Doctoral Dissertations

Date of Award

12-1999

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Gary S. Sayler

Committee Members

Terry W. Shultz, Steven W. Wilhelm, James T. Fleming

Abstract

Agent Orange herbicide contaminated soils were utilized in enrichment culture studies to isolate 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5- trichlorophenoxyacetic acid (2,4,5-T) degrading bacteria. HPLC analyses of these soils demonstrated the presence of 22.4 mg/kg 2,4-D and 73.8 mg/kg 2,4,5-T. Two bacteria, Burkholderia species strain JRBl and Burkholderia species strain JR7B3, were isolated from these soils. These strains were able to mineralize 2,4-D and 2,4,5-T, respectively. Similar enrichment culture studies were unsuccessful in identifying dibenzofuran (DBF) and dibenzo-p-dioxin (DD) degrading bacteria. PGR experiments utilizing known genetic sequences from other 2,4-D and 2,4,5-T degrading bacteria have shown that these organisms contain gene sequences corresponding to the tfdA,B,C, E, and R genes (2,4-D degrading strain) and the tftA,C,and E genes (2,4,5-T degrading strain). These results are the first to indicate that both 2,4-D and 2,4,5-T metabolic pathways can exist in Agent Orange contaminated soils. Burkholderia species strain JR7B3 represents the first bacterium isolated directly from 2,4,5-T contaminated soil, which contains genes specific for 2,4,5-T degradation. Future studies comparing this environmental isolate with Burkholderia cepacia AC 1100 and other 2,4,5-T degrading bacteria will provide valuable information regarding the evolution, abundance, and activity of these ift genes in association with contaminated sites.

Growing cell assays coupled with messenger RNA analyses were utilized to examine the ability of these bacteria to degrade 2,4-D and 2,4,5-T in the presence of metabolic intermediates, which may affect catabolism of these compounds in Agent Orange contaminated soils. Degradation of 2,4-D by Burkholderia species strain JRBl was enhanced in the presence of glucose and RNA hybridization studies demonstrated a two-fold increase of ifdB mRNA transcripts. Addition of succinate resulted in a buildup of 2,4-dichlorophenol (2,4-DCP), a cell death phase, delayed degradation of 2,4-D, and a two-fold decrease of tfdB mRNA transcripts. Growth on 2,4,5-T by a bacterial consortium containing Burkholderia species strain JR7B3 and Burkholderia species strain JR7B2 in the presence of glucose and succinate resulted in increased 2,4,5- trichlorophenol (2,4,5-TCP) accumulation. Burkholderia species strain JR7B3 grown auxotrophically on 2,4,5-T in the presence of glucose resulting in 2,4,5-TCP concentrations of 22.4 mg/L, and no subsequent degradation or growth occurred. When succinate was provided as a co-carbon source 2,4,5-TCP concentrations remained below 300 pg/L and 2,4,5-T was slowly metabolized. 10 mg/L and 25 mg/L 2,4-DCP amendments induced degradation of 2,4,5-T relative to cultures containing only 2,4,5-T whereas 500 mg/L 2,4-D inhibited 2,4,5-T degradation for this strain.

Sphingomonas species strain RWl was chosen as a model bacterium to examine DBF degradation in the presence of metabolic intermediates of 2,4-D and 2,4,5-T. Random arbitrarily primed reverse transcriptase-PCR (RAP-PCR) was used to detect a mRNA transcript, putatively designated OBI, which was upregulated in the presence of DBF versus acetate. This previously unidentified gene sequence, OBI, was found to be located downstream of the dxnB gene and is clustered with other genes involved in DBF/DD metabolism. Bacterial growth experiments demonstrated that 2,4-DCP and 2,4,5-TCP inhibited growth when present at concentrations of 10 mg/L, and that 500 mg/L 2,4-D and 500 mg/L 2,4,5-T delayed growth of Sphingomonas species strain RWl on acetate. Bacterial cultures containing DBF and 2,4,5-T had a lower overall biomass relative to cultures grown with only DBF. Cultures containing acetate and 2,4-D or DBF and 2,4-D had increased overall biomass and metabolized all of the 2,4-D present. During growth on DBF/2,4-D and acetate/2,4-D 2,4-dichlorophenol concentrations were maintained below 1.0 mg/L. However, during growth on acetate/2,4,5-T, 2,4,5- trichlorophenol concentrations reached a maximum of 11.9 mg/L. RNA hybridization studies showed that addition of 2,4-D, 2,4-DCP, and 2,4,5-TCP to acetate and DBF growing cells resulted in decreased dxnAl, dbfB and OBI transcript abundance. These results illustrate that strain RWl has the capacity to completely metabolize 2,4-D and 2,4- DCP in the presence of either DBF or acetate. Strain RWl can also cometabolize 2,4,5-T to a minor degree, resulting in production of 2,4,5-TCP, which was inhibitory to utilization of DBF as a growth substrate.

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