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. 2003 Mar;185(6):1911-22.
doi: 10.1128/jb.185.6.1911-1922.2003.

Additional Targets of the Bacillus Subtilis Global Regulator CodY Identified by Chromatin Immunoprecipitation and Genome-Wide Transcript Analysis

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Free PMC article

Additional Targets of the Bacillus Subtilis Global Regulator CodY Identified by Chromatin Immunoprecipitation and Genome-Wide Transcript Analysis

Virginie Molle et al. J Bacteriol. .
Free PMC article

Abstract

Additional targets of CodY, a GTP-activated repressor of early stationary-phase genes in Bacillus subtilis, were identified by combining chromatin immunoprecipitation, DNA microarray hybridization, and gel mobility shift assays. The direct targets of CodY newly identified by this approach included regulatory genes for sporulation, genes that are likely to encode transporters for amino acids and sugars, and the genes for biosynthesis of branched-chain amino acids.

Figures

FIG. 1.
FIG. 1.
Expression of lacZ fusions to promoters of putative CodY target genes in codY+ and ΔcodY strains. Strains were grown in minimal glucose-glutamine medium containing a mixture of 16 additional amino acids (see Materials and Methods), and samples were removed at indicated times after inoculation for assays of β-galactosidase activity. Isogenic codY+ and ΔcodY strains carrying each gene disruption were FU384 and FU385 for ilvB, FU386 and FU387 for ilvD, FU388 and FU389 for ybgE, FU390 and FU391 for yufN, FU392 and FU393 for yufO, FU394 and FU395 for yurP, FU396 and FU397 for yurN, FU398 and FU399 for ykfA, and FU400 and FU401 for yhdG. Circles and squares denote codY+ and ΔcodY strains, respectively, whereas open and closed symbols represent the OD600 and β-galactosidase activity, respectively.
FIG. 2.
FIG. 2.
Expression of a yufN-lacZ fusion integrated at the amyE locus. Strains FU407 (codY+) and FU408 (ΔcodY) were grown in minimal glucose-glutamine medium containing 16 additional amino acids (see Materials and Methods). Samples removed at the indicated times after inoculation of the culture were assayed for β-galactosidase activity. Circles and squares denote codY+ and ΔcodY strains, respectively, whereas open and closed symbols represent the OD600 and β-galactosidase activity, respectively.
FIG. 3.
FIG. 3.
Gel mobility shift analysis of CodY binding to putative target regulatory regions. The regulatory regions of potential target genes were amplified by PCR by using radioactive primers, incubated with purified CodY-His6 protein at various concentrations, and analyzed by nondenaturing polyacrylamide gel electrophoresis (see Materials and Methods for details). The lengths of the PCR products in base pairs were as follows: ilvB, 453; ilvD, 525; ybgE, 446; yhdG, 345; yufN, 492; yufO, 199; ykfA, 340; and yurP, 321. In each panel, the position of unshifted DNA is seen in the leftmost lane, which contained no CodY. Where indicated, GTP was included in the reaction mixture at a concentration of 2 mM.

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