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Intermediate Levels of Bacillus subtilis CodY Activity Are Required for Derepression of the Branched-Chain Amino Acid Permease, BraB.

Belitsky BR, Brinsmade SR, Sonenshein AL - PLoS Genet. (2015)

Bottom Line: However, under conditions of reduced CodY activity, CodY-mediated repression was relieved to a greater extent than ScoC-mediated repression was increased, leading to elevated braB expression.We conclude that restricting increased expression of braB to conditions of moderate nutrient limitation is the raison d'être of the feed-forward regulatory loop formed by CodY and ScoC at the braB promoter.The increase in BraB expression only at intermediate activities of CodY may facilitate the uptake of BCAA when they are not in excess but prevent unneeded BraB synthesis when other BCAA transporters are active.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
The global transcriptional regulator, CodY, binds strongly to the regulatory region of the braB gene, which encodes a Bacillus subtilis branched-chain amino acid (BCAA) permease. However, under conditions that maximize CodY activity, braB expression was similar in wild-type and codY mutant cells. Nonetheless, expression from the braB promoter was significantly elevated in cells containing partially active mutant versions of CodY or in wild-type cells under growth conditions leading to intermediate levels of CodY activity. This novel pattern of regulation was shown to be due to two opposing mechanisms, negative and positive, by which CodY affects braB expression. A strong CodY-binding site located downstream of the transcription start point conferred negative regulation by direct interaction with CodY. Additionally, sequences upstream and downstream of the promoter were required for repression by a second pleiotropic B. subtilis regulator, ScoC, whose own expression is repressed by CodY. ScoC-mediated repression of braB in codY mutants cells was as efficient as direct, CodY-mediated repression in wild-type cells under conditions of high CodY activity. However, under conditions of reduced CodY activity, CodY-mediated repression was relieved to a greater extent than ScoC-mediated repression was increased, leading to elevated braB expression. We conclude that restricting increased expression of braB to conditions of moderate nutrient limitation is the raison d'être of the feed-forward regulatory loop formed by CodY and ScoC at the braB promoter. The increase in BraB expression only at intermediate activities of CodY may facilitate the uptake of BCAA when they are not in excess but prevent unneeded BraB synthesis when other BCAA transporters are active.

No MeSH data available.


Gel shift assays of CodY binding to braB fragments.The braB242p+ (A), braB156p+ (B), braB156p1 (C), braB144p+ (D), braB144p3 (E), braB144p2 (F), and braB144p2/3 (G) DNA fragments labeled on the template strand were incubated with increasing amounts of purified CodY in the presence of 10 mM ILV. The fragments were obtained by PCR with oligonucleotides oBB67 and oBB102 (A), oBB358 and oBB102 (B, C), and oBB422 and oBB102 (D-G) and their positions in the gel are indicated by right-pointing arrows. The unspecific DNA fragment, present in some panels, is indicated by a left-pointing arrow. CodY concentrations used (nM of monomer) are reported below each lane; concentrations corresponding to the apparent KD for binding are underlined.
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pgen.1005600.g004: Gel shift assays of CodY binding to braB fragments.The braB242p+ (A), braB156p+ (B), braB156p1 (C), braB144p+ (D), braB144p3 (E), braB144p2 (F), and braB144p2/3 (G) DNA fragments labeled on the template strand were incubated with increasing amounts of purified CodY in the presence of 10 mM ILV. The fragments were obtained by PCR with oligonucleotides oBB67 and oBB102 (A), oBB358 and oBB102 (B, C), and oBB422 and oBB102 (D-G) and their positions in the gel are indicated by right-pointing arrows. The unspecific DNA fragment, present in some panels, is indicated by a left-pointing arrow. CodY concentrations used (nM of monomer) are reported below each lane; concentrations corresponding to the apparent KD for binding are underlined.

Mentions: In gel-shift experiments, CodY bound to DNA fragments containing only sites III and I (braB156) or only site II (braB144) with apparent dissociation constants (KD) of ∼75 nM and ∼4 nM, respectively, compared with ∼3 nM for the full-length fragment, braB242 (Fig 4A, 4B and 4D) (KD reflects the CodY concentration needed to shift 50% of DNA fragments under conditions of vast CodY excess over DNA). Complexes with lower mobility were formed at higher concentrations of CodY for all fragments, indicating apparent changes in stoichiometry of CodY binding (Fig 4).


