Transcriptional analysis of the tutE tutFDGH gene cluster from Thauera aromatica strain T1

Abstract

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.

FIG. 1

Restriction map of the region of cosmid clone 13-6-4 that contains the tutE tutFDGH genes. The five identified open reading frames are indicated with thick arrows. Thin arrows above the map indicate the major transcriptional start sites identified. T, potential terminator site. The probes used for Northern analysis are also indicated below the map with thick lines. Potential transcripts (from the starts to the terminators) are shown below the probes with thin lines. Restriction site abbreviations: B, BamHI; C, ClaI; N, NcoI; P, PstI; R, EcoRI; Sa, SacII; Sc, SacI. Sites blocked by methylation are omitted.

FIG. 2

Northern analysis of total RNA isolated from cells grown under denitrifying conditions with either toluene (T) or pyruvate (P) as the carbon source and visualized with gene-specific probes derived from tutE, tutF, tutD, tutG, or tutH. Samples of digoxigenin-labeled RNA were included to serve as size markers (M) and are labeled (in kilobases) on the sides.

FIG. 3

Primer extension analysis to map the transcriptional start sites of the tutE and tutF genes. End-labeled primer E-PE3 was used to identify the tutE start of transcription and end-labeled primer F-PE1 was used to identify the tutF start of transcription. The same primers were used to generate the sequencing ladder by the dideoxy method (lanes marked G, A, T, and C). The sequence encompassing the major transcriptional start (marked with an asterisk) is enlarged.