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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.


Determination of the braB transcription start point and CodY-binding regions.A. Primer extension analysis of the braB mRNA. Primer oBB102 annealing to the lacZ gene of the braB242-lacZ fusion was extended with reverse transcriptase using as the template total RNA from fusion-containing strains BB3076 (wt) and BB3079 (codY) grown in TSS + 16 aa medium. The A+G sequence of the template strand of pBB1593 determined from reactions primed with oBB102 is shown to the left. The apparent transcription start site of the braB gene is in bold and marked by the +1 notation. B. DNase I footprinting analysis of CodY binding to the braB regulatory region. The braB242p+ DNA fragment obtained by PCR with oligonucleotides oBB67 and oBB102 and labelled on the template strand was incubated with increasing amounts of purified CodY in the presence of 10 mM ILV and 2 mM GTP and then with DNase I. The protected areas are indicated by vertical lines and the corresponding sequences are reported; the protected nucleotides are italicized. The apparent transcription start site and direction of transcription are shown by a bent arrow. 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. C. Same as B, the gel was run longer to improve the resolution of the upstream CodY-binding sites III and I.
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pgen.1005600.g002: Determination of the braB transcription start point and CodY-binding regions.A. Primer extension analysis of the braB mRNA. Primer oBB102 annealing to the lacZ gene of the braB242-lacZ fusion was extended with reverse transcriptase using as the template total RNA from fusion-containing strains BB3076 (wt) and BB3079 (codY) grown in TSS + 16 aa medium. The A+G sequence of the template strand of pBB1593 determined from reactions primed with oBB102 is shown to the left. The apparent transcription start site of the braB gene is in bold and marked by the +1 notation. B. DNase I footprinting analysis of CodY binding to the braB regulatory region. The braB242p+ DNA fragment obtained by PCR with oligonucleotides oBB67 and oBB102 and labelled on the template strand was incubated with increasing amounts of purified CodY in the presence of 10 mM ILV and 2 mM GTP and then with DNase I. The protected areas are indicated by vertical lines and the corresponding sequences are reported; the protected nucleotides are italicized. The apparent transcription start site and direction of transcription are shown by a bent arrow. 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. C. Same as B, the gel was run longer to improve the resolution of the upstream CodY-binding sites III and I.

Mentions: DNase I footprinting experiments showed that CodY protected two sites, I and II, within the 194-bp iscSB-braB intergenic region from positions -62 to -47 and +11 to +50 of the template DNA strand with respect to the braB transcription start point, respectively (Figs 1A, 2B and 2C). Site II is much stronger than site I. Binding to an additional very weak site, III, from positions –143 to –124, which is within the upstream iscSB gene, was observed only at high concentrations of CodY (≥200 nM) (Fig 2B and 2C).


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)

Determination of the braB transcription start point and CodY-binding regions.A. Primer extension analysis of the braB mRNA. Primer oBB102 annealing to the lacZ gene of the braB242-lacZ fusion was extended with reverse transcriptase using as the template total RNA from fusion-containing strains BB3076 (wt) and BB3079 (codY) grown in TSS + 16 aa medium. The A+G sequence of the template strand of pBB1593 determined from reactions primed with oBB102 is shown to the left. The apparent transcription start site of the braB gene is in bold and marked by the +1 notation. B. DNase I footprinting analysis of CodY binding to the braB regulatory region. The braB242p+ DNA fragment obtained by PCR with oligonucleotides oBB67 and oBB102 and labelled on the template strand was incubated with increasing amounts of purified CodY in the presence of 10 mM ILV and 2 mM GTP and then with DNase I. The protected areas are indicated by vertical lines and the corresponding sequences are reported; the protected nucleotides are italicized. The apparent transcription start site and direction of transcription are shown by a bent arrow. 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. C. Same as B, the gel was run longer to improve the resolution of the upstream CodY-binding sites III and I.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005600.g002: Determination of the braB transcription start point and CodY-binding regions.A. Primer extension analysis of the braB mRNA. Primer oBB102 annealing to the lacZ gene of the braB242-lacZ fusion was extended with reverse transcriptase using as the template total RNA from fusion-containing strains BB3076 (wt) and BB3079 (codY) grown in TSS + 16 aa medium. The A+G sequence of the template strand of pBB1593 determined from reactions primed with oBB102 is shown to the left. The apparent transcription start site of the braB gene is in bold and marked by the +1 notation. B. DNase I footprinting analysis of CodY binding to the braB regulatory region. The braB242p+ DNA fragment obtained by PCR with oligonucleotides oBB67 and oBB102 and labelled on the template strand was incubated with increasing amounts of purified CodY in the presence of 10 mM ILV and 2 mM GTP and then with DNase I. The protected areas are indicated by vertical lines and the corresponding sequences are reported; the protected nucleotides are italicized. The apparent transcription start site and direction of transcription are shown by a bent arrow. 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. C. Same as B, the gel was run longer to improve the resolution of the upstream CodY-binding sites III and I.
Mentions: DNase I footprinting experiments showed that CodY protected two sites, I and II, within the 194-bp iscSB-braB intergenic region from positions -62 to -47 and +11 to +50 of the template DNA strand with respect to the braB transcription start point, respectively (Figs 1A, 2B and 2C). Site II is much stronger than site I. Binding to an additional very weak site, III, from positions –143 to –124, which is within the upstream iscSB gene, was observed only at high concentrations of CodY (≥200 nM) (Fig 2B and 2C).

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.