Fig 1.
REPSA section of FadR-dependent IISRE cleavage-resistant DNA species.
Shown are LICOR Odyssey images of restriction endonuclease cleavage protection assays during Rounds 1 through 5 of REPSA selection with 6 nM FadR protein. The presence of FadR or IISRE FokI (F) or BpmI (B) is indicated above each lane. Lanes include: (+/-) total DNA control, (-/F or -/B) IISRE cleavage control, and (+/F or +/B) IISRE selection with FadR. The electrophoretic mobility of the intact (T) and cleaved (X) selection template, as well as the IRD7_ST2R primer (P), are indicated at right of figure.
Fig 2.
Validation of REPSA-selected FadR-binding DNA species.
Shown are LICOR images of EMSAs containing pooled DNA from either Round 1 (left lanes) or Round 5 (right lanes) of REPSA selection and increasing concentrations of FadR protein (from left to right: 0, 0.6, 6, 60, or 600 nM FadR). The electrophoretic mobility of a single protein-DNA complex (S) as well as uncomplexed ST2R24 selection template (T) and IRD7_ST2R primer (P) are indicated at right of figure.
Fig 3.
Sequence logos of REPSA-selected FadR-binding sequences.
Sequence logos were determined using MEME software with inputs of 1000 Round 5 DNA sequences. (A) MEME performed with no filters. (B) Palindromic filter.
Fig 4.
Quantitative EMSA analysis of FadR-binding to consensus and mutant sequences.
Shown are LICOR images of IRD700-labeled FadR consensus or point-mutated DNAs, as indicated, incubated with a twofold (wt, m1, m3, m4, m5, m7) or tenfold (m2, m6) titration of FadR protein, as indicated. (S) FadR-DNA complex, (T) uncomplexed DNA. (A) ST2_FadR_R5_wt consensus DNA; 0, 0.038, 0.075, 0.15, 0.3, 0.6, 1.2 nM FadR. (B) ST2_FadR_R5_m1 mutant DNA; 0, 0.38, 0.75, 1.5, 3.0, 6.0, 12 nM FadR. (C) ST2_FadR_R5_m2 mutant DNA; 0, 0.06, 0.6, 6, 60, or 600 nM FadR. (D) ST2_FadR_R5_m3 mutant DNA; 0, 3.8, 7.5, 15, 30, 60, 120 nM FadR. (E) ST2_FadR_R5_m4 mutant DNA; 0, 0.75, 1.5, 3.0, 6.0, 12, 24 nM FadR. (F) ST2_FadR_R5_m5 mutant DNA; 0, 7.5, 15, 30, 60, 120, 240 nM FadR. (G) ST2_FadR_R5_m6 mutant DNA; 0, 0.06, 0.6, 6, 60, or 600 nM FadR. (H) ST2_FadR_R5_m7 mutant DNA; 0, 7.5, 15, 30, 60, 120, 240 nM FadR. Binding site sequence and KD values are indicated below each panel. Lowercase nucleotides indicate mutation from consensus FadR sequence.
Table 1.
FIMO of best possible match TTGGACTTAGTCCAA.
Fig 5.
Bioinformatic identification of T. thermophilus HB8 promoters potentially regulated by FadR.
Shown are sequences +/- 200 bp of the FadR-binding sequence of a target gene identified through FIMO analysis as being potentially regulated by FadR (see Table 1). Longest open reading frames with identical orientation as the target gene are indicated with blue nucleotides. Open reading frames with opposite orientation are indicated with green nucleotides. Black nucleotides indicate intergenic regions. Potential promoter elements (-30 and -10 boxes, +1 start site of transcription) were identified using Softberry BPROM and are indicated with blue highlighting. FadR-binding sites are indicated with yellow highlighting. Regions of overlap between FadR-binding sites and promoter elements are indicated by green highlighting. Underlining and italics indicate core promoter elements and FadR-binding sites, respectively, identified previously by Agari et al [7].
Table 2.
Potential FadR-regulated genes.
Fig 6.
EMSA analysis of FadR-binding to REPSA-identified promoter sequences through a titration of FadR concentrations.
Shown are LICOR images of IRD700-labeled DNA probes containing FadR-binding sites from the REPSA-identified promoter regions, as indicated, incubated with 0, 0.38, 0.75, 1.5, 3, 6, or 12 nM FadR protein. (S) FadR-DNA complex, (T) uncomplexed DNA. (A) ST2_FadR_TTHA0890 DNA. (B) ST2_FadR_TTHA0402 DNA. (C) ST2_FadR_TTHA0604 DNA. (D) ST2_FadR_TTHA0846 DNA. (E) ST2_FadR_TTHA1118 DNA. (F) ST2_FadR_TTHB017 DNA. (G) ST2_FadR_TTHA0390 DNA. (H) ST2_FadR_TTHA1463 DNA. (I) ST2_FadR_TTHA1144 DNA. (J) ST2_FadR_TTHA0103 DNA. Binding site sequence and KD values are indicated below each panel. Lowercase nucleotides indicate mutation from consensus FadR sequence.
Table 3.
BLASTn analysis of orthologous FadR sites.
Table 4.
FadR clusters of co-regulated orthologous operons.