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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.


Determination of braB ScoC-binding regions.DNase I footprinting analysis of ScoC binding to the braB regulatory region. The braB242p+ or braB162p+ DNA fragment obtained by PCR with oligonucleotides oBB67 and oBB102 and labelled on the template strand was incubated with increasing amounts of purified ScoC and then with DNase I. See the legend to Fig 2B for additional details.
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pgen.1005600.g007: Determination of braB ScoC-binding regions.DNase I footprinting analysis of ScoC binding to the braB regulatory region. The braB242p+ or braB162p+ DNA fragment obtained by PCR with oligonucleotides oBB67 and oBB102 and labelled on the template strand was incubated with increasing amounts of purified ScoC and then with DNase I. See the legend to Fig 2B for additional details.

Mentions: In DNase I footprinting experiments, ScoC protected two sites, I and II, within the iscSB-braB intergenic region from positions -79 to -68 and +43 to +57 of the template DNA strand with respect to the braB transcription start point, respectively (Figs 1A and 7). A short, weakly protected region, site III (possibly a part of site II), was also detected from positions +16 to +20. Binding of ScoC to the downstream sites II and III was independent of the presence of the upstream site I on the same DNA fragment (Fig 7).


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 braB ScoC-binding regions.DNase I footprinting analysis of ScoC binding to the braB regulatory region. The braB242p+ or braB162p+ DNA fragment obtained by PCR with oligonucleotides oBB67 and oBB102 and labelled on the template strand was incubated with increasing amounts of purified ScoC and then with DNase I. See the legend to Fig 2B for additional details.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005600.g007: Determination of braB ScoC-binding regions.DNase I footprinting analysis of ScoC binding to the braB regulatory region. The braB242p+ or braB162p+ DNA fragment obtained by PCR with oligonucleotides oBB67 and oBB102 and labelled on the template strand was incubated with increasing amounts of purified ScoC and then with DNase I. See the legend to Fig 2B for additional details.
Mentions: In DNase I footprinting experiments, ScoC protected two sites, I and II, within the iscSB-braB intergenic region from positions -79 to -68 and +43 to +57 of the template DNA strand with respect to the braB transcription start point, respectively (Figs 1A and 7). A short, weakly protected region, site III (possibly a part of site II), was also detected from positions +16 to +20. Binding of ScoC to the downstream sites II and III was independent of the presence of the upstream site I on the same DNA fragment (Fig 7).

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.