Interaction of Bacillus subtilis CodY with GTP

Abstract

Many of the adaptive mechanisms that allow Bacillus subtilis to adjust to changes in nutrient availability are controlled by CodY. Binding of CodY to its target genes is stimulated by interaction with its effectors, GTP and the branched-chain amino acids (BCAAs). Upon nutrient limitation, intracellular pools of these effectors are depleted and CodY can no longer repress genes required for adaptation. In vitro studies reported here explored in more detail the interaction of CodY with GTP. DNase I footprinting experiments indicated that CodY has an affinity for GTP in the millimolar range. Further, CodY was shown to interact specifically with GTP and dGTP; no other naturally occurring nucleotides that were tested, including ppGpp and pppGpp, resulted in DNA protection. Two nonhydrolyzable analogs of GTP were fully able to activate CodY binding to target DNA, demonstrating that GTP hydrolysis is not necessary for CodY-dependent regulation. GTP and the BCAAs were shown to act additively to increase the affinity of CodY for DNA; increased protection was observed in DNase I footprinting experiments when both effectors were present, compared to either effector alone, and in in vitro transcription reactions, transcriptional repression by CodY was stronger in the presence of both GTP and BCAAs than of BCAAs alone. Thus, interaction of CodY with GTP is specific and results in increased affinity for its target genes. This increase in affinity is independent of GTP hydrolysis and is augmented in the presence of BCAAs.

FIG. 1.

(A) DNase I footprinting analysis of CodY binding to the ilvB promoter region. Lane 1, no CodY protein or GTP; lane 2, 125 nM CodY and no GTP; lanes 3 to 6, 125 nM CodY and increasing amounts of GTP. The bracket on the left shows the regions compared by densitometric analysis and corresponds to a portion of high-affinity region II of reference . The values under the lanes indicate the percent protected ilvB promoter DNA, compared to the leftmost lane, as assayed by densitometry. A sequencing ladder is shown at the right. (B) Plot of percent protection of ilvB promoter DNA versus GTP concentration in the presence of 125 nM CodY. We have assumed a linear relationship between GTP concentration and CodY binding but have not ruled out other potential kinetic relationships.

FIG. 2.

DNase I footprinting analysis of CodY binding to the ilvB promoter in the presence of GTP. Leftmost lane, no CodY, no GTP; other lanes, various concentrations of CodY (0.1 to 3.2 μM) with or without GTP (4 mM). The bracket on the left shows the regions compared by densitometric analysis and corresponds to a portion of high-affinity region II of reference . The values under the lanes indicate the percent protected ilvB promoter DNA, compared to the leftmost lane, as assayed by densitometry.

FIG. 3.

(A) DNase I footprinting analysis of ilvB promoter binding by CodY in the presence of various nucleotides. Lane 1, 125 nM CodY without nucleotides; other lanes, 125 nM CodY with 2, 5, or 10 mM nucleotides, as represented by the increasing size of the wedge. (B) DNase I footprinting analysis of ilvB promoter binding by CodY in the presence of various nucleotides. Lane 1, 125 nM CodY without nucleotides; other lanes, 125 nM CodY with 2, 5, or 10 mM nucleotides, as represented by the increasing size of the wedge. (C) DNase I footprinting analysis of ilvB promoter binding by CodY in the presence of various nucleotides. Lane 1, 150 nM CodY without nucleotides; other lanes, 150 nM CodY with 1, 2, or 4 mM nucleotides, as represented by the increasing size of the wedge.

FIG. 4.

DNase I footprinting analysis of ilvB promoter binding by CodY in the presence of nonhydrolyzable analogs of GTP. Lane 1, no CodY or nucleotides; lane 2, 125 nM CodY without effectors; other lanes, 125 nM CodY with 2, 5, or 10 mM nucleotides, as represented by the increasing size of the wedge.

FIG. 5.

DNase I footprinting analysis of the additive effects of GTP and isoleucine on CodY binding to the ilvB promoter. Lane 1, no CodY or effectors; lane 2, 20 nM CodY without effectors; other lanes, 20 nM CodY and effectors as indicated. When the GTP concentration was varied, it was at 0.5, 1, 2, or 4 mM, as represented by the increasing size of the wedge. When the isoleucine concentration was varied, it was at 2.5, 5, or 10 mM, as represented by the increasing size of the wedge. The bracket on the left shows the regions compared by densitometric analysis and corresponds to a portion of high-affinity region II of reference . The values under the lanes indicate the percent protected ilvB promoter DNA, compared to the leftmost lane, as assayed by densitometry.

FIG. 6.

DNase I footprinting analysis of the additive effects of GTP and BCAAs on CodY binding to the ilvB promoter. First set of lanes, various CodY concentrations (0, 50, 100, 300, and 500 nM) in the absence of effectors (−); second set of lanes, various CodY concentrations in the presence of 2 mM GTP (+G); third set of lanes, various CodY concentrations in the presence of 10 mM BCAAs (+B); fourth set of lanes, various CodY concentrations in the presence of 2 mM GTP and 10 mM BCAAs (+G +B). A sequencing ladder is shown at the right.

FIG. 7.

In vitro transcription analysis of ilvB expression in the presence of either BCAAs alone or BCAAs and GTP as effectors. In vitro transcription reactions were performed with 200 nM CodY when indicated and a 453-bp fragment of the ilvB promoter. Various concentrations of BCAAs were added as indicated, in the presence or absence of 2 mM GTP. Each tube additionally contained 150 μM GTP as part of the transcription reaction mixture. A promoter known not to be CodY regulated, veg, was used as a control in these assays.