Intermediate Levels of Bacillus subtilis CodY Activity Are Required for Derepression of the Branched-Chain Amino Acid Permease, BraB.

Belitsky BR, Brinsmade SR, Sonenshein AL - PLoS Genet. (2015)

Gel shift assays of CodY binding to braB fragments.The braB242p+ (A), braB156p+ (B), braB156p1 (C), braB144p+ (D), braB144p3 (E), braB144p2 (F), and braB144p2/3 (G) DNA fragments labeled on the template strand were incubated with increasing amounts of purified CodY in the presence of 10 mM ILV. The fragments were obtained by PCR with oligonucleotides oBB67 and oBB102 (A), oBB358 and oBB102 (B, C), and oBB422 and oBB102 (D-G) and their positions in the gel are indicated by right-pointing arrows. The unspecific DNA fragment, present in some panels, is indicated by a left-pointing arrow. CodY concentrations used (nM of monomer) are reported below each lane; concentrations corresponding to the apparent KD for binding are underlined.
© Copyright Policy
Related In: Results  -  Collection

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pgen.1005600.g004: Gel shift assays of CodY binding to braB fragments.The braB242p+ (A), braB156p+ (B), braB156p1 (C), braB144p+ (D), braB144p3 (E), braB144p2 (F), and braB144p2/3 (G) DNA fragments labeled on the template strand were incubated with increasing amounts of purified CodY in the presence of 10 mM ILV. The fragments were obtained by PCR with oligonucleotides oBB67 and oBB102 (A), oBB358 and oBB102 (B, C), and oBB422 and oBB102 (D-G) and their positions in the gel are indicated by right-pointing arrows. The unspecific DNA fragment, present in some panels, is indicated by a left-pointing arrow. CodY concentrations used (nM of monomer) are reported below each lane; concentrations corresponding to the apparent KD for binding are underlined.
Mentions: In gel-shift experiments, CodY bound to DNA fragments containing only sites III and I (braB156) or only site II (braB144) with apparent dissociation constants (KD) of ∼75 nM and ∼4 nM, respectively, compared with ∼3 nM for the full-length fragment, braB242 (Fig 4A, 4B and 4D) (KD reflects the CodY concentration needed to shift 50% of DNA fragments under conditions of vast CodY excess over DNA). Complexes with lower mobility were formed at higher concentrations of CodY for all fragments, indicating apparent changes in stoichiometry of CodY binding (Fig 4).

Bottom Line: However, under conditions of reduced CodY activity, CodY-mediated repression was relieved to a greater extent than ScoC-mediated repression was increased, leading to elevated braB expression.We conclude that restricting increased expression of braB to conditions of moderate nutrient limitation is the raison d'être of the feed-forward regulatory loop formed by CodY and ScoC at the braB promoter.The increase in BraB expression only at intermediate activities of CodY may facilitate the uptake of BCAA when they are not in excess but prevent unneeded BraB synthesis when other BCAA transporters are active.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
The global transcriptional regulator, CodY, binds strongly to the regulatory region of the braB gene, which encodes a Bacillus subtilis branched-chain amino acid (BCAA) permease. However, under conditions that maximize CodY activity, braB expression was similar in wild-type and codY mutant cells. Nonetheless, expression from the braB promoter was significantly elevated in cells containing partially active mutant versions of CodY or in wild-type cells under growth conditions leading to intermediate levels of CodY activity. This novel pattern of regulation was shown to be due to two opposing mechanisms, negative and positive, by which CodY affects braB expression. A strong CodY-binding site located downstream of the transcription start point conferred negative regulation by direct interaction with CodY. Additionally, sequences upstream and downstream of the promoter were required for repression by a second pleiotropic B. subtilis regulator, ScoC, whose own expression is repressed by CodY. ScoC-mediated repression of braB in codY mutants cells was as efficient as direct, CodY-mediated repression in wild-type cells under conditions of high CodY activity. However, under conditions of reduced CodY activity, CodY-mediated repression was relieved to a greater extent than ScoC-mediated repression was increased, leading to elevated braB expression. We conclude that restricting increased expression of braB to conditions of moderate nutrient limitation is the raison d'être of the feed-forward regulatory loop formed by CodY and ScoC at the braB promoter. The increase in BraB expression only at intermediate activities of CodY may facilitate the uptake of BCAA when they are not in excess but prevent unneeded BraB synthesis when other BCAA transporters are active.

No MeSH data available.