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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.


Expression of braB, bcaP, or scoC in mutants containing partially active versions of CodY.A and B. braB transcript abundance as determined by RNA-Seq (A) or quantitative, real-time RT-PCR (B). Cells were grown in TSS + 16 aa medium. The RNA-Seq values, expressed as reads per kilobase per million ORF (RPKMO) values, were taken from the Dataset S1 of Ref. [8]. The real-time RT-PCR results are expressed as braB transcript abundance relative to rpoC transcript abundance. Point mutant positions along the x-axis are arbitrary and do not imply a linear relationship. Data points are the means of at least two independent experiments, and the error bars show standard errors of the mean. C—G. Expression of braB-lacZ, bcaP-lacZ, and scoC-lacZ fusions in scoC+ or scoC  mutant cells containing partially active versions of CodY. Cells were grown in TSS + 16 aa medium. β-Galactosidase activity was assayed and expressed in Miller units.
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pgen.1005600.g005: Expression of braB, bcaP, or scoC in mutants containing partially active versions of CodY.A and B. braB transcript abundance as determined by RNA-Seq (A) or quantitative, real-time RT-PCR (B). Cells were grown in TSS + 16 aa medium. The RNA-Seq values, expressed as reads per kilobase per million ORF (RPKMO) values, were taken from the Dataset S1 of Ref. [8]. The real-time RT-PCR results are expressed as braB transcript abundance relative to rpoC transcript abundance. Point mutant positions along the x-axis are arbitrary and do not imply a linear relationship. Data points are the means of at least two independent experiments, and the error bars show standard errors of the mean. C—G. Expression of braB-lacZ, bcaP-lacZ, and scoC-lacZ fusions in scoC+ or scoC mutant cells containing partially active versions of CodY. Cells were grown in TSS + 16 aa medium. β-Galactosidase activity was assayed and expressed in Miller units.

Mentions: The analysis of Dataset S1 of Ref. (8) indicates that expression of braB determined by RNA-Seq experiments was up to 4.1-fold higher in three strains containing partially active versions of CodY, F71Y, R61K, or R61H (Fig 5A). The results of the RNA-Seq experiments were confirmed and extended by real-time RT-PCR and by analyzing expression of the braB242-lacZ transcriptional fusion in a larger collection of partial codY mutants (Fig 5B and 5C). The up-and-down expression pattern of the braB fusion, in which maximal activity was seen in mutants with intermediate levels of CodY activity, was in drastic contrast to the plateau-reaching expression pattern of the previously characterized CodY-repressed bcaP283-lacZ fusion (Fig 5F) [2] and all other CodY-regulated genes [2].


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)

Expression of braB, bcaP, or scoC in mutants containing partially active versions of CodY.A and B. braB transcript abundance as determined by RNA-Seq (A) or quantitative, real-time RT-PCR (B). Cells were grown in TSS + 16 aa medium. The RNA-Seq values, expressed as reads per kilobase per million ORF (RPKMO) values, were taken from the Dataset S1 of Ref. [8]. The real-time RT-PCR results are expressed as braB transcript abundance relative to rpoC transcript abundance. Point mutant positions along the x-axis are arbitrary and do not imply a linear relationship. Data points are the means of at least two independent experiments, and the error bars show standard errors of the mean. C—G. Expression of braB-lacZ, bcaP-lacZ, and scoC-lacZ fusions in scoC+ or scoC  mutant cells containing partially active versions of CodY. Cells were grown in TSS + 16 aa medium. β-Galactosidase activity was assayed and expressed in Miller units.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005600.g005: Expression of braB, bcaP, or scoC in mutants containing partially active versions of CodY.A and B. braB transcript abundance as determined by RNA-Seq (A) or quantitative, real-time RT-PCR (B). Cells were grown in TSS + 16 aa medium. The RNA-Seq values, expressed as reads per kilobase per million ORF (RPKMO) values, were taken from the Dataset S1 of Ref. [8]. The real-time RT-PCR results are expressed as braB transcript abundance relative to rpoC transcript abundance. Point mutant positions along the x-axis are arbitrary and do not imply a linear relationship. Data points are the means of at least two independent experiments, and the error bars show standard errors of the mean. C—G. Expression of braB-lacZ, bcaP-lacZ, and scoC-lacZ fusions in scoC+ or scoC mutant cells containing partially active versions of CodY. Cells were grown in TSS + 16 aa medium. β-Galactosidase activity was assayed and expressed in Miller units.
Mentions: The analysis of Dataset S1 of Ref. (8) indicates that expression of braB determined by RNA-Seq experiments was up to 4.1-fold higher in three strains containing partially active versions of CodY, F71Y, R61K, or R61H (Fig 5A). The results of the RNA-Seq experiments were confirmed and extended by real-time RT-PCR and by analyzing expression of the braB242-lacZ transcriptional fusion in a larger collection of partial codY mutants (Fig 5B and 5C). The up-and-down expression pattern of the braB fusion, in which maximal activity was seen in mutants with intermediate levels of CodY activity, was in drastic contrast to the plateau-reaching expression pattern of the previously characterized CodY-repressed bcaP283-lacZ fusion (Fig 5F) [2] and all other CodY-regulated genes [2].

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.