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The denitrifying strain T1, identified as Thauera aromatica, is able to grow with toluene serving as its sole carbon source. Previous work identified two genes, tutD and tutE, that are involved in toluene metabolism. Two small open reading frames, tutF and tutG, which may also play a role in toluene metabolism, were also identified. The present work examines the transcriptional organization and regulation of these toluene utilization genes. Northern analysis indicates that the four genes are organized into two operons, tutE and tutFDG, and that both operons are regulated in response to toluene. Primer extension analysis has identified major transcriptional start sites located 177 bp upstream of the tutE translational start and 76 bp upstream of the tutF translational start. Furthermore, a fifth gene, tutH, has been identified immediately downstream of tutG. It is transcribed from the same start site as tutFDG and is predicted to code for a 286-amino-acid protein with a calculated molecular mass of about 31,800 Da. The TutH protein is predicted to have an ATP/GTP binding domain and is similar to the NorQ/NirQ family of proteins.

T1 is a denitrifying bacterium isolated for its ability to grow with toluene serving as the sole carbon source. Mutants of this strain that have defects in the toluene utilization pathway have been isolated and have been separated into classes based on growth phenotypes. A cosmid clone has been isolated by complementing the tutB16 (for toluene utilization) mutation. The complementing gene has been localized to a 3.3-kb DNA fragment. An additional open reading frame upstream of the tutB gene has also been identified and is designated tutC. The nucleotide sequence and the predicted amino acid translation of the 6.4-kb DNA fragment that contains these genes are presented. The tutB and tutC gene products of strain T1 have homology to members of the two-component sensor-regulator family and are proposed to play a role in the regulation of toluene metabolic genes of strain T1. To our knowledge, this is the first published report of the isolation of mutants defective in anaerobic aromatic hydrocarbon degradation. Additionally, we report for the first time the cloning of genes involved in an anaerobic aromatic hydrocarbon degradation pathway.