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Dissertation
Presence of the pcpB gene sequences in indigenous bacteria that do not degrade pentachlorophenol: analysis and implications
Doctor of Philosophy (Ph.D.), Drexel University
1998
DOI:
https://doi.org/10.17918/00010311
Abstract
Detection of indigenous degradation capability in bacteria from a pentachlorophenol (PCP) contaminated site was sought using a gene probe based on the pcpB gene which encodes for PCP-4-monooxygenase of Sphingomonas chlorophenolica. The premise of this research was to assess the presence of the gene and its correlation to PCP degradative capability in soil bacteria from the site. Bacteria isolated from the site showed the presence of microbial diversity (based on colony morphology) even in heavily contaminated samples. Strains were selected that could grow in the presence of 50 [mu]g/ml PCP and were hybridized to the pcpB gene probe. Two isolates, B2 and B4, contained sequences highly similar (99% identity) to the pcpB gene. These two strains were not able to degrade PCP. An attempt to induce PCP degradation in B2 and B4 by providing a soil matrix support (1% w/v) failed. PCR analysis of the two other genes, pcpD (PCP-4-monooxygenase reductase gene) and pcpR (LTTR type transcriptional activator gene) thought to associate with pcpB during degradation in S. chlorophenolica, was conducted. Neither of the two strains contained the pcpD and pcpR genes. Interestingly, the pcpD primers were able to amplify a gene fragment at high annealing temperature (63[degrees]C) from one of the isolates, B2. However, the PCR product sequence showed very little homology to pcpD (or any other gene). Thus, biodegradation inferences based on PCR analysis, especially for environmental samples, can be misleading. Identification based on fatty acid methyl ester profile (SherlockTM), substrate utilization (BIOLOGTM) and 16S rDNA sequencing showed that B2 and B4 were different from each other and from S. chlorophenolica and actually belonged to separate sub-divisions of the purple bacteria. The presence of pcpB sequences in these non-degraders indicates that growth and hybridization data alone were insufficient to predict degradation capability, that pcpB might have an alternative function in soil bacteria and that there was no cause-and-effect relationship between the presence of PCP and pcpB. This study shows the limitation of gene probe hybridization and PCR in feasibility studies for detection of indigenous degradation capability.
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Details
- Title
- Presence of the pcpB gene sequences in indigenous bacteria that do not degrade pentachlorophenol
- Creators
- Vandana Mahesh Saboo
- Contributors
- Michael A. Gealt (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- x, 139 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- School of Environmental Science, Engineering, and Policy (1997-2002); Drexel University
- Other Identifier
- 991021888935904721