Aerobic and anaerobic toluene degradation by a newly isolated denitrifying bacterium, Thauera sp. strain DNT-1

Abstract

A newly isolated denitrifying bacterium, Thauera sp. strain DNT-1, grew on toluene as the sole carbon and energy source under both aerobic and anaerobic conditions. When this strain was cultivated under oxygen-limiting conditions with nitrate, first toluene was degraded as oxygen was consumed, while later toluene was degraded as nitrate was reduced. Biochemical observations indicated that initial degradation of toluene occurred through a dioxygenase-mediated pathway and the benzylsuccinate pathway under aerobic and denitrifying conditions, respectively. Homologous genes for toluene dioxygenase (tod) and benzylsuccinate synthase (bss), which are the key enzymes in aerobic and anaerobic toluene degradation, respectively, were cloned from genomic DNA of strain DNT-1. The results of Northern blot analyses and real-time quantitative reverse transcriptase PCR suggested that transcription of both sets of genes was induced by toluene. In addition, the tod genes were induced under aerobic conditions, whereas the bss genes were induced under both aerobic and anaerobic conditions. On the basis of these results, it is concluded that strain DNT-1 modulates the expression of two different initial pathways of toluene degradation according to the availability of oxygen in the environment.

FIG. 1.

Phylogenetic position of Thauera sp. strain DNT-1 among related aromatic compound-degrading Thauera and Azoarcus strains based on 16S rDNA sequence comparisons. The numbers are bootstrap values for branches based on 1,000 replicates. The bar shows 1 nucleotide substitution per 10,000 nucleotides.

FIG. 2.

Growth of Thauera sp. strain DNT-1 on toluene under aerobic (A), oxygen-limiting (B), and anaerobic (C) conditions, with 21, 2, and 0% oxygen in the gas phase of culture vessels, respectively. The amount of the metabolite was quantified by the area values of the peaks calculated by the recorder. The point of complete depletion of oxygen and the start of anaerobic degradation are indicated by arrows. Symbols in the top graphs: open circles, oxygen; open squares, nitrate; open triangles, nitrite. Symbols in the bottom graphs: open circles, toluene; closed circles, growth; closed diamonds, metabolite.

FIG. 3.

Compositions and putative functions of tod (A) and bss (B) genes of Thauera sp. strain DNT-1 aligned with some of the most similar genes found in other aromatic compound-degrading bacteria. The percentages below the ORFs indicate the similarity of amino acid sequences to the corresponding ORF of strain DNT-1.

FIG. 4.

Northern blot analyses of todA and bssA genes expressed in Thauera sp. strain DNT-1 grown under aerobic and anaerobic conditions with toluene (Tol) or succinate (Suc) as the sole carbon source. 16S and 23S rRNA bands visualized with ethidium bromide are shown under each lane to confirm that equal amounts of total RNA were loaded.

FIG. 5.

Kinetic PCR curves of real-time QRT-PCR targeting todA and bssA genes. Relative fluorescence output is plotted versus PCR cycle number. Kinetic curves for cells grown aerobically in toluene (line 1) or succinate (line 2) and for cells grown anaerobically in toluene (line 3) or succinate (line 4) are shown.