Microbial and molecular basis of polycyclic aromatic hydrocarbon biodegradation in contaminated soils

Author

Yeonghee Ahn

Date of Award

12-1996

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Gary S. Sayler

Committee Members

Jayant Joshi, Bert Lampson, Dwayne Savage, David White

Abstract

Studies were undertaken to investigate the microbial and molecular basis of polycyclic aromatic hydrocarbon (PAH) biodegradation in contaminated soils. A culture collection of bacteria isolated from PAH-contaminated soils was characterized genotypically and phenotypically. The collection consisted of 141 putative PAH-degraders originally isolated using the PAH spray plate and the naphthalene vapor plate methods. The characterizations were designed to analyze genotypes and phenotypes of PAH-degraders, to screen HMW (high molecular weight) PAH degraders as sources for understanding of HMW PAH degradation, and to screen nonNAH7-hybridizing PAH- degraders as sources for the future development of new gene probes. The methods used to achieve the purpose were as follows: PAH spray plate method, ¹⁴C-PAH mineralization assay, indigo test, and DNA hybridization. NAH7-derived gene probes were used in the hybridization. The results of the analyses showed that number of PAH-degraders with NAH7-like genotype was dominant in the contaminated soils and that the nahA gene probe could be used to determine the microbial potential to degrade not only naphthalene but also 3- or 4-ring PAHs. The results support the continued use of the NAH7-derived gene probes for contaminated soils to monitor genetic potential of in situ microorganisms to degrade PAHs. However, some PAH-degraders did not hybridize with NAH7-derived probes, showing the limitation of NAH7-derived gene probes. This suggested that considering the numerous unculturable microorganisms in nature, NAH7-derived gene probes might underestimate in situ microbial potential to catabolize PAHs. Therefore it is necessary to develop new gene probes for PAH-degraders to better understand in situ microbial potential to catabolize PAHs.

Strain ASAN3 was chosen for further study by virtue of its non-hybridization with NAH7-derived gene probes and ability to degrade a broad range of PAHs including benzo[a]pyrene (BaP). A8AN3 was determined to be most similar to Sphingomonas paucimobilis based on phenotypic and chemotaxonomic traits. The 16S rDNA of A8AN3 was partially sequenced to determine the phylogenetic relationship of the strain with bacteria currently in the Ribosomal Database Project database. Comparative sequence analysis of the 16S rDNA suggested that A8AN3 was closest to S. adhaesiva in the database.

To investigate PAH-degradative genes in A8AN3 at the molecular level, random Tn5-mutagenesis was performed and gene libraries were constructed. When Tn5 mutagenesis was performed, a mutant strain of A8AN3 was obtained which lost the ability to convert indole to indigo (Ind^-). Furthermore, the mutant could no longer mineralize BaP. Southern blot analyses suggested that Tn5 was inserted in the chromosome of the mutant. Based on the phenotype of the mutant and the result of Southern blot analysis, Tn5 is likely inserted in the initial dioxygenase genes or upstream of the genes in an operon. As a result of constructing the gene libraries of A8AN3 and the Ind mutant, two different genes (cdo1 and cdo2) of extradiol dioxygenase and short flanking-regions of Tn5 were isolated from the libraries. The flanking regions can be used for future study to isolate genes encoding initial dioxygenase which attacks BaP. Southern blot analyses suggested that cdo1 gene was isolated from chromosomal DNA and cdo2 gene was from plasmid DNA in A8AN3. Nucleotide sequences of the two genes were determined. Analyses of their deduced amino acid sequences showed that the chromosome-encoded extradiol dioxygenase (CDO1) was most closely related (92.2% identity) to XylE of S. yanoikuyae B1. The plasmid-encoded extradiol dioxygenase (CDO2) had also most similarity (74.9% identity) to XylE of S. yanoikuyae B1 although the similarity was less than chromosome-encoded CDOI. A dendrogram produced by comparison of amino acid sequences showed considerable diversity of Sphingomonas extradiol dioxygenases from extradiol dioxygenases of the other strains.

Recommended Citation

Ahn, Yeonghee, "Microbial and molecular basis of polycyclic aromatic hydrocarbon biodegradation in contaminated soils. " PhD diss., University of Tennessee, 1996.
https://trace.tennessee.edu/utk_graddiss/9657