Article Figures & Data
Figures
- FIG. 1.
Amino acid sequence alignment of T. thermophilus σE with representative homologous proteins. The amino acid sequences of T. thermophilus σE (TT), Pseudomonas fluorescens PfO-1 Pfl_3746 (PF), Deinococcus geothermalis DSM 11300 Dgeo_0606 (DG), Frankia alni ACN14a FRAAL3632 (FA), and Roseiflexus castenholzii DSM 13941 RcasDRAFT_1056 (RC) were aligned using Clustal W (28). Identical residues in all sequences (*), conserved substitutions (:), and semiconserved substitutions (.) are indicated. The e values obtained with the BLAST search are indicated on the right. The regions similar to regions 2 and 4 of σ70-type σ factors, identified in the conserved domain database search, are indicated.
- FIG. 2.
Three clones of the wild-type (closed circles) and of the ΔsigE (open circles) strains of T. thermophilus were each grown in a rich medium, and the A 600 values obtained at the indicated times are expressed as means ± standard deviations (SD), respectively. The expression levels of the T. thermophilus sigE (ΤΤΗΒ211) (open triangles) and TTHB212 (closed triangles) mRNAs in the wild-type strain were investigated at the indicated times using GeneChip technology, as described previously (23), and are expressed as normalized intensities ± SD.
- FIG. 3.
Expression of T. thermophilus σE and purification of T. thermophilus RNAP-σE holoenzyme (EσE). N-terminal His-tagged T. thermophilus σE (His-σE) was synthesized by means of E. coli coupled transcription-translation cell-free protein synthesis in the absence (lanes 7 and 8) or presence (lanes 9 and 10) of the T. thermophilus RNAP core enzyme. After the reaction, each sample was fractionated into a supernatant (S) and a precipitate (P), which were analyzed by SDS-PAGE. Control samples without the sigE gene are shown (lanes 3 to 6). The EσE synthesized in vitro was purified and analyzed by SDS-PAGE (lane 11). Lane 1, molecular mass markers in kDa; lane 2, T. thermophilus RNAP core enzyme.
- FIG. 4.
(A) Upstream sequence of the T. thermophilus sigE (TTHB211) gene, which was used as the template for the runoff transcription assays. The numerals represent genome positions in megaplasmid pTT27. The transcriptional start site of EσE is capitalized and that of EσA is underlined. (B) Runoff transcription assay performed with T. thermophilus EσE and templates a, b, c, d, and e is shown in panel A. After the reaction, samples were analyzed by PAGE, followed by autoradiography. Lane 1, [α-32P]dCTP-labeled MspI fragments of pBR322. (C) The runoff transcription assay was performed with T. thermophilus EσE (σE) (lanes 2, 4, 6, 9, and 11) or EσA (σA) (lanes 3, 5, 7, 10, and 12) involving the DNA fragment containing the region upstream of TTHB211 (TTHB211_up), TTHB210 (TTHB210_up), or TTHB213 (TTHB213_up), the T. thermophilus 16S rRNA gene (16S rRNA), or the E. coli consensus promoter (full con) as a template. After the reaction, samples were analyzed by PAGE, followed by autoradiography. Lanes 1 and 8, [α-32P]dCTP-labeled MspI fragments of pBR322.
- FIG. 5.
(A) Identification of the transcription start site. The RNA transcribed by T. thermophilus EσE (lane 5) or EσA (lane 6) from the genes containing the sequences upstream of TTHB210 (TTHB210_up), TTHB211 (TTHB211_up), and TTHB213 (TTHB213_up), respectively, was reverse transcribed, and the nucleotide sequence of the template DNA was determined by the dideoxy-mediated chain termination method (lanes 1 to 4). After the reaction, samples were analyzed by PAGE, followed by autoradiography. The sample volume applied to lane 6 for TTHB211_up was twofold more than that applied to lane 5 for TTHB211_up. The 3′ terminus of the cDNA is indicated by an asterisk. (B) The nucleotide sequence alignment of predicted promoter regions recognized by T. thermophilus EσE found in TTHB210_up, TTHB211_up, and TTHB213_up is shown. The transcription start site of EσE is capitalized and that of EσA is underlined. The conserved bases are boxed, and the consensus sequence (Consensus) is indicated. The predicted −10 and −35 regions are shown in bold. Nucleotides y, k, m, w, and s represent t or c, g or t, a or c, a or t, and g or c, respectively. Nucleotide n represents g, a, t, or c. (C) Schematic view of the T. thermophilus σE regulon found in this study. The arrowhead indicates the ORF. The arrows indicate the promoter positions and directions of transcription by EσE (σE; solid line) or EσA (σA; broken line).
Additional Files
Supplemental material
Files in this Data Supplement:
- Supplemental file 2 -
Fig. S1, raw data used for the creation of Fig. 4B and C
PDF, 5MB. - Supplemental file 1
-
Table S1, genes exhibiting decreased expression in the DsigE strain compared with in the wild type
Zipped Excel document, 6K.
- Supplemental file 2 -
Fig. S1, raw data used for the creation of Fig. 4B and C
