A Novel Approach for Transcription Factor Analysis Using SELEX with High-Throughput Sequencing (TFAST)

<div><h3>Background</h3><p>In previous work, we designed a modified aptamer-free SELEX-seq protocol (afSELEX-seq) for the discovery of transcription factor binding sites. Here, we present original software, TFAST, designed to analyze afSELEX-seq data, validated against our previously generated afSELEX-seq dataset and a model dataset. TFAST is designed with a simple graphical interface (Java) so that it can be installed and executed without extensive expertise in bioinformatics. TFAST completes analysis within minutes on most personal computers.</p> <h3>Methodology</h3><p>Once afSELEX-seq data are aligned to a target genome, TFAST identifies peaks and, uniquely, compares peak characteristics between cycles. TFAST generates a hierarchical report of graded peaks, their associated genomic sequences, binding site length predictions, and dummy sequences.</p> <h3>Principal Findings</h3><p>Including additional cycles of afSELEX-seq improved TFAST's ability to selectively identify peaks, leading to 7,274, 4,255, and 2,628 peaks identified in two-, three-, and four-cycle afSELEX-seq. Inter-round analysis by TFAST identified 457 peaks as the strongest candidates for true binding sites. Separating peaks by TFAST into classes of worst, second-best and best candidate peaks revealed a trend of increasing significance (e-values 4.5×10¹², 2.9×10⁻⁴⁶, and 1.2×10⁻⁷³) and informational content (11.0, 11.9, and 12.5 bits over 15 bp) of discovered motifs within each respective class. TFAST also predicted a binding site length (28 bp) consistent with non-computational experimentally derived results for the transcription factor PapX (22 to 29 bp).</p> <h3>Conclusions/Significance</h3><p>TFAST offers a novel and intuitive approach for determining DNA binding sites of proteins subjected to afSELEX-seq. Here, we demonstrate that TFAST, using afSELEX-seq data, rapidly and accurately predicted sequence length and motif for a putative transcription factor's binding site.</p> </div

Model describing the differential effect of <i>E. coli</i> and <i>P. mirabilis</i> metabolism on the C/N ratio within the urinary tract.

<p>The urinary tract environment has a low C/N ratio due to the dilute mixture of amino acids and peptides as the primary carbon source and the abundance of urea in urine providing a substantial nitrogen contribution. <i>E. coli</i> is unable to utilize or sense the nitrogen sequestered in urea because it lacks urease, which liberates ammonia from urea. In contrast, <i>P. mirabilis</i> is urease positive; consequently, <i>P. mirabilis</i> senses a physiologically lower C/N ratio than <i>E. coli</i>. This results in <i>E. coli</i> activation of the glutamine synthetase and glutamate oxo-glutarate aminotransferase system (GS/GOGAT) to assimilate nitrogen while <i>P. mirabilis</i> assimilates nitrogen, via glutamate dehydrogenase (Gdh) due to the apparent excess nitrogen available from ammonia produced by urea hydrolysis. This difference in physiological nitrogen availability explains the dramatic difference between <i>E. coli</i> and <i>P. mirabilis</i> central carbon pathway requirements for fitness during urinary tract infection.</p

Fluorescence difference in gel electrophoresis (2D-DIGE) of UPEC cytoplasmic proteins during growth in urine.

<p>Soluble proteins (50 µg) from <i>E. coli</i> CFT073 cultured in urine were labeled with Cy3 (green), from CFT073 grown in LB with Cy5 (red), and the pooled internal standard representing an equal amount of urine and LB soluble proteins with Cy2 (blue). The labeled proteins (150 µg) were pooled and applied to a pH 4–7 IPG strip and second dimension 10% SDS-PAGE. Green spots indicate protein features induced in urine; red spots represent proteins induced in LB medium.</p

UPEC acquires amino acids and requires gluconeogenesis and the TCA cycle for fitness <i>in vivo</i>.

<p>Peptide substrate-binding protein genes <i>dppA</i> and <i>oppA</i> are required to import di- and oligopeptides into the cytoplasm from the periplasm. Short peptides are degraded into amino acids in the cytoplasm and converted into pyruvate and oxaloacetate. Pyruvate is converted into acetyl-CoA and enters the TCA cycle to replenish intermediates and generate oxaloacetate. Oxaloacetate is converted to phosphoenolpyruvate by the <i>pckA</i> gene product during gluconeogenesis. Mutations in the indicated genes <i>dppA</i>, <i>oppA</i>, <i>pckA</i>, <i>sdhB</i>, and <i>tpiA</i> demonstrated fitness defects <i>in vivo</i>.</p

TFAST identifies peaks with discoverable motifs from afSELEX-seq data.

<p>TFAST and MACS were used to pick and evaluate peaks from our data set. (<b>A</b>) TFAST picked a total of 2,628 peaks, of which 2,197 covered 96% of the peaks identified by MACS in the final cycle of afSELEX-seq. Positional weight matrices generated in MEME instructed to search for a 15 bp motif using 200 sequences from (<b>B</b>) the 457 “Best” weight (most enriched) peaks, (<b>C</b>) the 888 next-best weight (second most enriched) peaks, (<b>D</b>) the 1,283 worst weight (least enriched) peaks, (<b>E</b>) all peaks called by TFAST pooled together and (<b>F</b>) 200 peaks called by MACS with the lowest false discovery rate (FDR). Sets of peaks from (<b>B–F</b>) were subjected again to analysis by MEME under the similar conditions but with the inclusion of a zero-order background Markov model to generate (<b>G–K</b>). E-value (the chance that a motif arose from a dataset by chance) and bit score (the total information content of a positional weight matrix) are shown below each logo.</p

Diagram of central metabolism and map of the specific pathways disrupted by targeted mutations in uropathogenic <i>E. coli</i> and <i>P. mirabilis</i>.

