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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'ĂȘtre 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.


Role of braB CodY-binding sites in CodY binding.The braB156p+ (A) or braB144p+, braB144p2, and braB144p3 (B) DNA fragments obtained by PCR with oligonucleotides oBB67 and oBB102 and labelled on the template strand were incubated with increasing amounts of purified CodY in the presence of 10 mM ILV and then with DNase I. The protected areas are indicated by vertical lines. CodY concentrations used (nM of monomer) are indicated below each lane. The A + G sequencing ladder of the template DNA strand is shown in the flanking lanes.
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pgen.1005600.g003: Role of braB CodY-binding sites in CodY binding.The braB156p+ (A) or braB144p+, braB144p2, and braB144p3 (B) DNA fragments obtained by PCR with oligonucleotides oBB67 and oBB102 and labelled on the template strand were incubated with increasing amounts of purified CodY in the presence of 10 mM ILV and then with DNase I. The protected areas are indicated by vertical lines. CodY concentrations used (nM of monomer) are indicated below each lane. The A + G sequencing ladder of the template DNA strand is shown in the flanking lanes.

Mentions: Binding of CodY to upstream braB sites occurred independently of the presence of the downstream site and vice versa (Figs 1 and 3; see below for generation of the truncated fragments), similar to the case for other genes containing multiple CodY-binding sites within their regulatory regions [2, 25, 26].


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)

Role of braB CodY-binding sites in CodY binding.The braB156p+ (A) or braB144p+, braB144p2, and braB144p3 (B) DNA fragments obtained by PCR with oligonucleotides oBB67 and oBB102 and labelled on the template strand were incubated with increasing amounts of purified CodY in the presence of 10 mM ILV and then with DNase I. The protected areas are indicated by vertical lines. CodY concentrations used (nM of monomer) are indicated below each lane. The A + G sequencing ladder of the template DNA strand is shown in the flanking lanes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608796&req=5

pgen.1005600.g003: Role of braB CodY-binding sites in CodY binding.The braB156p+ (A) or braB144p+, braB144p2, and braB144p3 (B) DNA fragments obtained by PCR with oligonucleotides oBB67 and oBB102 and labelled on the template strand were incubated with increasing amounts of purified CodY in the presence of 10 mM ILV and then with DNase I. The protected areas are indicated by vertical lines. CodY concentrations used (nM of monomer) are indicated below each lane. The A + G sequencing ladder of the template DNA strand is shown in the flanking lanes.
Mentions: Binding of CodY to upstream braB sites occurred independently of the presence of the downstream site and vice versa (Figs 1 and 3; see below for generation of the truncated fragments), similar to the case for other genes containing multiple CodY-binding sites within their regulatory regions [2, 25, 26].

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'ĂȘtre 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.