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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.


The sequence of the braB regulatory region and map of the promoter fragments used.A. The sequence (5’ to 3’) of the coding (non-template) strand of the braB regulatory region within the braB242-lacZ fusion. Coordinates are reported with respect to the transcription start point. The upstream boundary of the braB184, braB162, and braB144 fusions at positions –87, -65, and -47, respectively, are indicated by vertical arrows above the sequence. The vertical arrows below the sequence indicate the junction points, at position +11 and +36, between the braB and lacZ sequences. The likely translation initiation codon, -10 and -35 promoter regions, and apparent transcription start point are in boldface. The directions of transcription and translation are indicated by the horizontal arrows. The sequences on the template strand that were protected by CodY or ScoC in DNase I footprinting experiments are underlined or shown by dotted horizontal lines below the sequence, respectively. The sequences of CodY-binding motifs are italicized and shown in Table 1. The mutated nucleotides are shown in lowercase above the sequence. B. Schematic maps of the braB fragments used to construct lacZ fusions or in DNA-binding experiments. The coordinates indicate the boundaries of different fusions with respect to the braB transcription start point. The location of the apparent transcription start point is indicated by the bent arrow. CodY- and ScoC-binding sites determined in DNase I footprinting experiments are shown as clear or shaded rectangles, respectively.
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pgen.1005600.g001: The sequence of the braB regulatory region and map of the promoter fragments used.A. The sequence (5’ to 3’) of the coding (non-template) strand of the braB regulatory region within the braB242-lacZ fusion. Coordinates are reported with respect to the transcription start point. The upstream boundary of the braB184, braB162, and braB144 fusions at positions –87, -65, and -47, respectively, are indicated by vertical arrows above the sequence. The vertical arrows below the sequence indicate the junction points, at position +11 and +36, between the braB and lacZ sequences. The likely translation initiation codon, -10 and -35 promoter regions, and apparent transcription start point are in boldface. The directions of transcription and translation are indicated by the horizontal arrows. The sequences on the template strand that were protected by CodY or ScoC in DNase I footprinting experiments are underlined or shown by dotted horizontal lines below the sequence, respectively. The sequences of CodY-binding motifs are italicized and shown in Table 1. The mutated nucleotides are shown in lowercase above the sequence. B. Schematic maps of the braB fragments used to construct lacZ fusions or in DNA-binding experiments. The coordinates indicate the boundaries of different fusions with respect to the braB transcription start point. The location of the apparent transcription start point is indicated by the bent arrow. CodY- and ScoC-binding sites determined in DNase I footprinting experiments are shown as clear or shaded rectangles, respectively.

Mentions: The unexpected absence of an effect of a codY mutation on expression of a gene with a strong CodY-binding site in its putative regulatory region led us to analyze braB transcription in more detail. A primer extension experiment established that the 5’ end of the braB mRNA is located 72 bp upstream of the initiation codon. The sequences TTGACT and TATAAT, with one and no mismatches to the –35 and –10 regions of σA-dependent promoters, respectively, and a 16-bp spacer region, can be identified upstream of the 5’ end location, suggesting that this position does in fact correspond to the transcription start point (Fig 1A). (Since B. subtilis σA-dependent promoters rarely have a 16-bp spacer, our assignment of the -10 and -35 regions may be off by 1 or 2 bp.) A mutation, T(-29)C, located immediately downstream of the likely -35 region, reduced expression of a braB-lacZ fusion 6-fold (1.97±0.35 Miller units, see below), consistent with our assignment of the promoter.


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)

The sequence of the braB regulatory region and map of the promoter fragments used.A. The sequence (5’ to 3’) of the coding (non-template) strand of the braB regulatory region within the braB242-lacZ fusion. Coordinates are reported with respect to the transcription start point. The upstream boundary of the braB184, braB162, and braB144 fusions at positions –87, -65, and -47, respectively, are indicated by vertical arrows above the sequence. The vertical arrows below the sequence indicate the junction points, at position +11 and +36, between the braB and lacZ sequences. The likely translation initiation codon, -10 and -35 promoter regions, and apparent transcription start point are in boldface. The directions of transcription and translation are indicated by the horizontal arrows. The sequences on the template strand that were protected by CodY or ScoC in DNase I footprinting experiments are underlined or shown by dotted horizontal lines below the sequence, respectively. The sequences of CodY-binding motifs are italicized and shown in Table 1. The mutated nucleotides are shown in lowercase above the sequence. B. Schematic maps of the braB fragments used to construct lacZ fusions or in DNA-binding experiments. The coordinates indicate the boundaries of different fusions with respect to the braB transcription start point. The location of the apparent transcription start point is indicated by the bent arrow. CodY- and ScoC-binding sites determined in DNase I footprinting experiments are shown as clear or shaded rectangles, respectively.
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Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4608796&req=5

pgen.1005600.g001: The sequence of the braB regulatory region and map of the promoter fragments used.A. The sequence (5’ to 3’) of the coding (non-template) strand of the braB regulatory region within the braB242-lacZ fusion. Coordinates are reported with respect to the transcription start point. The upstream boundary of the braB184, braB162, and braB144 fusions at positions –87, -65, and -47, respectively, are indicated by vertical arrows above the sequence. The vertical arrows below the sequence indicate the junction points, at position +11 and +36, between the braB and lacZ sequences. The likely translation initiation codon, -10 and -35 promoter regions, and apparent transcription start point are in boldface. The directions of transcription and translation are indicated by the horizontal arrows. The sequences on the template strand that were protected by CodY or ScoC in DNase I footprinting experiments are underlined or shown by dotted horizontal lines below the sequence, respectively. The sequences of CodY-binding motifs are italicized and shown in Table 1. The mutated nucleotides are shown in lowercase above the sequence. B. Schematic maps of the braB fragments used to construct lacZ fusions or in DNA-binding experiments. The coordinates indicate the boundaries of different fusions with respect to the braB transcription start point. The location of the apparent transcription start point is indicated by the bent arrow. CodY- and ScoC-binding sites determined in DNase I footprinting experiments are shown as clear or shaded rectangles, respectively.
Mentions: The unexpected absence of an effect of a codY mutation on expression of a gene with a strong CodY-binding site in its putative regulatory region led us to analyze braB transcription in more detail. A primer extension experiment established that the 5’ end of the braB mRNA is located 72 bp upstream of the initiation codon. The sequences TTGACT and TATAAT, with one and no mismatches to the –35 and –10 regions of σA-dependent promoters, respectively, and a 16-bp spacer region, can be identified upstream of the 5’ end location, suggesting that this position does in fact correspond to the transcription start point (Fig 1A). (Since B. subtilis σA-dependent promoters rarely have a 16-bp spacer, our assignment of the -10 and -35 regions may be off by 1 or 2 bp.) A mutation, T(-29)C, located immediately downstream of the likely -35 region, reduced expression of a braB-lacZ fusion 6-fold (1.97±0.35 Miller units, see below), consistent with our assignment of the promoter.

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.