<p>Carbon sources or biochemical intermediates shared between pathways are indicated in capital letters or abbreviated: G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; G3P, glyceraldehyde-3-phosphate; 6PGN, 6-phosphogluconate. Reactions are denoted with arrows. Specific reactions (red arrows) were targeted by deletion or insertion in <i>E. coli</i> CFT073 or <i>P. mirabilis</i> HI4320, respectively. In glycolysis: <i>pgi</i>, glucose-6-phosphate isomerase; <i>pfkA</i>, 6-phosphofructokinase transferase; <i>tpiA</i>, triosephosphate isomerase; <i>pykA</i>, pyruvate kinase; in pentose phosphate pathway: gnd, 6-phosphogluconate dehydrogenase; <i>talB</i>, transaldolase; in Entner-Duodoroff pathway: <i>edd</i>, 6-phosphogluconate dehydratase; in gluconeogenesis: <i>pckA</i>, phosphoenolpyruvate carboxykinase; and in the TCA cycle: <i>sdhB</i>, succinate dehydrogenase; <i>fumC</i>, fumarate hydratase; <i>frdA</i>, fumarate reductase.</p

Growth of central metabolism mutants <i>in vitro</i> and <i>in vivo</i>.

<p>Growth of central metabolism mutants <i>in vitro</i> and <i>in vivo</i>.</p

<i>In vivo</i> fitness of UPEC central metabolism mutants.

<p>Individual female mice were transurethrally inoculated with 2×10⁸ CFU of a 1∶1 mixture of wild-type and mutant bacteria. <i>In vivo</i> fitness at 48 hpi for UPEC mutants defective in: (A,B) glycolysis, (C) pentose phosphate pathway, (D) Entner-Doudoroff pathway, (E) TCA cycle, and (F) gluconeogenesis. At 48 hpi, bladders and kidneys were aseptically removed, homogenized, and plated on LB or LB containing kanamycin to determine viable counts of wild-type and mutant strains, respectively. Each dot represents the log CFU/g from an individual animal. Bars represent the median CFU/g, and the limit of detection is 200 CFU. Significant differences in colonization levels (<i>P</i><0.05) are indicated and were determined using a two-tailed Wilcoxon matched pairs test.</p

Contribution of the TCA cycle and gluconeogenesis during UTI.

<p>Competitive indices (CI) were determined following co-challenge infections of female CBA/J mice with a 1∶1 ratio of either wild-type (A) <i>E. coli</i> CFT073 or (B) <i>P. mirabilis</i> HI4320 and their respective mutants in the following genes: <i>sdhB</i>, succinate dehydrogenase; <i>fumC</i>, fumarate hydratase; <i>frdA</i>, fumarate reductase; and <i>pckA</i>, phosphoenolpyruvate carboxykinase. <i>E. coli</i> was cultured from bladders and kidneys at 48 hpi. <i>P. mirabilis</i> was cultured from organs at 7 dpi. Each dot represents bladder (closed symbols) and kidneys (open symbols) from an individual animal. Bars indicate the median CI. Significant differences in colonization (*P<0.05) were determined by the Wilcoxon signed-rank test. A CI<1 indicates a fitness defect. Growth of (C, E) <i>E. coli</i> CFT073 and (D, F) <i>P. mirabilis</i> HI4320 wild-type and mutant strains in: <i>sdhB</i>, <i>fumC</i>, <i>frdA</i>, and <i>pckA</i> in LB medium (C, D) or defined medium containing 0.2% glucose (E, F) as the carbon source. A representative growth curve is shown for each panel.</p

Polymicrobial infection alters central metabolism requirements for <i>E. coli</i> and <i>P. mirabilis</i>.

<p>(A) Competitive indices (CI) were determined following co-challenge infections of female CBA/J mice with a 1∶1 ratio of wild-type: mutant bacteria for <i>gnd</i> (oxidative pentose phosphate pathway) and <i>pckA</i> (gluconeogenesis) for <i>P. mirabilis</i> at 48 hpi. Each dot represents bladder (closed symbols) and kidneys (open symbols) from an individual animal. Competitive indices (CI) were determined 48 hpi for mixed infections of (B) wild-type <i>E. coli</i> CFT073 and <i>P. mirabilis</i> HI4320 <i>gnd</i>, (C) wild-type HI4320 and CFT073 <i>gnd</i>, and (D) HI4320 <i>gnd</i> and CFT073 <i>gnd</i> mutant constructs. Each circle represents bladder or kidneys from an individual animal. In (A–D) bars indicate the median CI and significant differences in colonization (*) (P<0.05) were determined by Wilcoxon signed-rank test. (E) <i>In vivo</i> CI at 48 h and 7 d post-infection. (F) CI during logarithmic growth in LB medium. For (A–F) a CI<1 indicates a fitness defect. For mixed infections CFU/ml were determined following plating of serial dilutions on LB agar with and without tetracycline. CFU from tetracycline-containing plates (<i>P. mirabilis</i> are Tet^R) were subtracted from total CFU recovered on LB agar without antibiotics to determine CFU/ml for <i>E. coli</i> (Tet^S).</p