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Expression of the tutE tutFDGH gene cluster of Thauera aromatica strain T1 was examined by Northern and Western analysis in a wild-type strain and chromosomally deleted strains with or without in-frame deletion plasmids. While expression was observed when the wild-type strain was induced with toluene, various chromosomally deleted strains exhibited little or no expression of the tut genes. In contrast, both wild-type and chromosomally deleted strains expressed the tut genes when induced with benzylsuccinate. We conclude that benzylsuccinate is required for the full induction of the tutE tutFDGH gene cluster of T. aromatica strain T1.

Thauera aromatica T1 was isolated for its ability to use toluene as a sole carbon source under denitrifying conditions. A genetic approach was used to examine the roles of the tutF, tutD, and tutG gene products (part of a single operon) in the metabolism of toluene. The genes were individually deleted from the chromosome and each resulting mutant strain was unable to metabolize toluene. Plasmids carrying individual in-frame gene deletions failed to complement the corresponding chromosomal deletions but did complement chromosomal deletions downstream of the in-frame deletion. Hence, the tutF, tutD, and tutG genes are each essential for toluene metabolism in T. aromatica T1.

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.

The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene utilization pathway have been characterized. A clone has been isolated, and subclones which contain tutD and tutE, two genes in the T1 toluene metabolic pathway, have been generated. The tutD gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da. The tutE gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da. Two additional small open reading frames have been identified, but their role is not known. The TutE protein has homology to pyruvate formate-lyase activating enzymes. The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme. Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.

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.

T1, a dentrifying bacterium originally isolated for its ability to grow on toluene, can also metabolize 4-hydroxybenzoate and other aromatic compounds under denitrifying conditions. A cosmid clone carrying the three genes that code for the 4-chlorobenzoate dehalogenase enzyme complex isolated from the aerobic bacterium Pseudomonas sp. strain CBS3 was successfully conjugated into strain T1. The cloned enzyme complex catalyzes the hydrolytic dechlorination of 4-chlorobenzoate to 4-hydroxybenzoate. Since molecular oxygen is not required for the dehalogenation reaction, the transconjugate strain of T1 (T1-pUK45-10C) was able to grow on 4-chlorobenzoate in the absence of O2 under denitrifying conditions. 4-Chlorobenzoate was dehalogenated to 4-hydroxybenzoate, which was then further metabolized by strain T1. The dehalogenation and metabolism of 4-chlorobenzoate were nitrate dependent and were coupled to the production of nitrite and nitrogen gas. 4-Bromobenzoate was also degraded by this strain, while 4-iodobenzoate was not. Additionally, when T1-pUK45-10C was presented with a mixture of 4-chlorobenzoate and toluene, simultaneous degradation of the compounds was observed. These results illustrate that dechlorination and degradation of aromatic xenobiotics can be mediated by a pure culture in the absence of oxygen. Furthermore, it is possible to expand the range of xenobiotic substrates degradable by an organism, and it is possible that concurrent metabolism of these substrates can occur.