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Published on: December 6, 2017
Identification of CodY targets in Bacillus anthracis by genome-wide in vitro binding analysis
A Château1, W van Schaik, P Joseph
1Unité Toxines et Pathogénie Bactérienne, Département de Microbiologie, Institut Pasteur, Paris, France.
Abstract:
In Gram-positive bacteria, CodY is an important regulator of genes whose expression changes under conditions of nutrient limitation. Bacillus anthracis CodY represses or activates directly or indirectly approximately 500 genes. Affinity purification of CodY-DNA complexes was used to identify the direct targets of CodY. Of the 389 DNA binding sites that were copurified with CodY, 132 sites were in or near the regulatory regions governing the expression of 197 CodY-controlled genes, indicating that CodY controls many other genes indirectly. CodY-binding specificity was verified using electrophoretic mobility shift and DNase I footprinting assays for three CodY targets. Analysis of the bound sequences led to the identification of a B. anthracis CodY-binding consensus motif that was found in 366 of the 389 affinity-purified DNA regions. Regulation of the expression of the two genes directly controlled by CodY, sap and eag, encoding the two surface layer (S-layer) proteins, was analyzed further by monitoring the expression of transcriptional lacZ reporter fusions in parental and codY mutant strains. CodY proved to be a direct repressor of both sap and eag expression. Since the expression of the S-layer genes is under the control of both CodY and PagR (a regulator that responds to bicarbonate), their expression levels respond to both metabolic and environmental cues.
Insights
Bacillus anthracis CodY regulates nearly 500 genes, controlling nutrient limitation responses. This study identified direct CodY DNA targets and a consensus binding motif, revealing CodY
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Genetics
Background:
- CodY is a key transcriptional regulator in Gram-positive bacteria, responding to nutrient availability.
- Bacillus anthracis CodY influences the expression of approximately 500 genes, impacting various cellular processes.
- Understanding CodY's regulatory network is crucial for deciphering bacterial adaptation mechanisms.
Purpose of the Study:
- To identify the direct DNA targets of Bacillus anthracis CodY.
- To elucidate the CodY-binding specificity and consensus motif.
- To analyze the direct regulation of surface layer protein genes (sap and eag) by CodY.
Main Methods:
- Affinity purification of CodY-DNA complexes to identify binding sites.
- Electrophoretic mobility shift assays (EMSA) and DNase I footprinting to confirm binding specificity.
- Analysis of transcriptional lacZ reporter fusions in wild-type and codY mutant strains.
Main Results:
- Identified 389 CodY DNA binding sites, with 132 sites near regulatory regions of 197 genes.
- Determined a Bacillus anthracis CodY-binding consensus motif present in most purified DNA regions.
- Confirmed CodY as a direct repressor of sap and eag gene expression, encoding surface layer proteins.
Conclusions:
- CodY directly binds to specific DNA sequences to regulate gene expression in Bacillus anthracis.
- CodY controls a large regulon, including many genes indirectly, highlighting its broad regulatory role.
- The expression of surface layer proteins is co-regulated by CodY and PagR, integrating metabolic and environmental signals.
