmentation of natural microbial communities with microorganisms that are adapted or engineered to degrade target pollutants is one approach to in situ bioremediation. In order to assess the efficacy of this approach, one must be able to monitor the environmental fate of applied bioagents. Molecular-genetic methods that couple the polymerase chain reaction (PCR) to DNA hybridization allow one to unambiguously identify introduced microorganisms in complex environmental samples via detection of specific target-DNA sequences.1'3 These methods are also suitable for determining whether genes encoding degradative enzymes are retained by bioagents and/or transferred to indigenous microorganisms. Pseudomonas pseudoalcaligenes strain POB310 (POB310) uses 3- and 4-phenoxybenzoic acid (3-POB and 4-POB) as sole sources of carbon and energy.4 This ability suggests that POB310 may potentially be used as a bioremediation agent for the cleanup of sites containing diaryl ether compounds. The degradative pathway for 3-POB and 4-POB in POB310 relies on an enzyme, 4- POB-dioxygenase, that is encoded on a self-transmissible, unstable plasmid (pPOB). Previously, genes encoding the strain's 4-POB-dioxygenase as well as its 16S rRNA were cloned and the corresponding nucleic acid sequences derived.5 In this study, we have used the sequence data to construct PCR primers and oligonucleotide probes that facilitate the detection and identification of POB310 and the genes encoding 4-POB-dioxygenase in environmental samples. During our research, it became obvious that POB310 rapidly loses its ability to degrade phe- noxybenzoate compounds in non-selective growth conditions.5 Thus, we created a novel phe- noxybenzoate-degrading bacterium, P. sp. strain B13 (pD30.9), by transfer of the genes encoding 4-POB-dioxygenase into P. sp. strain B13. We hypothesized that the new strain might retain and express the genes more effectively than POB310 during environmental applications, since strain B13 has previously been observed to survive and degrade target pollutants in river, lake, and aquifer sediments as well as in activated sludge.6"9 This hypothesis was tested in soil microcosm experiments involving both POB310 and B13 (pD30.9), the genetically engineered microorganism (GEM). In these experiments, B13 (pD30.9) was able to persist at higher densities and degrade the target compound, 3-POB, more rapidly than POB310. MATERIALS AND METHODS Development and Testing of PCR Primers and Probes Sequence Analyses of the 16S rDNA ofPOBHO A partial sequence of the 16S rDNA of POB310 was provided courtesy of Dr. Edward Moore. GBF, Braunschweig, Germany. Analysis of the DNA sequence was performed with computer programs available through the GCG-Wisconsin Package (Genetics Computer Group, Madison, WI) and the Ribosomal Database Project server.10 PCR primers and internal probes were synthesized by National Biosciences, Inc., Minneapolis, MN. 50th Purdue Industrial Waste Conference Proceedings. 199.S, Ann Arbor Press, inc., Chelsea. Michigan 48118. Printed in U.S.A. 57 |
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