Project Abstract

Molecular Diversity of Toluene Ortho-Monooxygenase Genes

Principal Investigators

James M. Tiedje
Michigan State University
E-mail: tiedjej@pilot.msu.edu

Jizhong Zhou
Oak Ridge National Laboratory

Summary

Toluene ortho-monooxygenase appears to be widespread in nature and be one of the most effective enzymes in cometabolizing trichloroethylene (TCE). To understand their molecular diversity, the DNA fragments (~550 bp) containing the active site (binuclear iron centers) of the large subunit of the monooxygenase enzymes were cloned and sequenced from 28 toluene-degrading bacteria varying greatly in TCE cometabolizing activities. Large variations in both nucleotide and amino acid sequences were observed among these isolates, with similarities of 60-100%. Phylogenetic analyses revealed that 86% of the sequences grouped with toluene ortho-monooxygenase genes from Burkholderia sp. JS150 and B. cepacia G4, whereas 14% of the sequences were more closely related to the phenol hydroxylase genes from Pseudomonas and Acinetobacter species. Overall, the functional gene-based phylogeny appears to be consistent with the 16S rRNA gene-based phylogeny, suggesting that the horizontal transfer of monooxygenase genes among these bacteria was rare during their evolution. The amino acid sequences for the two binuclear iron centers are conserved among all of these species. These results suggest that the active site on the large subunit of the monooxygenases are not responsible for the differences of TCE cometabolism activities of these isolates.

Introduction

The major challenge for TCE bioremediation is that there are no known organisms that can use TCE as a primary substrate for energy and growth, but TCE has been found to undergo degradation through cometabolism or fortuitous transformation by enzymes developed and used by microorganisms for other purposes. Some important questions to in situ TCE bioremediation based on cometabolism are: (i) how to evaluate a site's population for TCE bioremediation potential; (ii) how to maintain TCE-degrading stable populations; and (iii) how to track and monitor the changes and the fates of TCE-degrading populations. To answer some of the questions, in this study, we will define molecular diversity of orthomonooxygenase genes and develop molecular signatures for their detection in the environments.

Research Objectives

The goal of this project is to determine molecular diversity of toluene ortho-monooxygenase genes, develop molecular signatures to detect TCE degrading bacteria, and use them to monitor TCE cooxidizers in contaminated Department of Defense sites. Special objectives are to:

analyze and compare the sequence divergence of the ortho-monooxygenase genes from the bacteria capable of rapidly degrading TCE with those from the strains unable to cometabolize TCE.
develop molecular signatures based on the ortho-monooxygenase genes to distinguish both the bacteria capable of degrading TCE and those unable to cometabolize TCE.
use the developed molecular methods to evaluate bioremediation strategies and to monitor TCE cooxidizers in contaminated Department of Defense sites.

Results

Technical accomplishments include:

PCR amplification primers were designed and amplification was successfully obtained from a variety of bacteria.
the orthomonoxygenase genes were partially sequenced and analyzed.
16 rRNA gene sequences were determined to establish the phylogenetic diversity of the selected toluene-degrading isolates.

We have designed a set of PCR amplification primers based on the sequences of oxygenase genes (TomD) from Burkholderia cepacia G4 and Pseudomonas sp. JS150. We successfully amplified TomD genes (about 1.4 kb) with these primers from some of the strains showing different levels of TCE degradation activities. We have also designed 8 internal sequencing primers, and we used these primers to sequence orthomonooxygenase genes from three 3 strains: MF-19, MF-141 and MF-62. However these primers did not work for most of the other strains in our collection. Thus, based on these sequences, we redesigned a new set of primers, which amplify about 700 bp of TomD genes. The amplified fragment span the active sites of orthomooxygenase genes. We successfully amplified orthomoxygenase genes from almost all of the strains with this new primer set, suggesting that these primers appear to be highly conserved and can be used as general primers for detecting monooxygenase genes. The amplified fragments were successfully sequenced with the same primers used for PCR amplification from 28 toluene-degrading bacteria varying greatly in TCE cometabolizing activities..

Future Directions

The sequence results suggested that it appears difficult to develop molecular signature to distinguish effective TCE degraders from poor TCE degraders because the sequence regions containing the active site are not different enough for differentiation. However, we found that the primers used are highly conserved, and they could be very useful in detecting bacteria containing ortho-monooxygenase genes. We will develop PCR-based quantitative methods based on these primers for detecting TCE-degrading bacteria in the environments, and then we will use them to monitor effective TCE cooxidizers in contaminated Department of Defense sites.