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.
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