Curation Information

Publication

Identification of CodY targets in Bacillus anthracis by genome-wide in vitro binding analysis.;Château A, van Schaik W, Joseph P, Handke LD, McBride SM, Smeets FM, Sonenshein AL, Fouet A;Journal of bacteriology 2013 Mar; 195(6):1204-13 [23292769]

TF

CodY [Q81WK7, view regulon]

Reported TF sp.

Bacillus subtilis subsp. subtilis str. SMY

Reported site sp.

Bacillus subtilis subsp. subtilis str. SMY

Created by

Elliot White

Curation notes

-

External databases

ArrayExpress (E-MEXP-1706) http://www.ebi.ac.uk/arrayexpress/arrays/E-MEXP-1706/.

Experimental Process

Experimenters ran 3 chip-seq experiments; sites that appeared in two of three were cross-referenced against a previously published micro-array to determine regulation.

ChIP assay conditions

Growth of the cultures was followed by measuring the absorbance at 600 nm (A600). B. anthracis precultures were grown overnight at 37°C in BHI (brain heart infusion) (Difco) medium supplemented with 0.5% glycerol, with rotary shaking at 150 rpm. Experimental cultures were grown at 37°C under a 5% CO2 atmosphere in R medium (27) supplemented with 0.6% sodium bicarbonate (RBic) (2). RBic was inoculated from an overnight culture to a starting A600 of 0.05. Aeration of the culture was achieved by continuous stirring with a magnetic bar at 450 rpm. For DNA extraction, the culture was grown at 37°C in BHI until it reached mid-exponential phase (A600 = 0.8). The antibiotics used were spectinomycin (100 μg/ml) and kanamycin (40 μg/ml).

ChIP notes

Genomic DNA was extracted from B. anthracis RTC10 as described in reference 31. Four samples of 10 μg of DNA each were dissolved in 500 μl of sterile deionized water and sheared by sonication. The method used was initially developed by Majerczyk et al. (25) and improved by Dineen et al. (24). In brief, adapters were ligated to the ends of the sheared DNA fragments and DNA fragments of 300 to 500 bp were purified. Amplification of the fragments was performed by PCR using primers that were complementary to the adapter sequences. The DNA fragments were incubated with purified His6-tagged B. anthracis CodY (50 or 100 nM) in the presence of the coeffectors, 5 mM GTP and 10 mM each isoleucine, leucine, and valine. A negative control consisted of carrying out the same experiment using purified Bacillus subtilis His10-aconitase (32) in place of B. anthracis CodY. Purification of CodY that was bound to its target genes was performed on a Talon Co2+ metal affinity resin (Clontech). The DNA fragments that copurified with the His-tagged proteins were sequenced bidirectionally by the Tufts University Nucleic Acid and Protein Core Facility using an Illumina genome analyzer II.

Transcription Factor Binding Sites

• Sites reported in the paper
• Sites matched in the genome
• High-throughput data

Gene Regulation

Regulated genes for each binding site are displayed below. Gene regulation diagrams show binding sites, positively-regulated genes, negatively-regulated genes, both positively and negatively regulated genes, genes with unspecified type of regulation. For each indvidual site, experimental techniques used to determine the site are also given.