Identification and characterization of preferred DNA-binding sites for the Thermus thermophilus transcriptional regulator FadR.

One of the primary transcriptional regulators of fatty acid homeostasis in many prokaryotes is the protein FadR. To better understand its biological function in the extreme thermophile Thermus thermophilus HB8, we sought to first determine its preferred DNA-binding sequences in vitro using the combinatorial selection method Restriction Endonuclease Protection, Selection, and Amplification (REPSA) and then use this information to bioinformatically identify potential regulated genes. REPSA determined a consensus FadR-binding sequence 5´-TTRNACYNRGTNYAA-3´, which was further characterized using quantitative electrophoretic mobility shift assays. With this information, a search of the T. thermophilus HB8 genome found multiple operons potentially regulated by FadR. Several of these were identified as encoding proteins involved in fatty acid biosynthesis and degradation; however, others were novel and not previously identified as targets of FadR. The role of FadR in regulating these genes was validated by physical and functional methods, as well as comparative genomic approaches to further characterize regulons in related organisms. Taken together, our study demonstrates that a systematic approach involving REPSA, biophysical characterization of protein-DNA binding, and bioinformatics can be used to postulate biological roles for potential transcriptional regulators

EMSA analysis of FadR-binding to REPSA-identified promoter sequences through a titration of FadR concentrations.

<p>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. (<b>A</b>) ST2_FadR_TTHA0890 DNA. (<b>B</b>) ST2_FadR_TTHA0402 DNA. (<b>C</b>) ST2_FadR_TTHA0604 DNA. (<b>D</b>) ST2_FadR_TTHA0846 DNA. (<b>E</b>) ST2_FadR_TTHA1118 DNA. (<b>F</b>) ST2_FadR_TTHB017 DNA. (<b>G</b>) ST2_FadR_TTHA0390 DNA. (<b>H</b>) ST2_FadR_TTHA1463 DNA. (<b>I</b>) ST2_FadR_TTHA1144 DNA. (<b>J</b>) ST2_FadR_TTHA0103 DNA. Binding site sequence and K_D values are indicated below each panel. Lowercase nucleotides indicate mutation from consensus FadR sequence.</p

Measurement for Self-Disposal of Wastes From Radioactive Material Research Facility

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REPSA section of FadR-dependent IISRE cleavage-resistant DNA species.

<p>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.</p

Quantitative EMSA analysis of FadR-binding to consensus and mutant sequences.

<p>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. (<b>A</b>) ST2_FadR_R5_wt consensus DNA; 0, 0.038, 0.075, 0.15, 0.3, 0.6, 1.2 nM FadR. (<b>B</b>) ST2_FadR_R5_m1 mutant DNA; 0, 0.38, 0.75, 1.5, 3.0, 6.0, 12 nM FadR. (<b>C</b>) ST2_FadR_R5_m2 mutant DNA; 0, 0.06, 0.6, 6, 60, or 600 nM FadR. (<b>D</b>) ST2_FadR_R5_m3 mutant DNA; 0, 3.8, 7.5, 15, 30, 60, 120 nM FadR. (<b>E</b>) ST2_FadR_R5_m4 mutant DNA; 0, 0.75, 1.5, 3.0, 6.0, 12, 24 nM FadR. (<b>F</b>) ST2_FadR_R5_m5 mutant DNA; 0, 7.5, 15, 30, 60, 120, 240 nM FadR. (<b>G</b>) ST2_FadR_R5_m6 mutant DNA; 0, 0.06, 0.6, 6, 60, or 600 nM FadR. (<b>H</b>) ST2_FadR_R5_m7 mutant DNA; 0, 7.5, 15, 30, 60, 120, 240 nM FadR. Binding site sequence and K_D values are indicated below each panel. Lowercase nucleotides indicate mutation from consensus FadR sequence.</p

EMSA analysis of FadR-binding to REPSA-identified promoter sequences through a titration of FadR concentrations.

<p>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. (<b>A</b>) ST2_FadR_TTHA0890 DNA. (<b>B</b>) ST2_FadR_TTHA0402 DNA. (<b>C</b>) ST2_FadR_TTHA0604 DNA. (<b>D</b>) ST2_FadR_TTHA0846 DNA. (<b>E</b>) ST2_FadR_TTHA1118 DNA. (<b>F</b>) ST2_FadR_TTHB017 DNA. (<b>G</b>) ST2_FadR_TTHA0390 DNA. (<b>H</b>) ST2_FadR_TTHA1463 DNA. (<b>I</b>) ST2_FadR_TTHA1144 DNA. (<b>J</b>) ST2_FadR_TTHA0103 DNA. Binding site sequence and K_D values are indicated below each panel. Lowercase nucleotides indicate mutation from consensus FadR sequence.</p

Potential FadR-regulated genes.

<p>Potential FadR-regulated genes.</p

FIMO of best possible match TTGGACTTAGTCCAA.

<p>FIMO of best possible match TTGGACTTAGTCCAA.</p

Validation of REPSA-selected FadR-binding DNA species.

<p>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.</p