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1.
Fig. 1.

Fig. 1. From: The Riboswitch Regulates a Thiamine Pyrophosphate ABC Transporter of the Oral Spirochete Treponema denticola .

Diagram showing the targeted mutagenesis of the tbpATd gene. Arrows indicate the relative positions of PCR primers for constructing the targeted mutagenesis vector tbpATd::erm, in which an 864-bp TDE0143 fragment is deleted and replaced by the erythromycin resistance cassette (Ermr). The sequences of these primers are listed in .

Jiang Bian, et al. J Bacteriol. 2011 Aug;193(15):3912-3922.
2.
Fig. 7.

Fig. 7. From: The Riboswitch Regulates a Thiamine Pyrophosphate ABC Transporter of the Oral Spirochete Treponema denticola .

Characterization of the T. denticola ΔtbpA mutant. (a) Western blotting of the T. denticola ΔtbpA mutant. The same amounts of wild-type T. denticola and T. denticola ΔtbpA whole lysates were analyzed by SDS-PAGE. The immunoblotting was conducted as described for . (b) RT-PCR analysis of the T. denticola ΔtbpA mutant. The transcripts of TDE0144 and TDE0145, the downstream genes of tbpATd, in wild-type T. denticola and T. denticola ΔtbpA were detected by RT-PCR.

Jiang Bian, et al. J Bacteriol. 2011 Aug;193(15):3912-3922.
3.
Fig. 8.

Fig. 8. From: The Riboswitch Regulates a Thiamine Pyrophosphate ABC Transporter of the Oral Spirochete Treponema denticola .

Growth curves of wild-type T. denticola (a) and T. denticola ΔtbpA (b). The two strains were cultivated in TA medium containing three different concentrations of TPP (100 nM, 10 μM, and 50 μM). Cell counting was repeated in duplicate with at least three independent samples, and the results are expressed as mean ± SEM.

Jiang Bian, et al. J Bacteriol. 2011 Aug;193(15):3912-3922.
4.
Fig. 4.

Fig. 4. From: The Riboswitch Regulates a Thiamine Pyrophosphate ABC Transporter of the Oral Spirochete Treponema denticola .

Sequence and secondary structure of Tdthi-box. (a) Comparison between the 5′-untranslated region (UTR) of tbpATd (Tdthi-box) and the TPP riboswitch motif box (RF00059) (). Diamonds represent the conserved residues required for the function and structure of TPP riboswitches. The underlined AUG is the translational start codon of tbpATd. (b) Diamonds represent the conserved residues identified in the riboswitches of thiM and thiC. The S/D sequence and the start codon are circled. The conserved TPP-sensing motif is boxed. The structure was predicted with the CONTRAfold program.

Jiang Bian, et al. J Bacteriol. 2011 Aug;193(15):3912-3922.
5.
Fig. 6.

Fig. 6. From: The Riboswitch Regulates a Thiamine Pyrophosphate ABC Transporter of the Oral Spirochete Treponema denticola .

The TbpATd level is repressed by high concentrations of TPP. Wild-type T. denticola was cultivated in TA medium containing different concentrations of TPP (100 nM, 10 μM, and 50 μM), and the same amount of whole-cell lysates was analyzed by SDS-PAGE. Polyclonal antibodies against TbpATd or FlaA, a flagellin protein of T. denticola, were used as probes. FlaA was used as an internal control.

Jiang Bian, et al. J Bacteriol. 2011 Aug;193(15):3912-3922.
6.
Fig. 5.

Fig. 5. From: The Riboswitch Regulates a Thiamine Pyrophosphate ABC Transporter of the Oral Spirochete Treponema denticola .

Characterization of a TPP-sensing riboswitch in T. denticola (Tdthi-box). (a) Construction of Tdthi-boxlacZ fusions for the β-galactosidase assays. thi-box-L represents the fragment from nucleotide −366 to +69 of Tdthi-box. (b) TPP repression β-galactosidase assays. The β-galactosidase activity was expressed as Miller units/107 cells, and the levels represent means ± standard errors of the means from three independent experiments. Plasmid pRS414 containing the thiC-box was used as a positive control and plasmid pRS414 () as a negative control.

Jiang Bian, et al. J Bacteriol. 2011 Aug;193(15):3912-3922.
7.
Fig. 9.

Fig. 9. From: The Riboswitch Regulates a Thiamine Pyrophosphate ABC Transporter of the Oral Spirochete Treponema denticola .

Schematic diagram showing the regulatory mechanism of TPP transport in T. denticola. T. denticola lacks a de novo TBS pathway, and it depends on the TPP transporter to take up exogenous TPP. The tbpABCTd operon encodes a TPP ABC transporter, which is regulated by a TPP-sensing riboswitch (Tdthi-box). When the intracellular TPP is insufficient, Tdthi-box folds into a structure that allows the transcription of the tbpABCTd operon, and more TPP transporter will be synthesized to take up exogenous TPP into the cells. When the intracellular TPP becomes sufficient, the free TPP binds to Tdthi-box and forms a stable aptamer, which in turn represses the transcription of the tbpABCTd operon and limits the uptake of exogenous TPP.

Jiang Bian, et al. J Bacteriol. 2011 Aug;193(15):3912-3922.
8.
Fig. 3.

Fig. 3. From: The Riboswitch Regulates a Thiamine Pyrophosphate ABC Transporter of the Oral Spirochete Treponema denticola .

Transcriptional analysis of the tbpABCTd operon. (a) Diagram showing the genes near TDE0143. The numbers and small arrows show the primers used for the RT-PCR analysis. (b) RT-PCR analysis. The lane numbers correspond to the primers labeled in panel a, and the sequences of these primers are described in . Lane M, 1.0-kb DNA ladder. (c) 5′-RLM-RACE analysis. The arrow shows the sequencing direction, and the asterisk indicates the identified transcriptional start site. (d) Comparison of the identified promoter sequence with the consensus sequence of the E. coli σ70 promoter. The conserved nucleotides are dotted.

Jiang Bian, et al. J Bacteriol. 2011 Aug;193(15):3912-3922.
9.
Fig. 2.

Fig. 2. From: The Riboswitch Regulates a Thiamine Pyrophosphate ABC Transporter of the Oral Spirochete Treponema denticola .

Structural characteristics of TbpATd. (a) Comparison of the overall structures of the E. coli TbpA protein (top) and TbpATd (bottom). The structural view of these two proteins is depicted by Cα trace with helices colored in blue, sheets in green, and the rest in gray. The TPP ligand is shown in a Van der Waals (VDW) representation. E. coli TbpA (Protein Data Bank ID 2QRY) () was selected for the structural modeling using the program Modeler 9v7 as previously described (). (b) Predicted thiamine-binding site with the amino acids and the bound TPP (red).

Jiang Bian, et al. J Bacteriol. 2011 Aug;193(15):3912-3922.

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