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 null 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 null 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.
Highlights
BraB is one of three permeases demonstrated to be involved in the uptake of branched-chain amino acids (BCAA) in Bacillus subtilis [1]
The unexpected absence of an effect of a codY null 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
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 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
Summary
BraB is one of three permeases demonstrated to be involved in the uptake of branched-chain amino acids (BCAA) in Bacillus subtilis [1]. The most efficient BCAA permease, BcaP, is subject to very strong transcriptional repression by CodY [2], a global regulator in B. subtilis and other Gram-positive bacteria [3, 4]. A strong CodY-binding site in the iscSB-braB intergenic region was detected in vitro during the global characterization of CodY-binding sites by IDAP-Seq [7]. The latter site is well-placed to serve as a potential site of regulation of braB. Transcription of neither braB nor iscSB was altered >2.0-fold by a null mutation in codY, as detected in DNA microarray or RNA-Seq experiments [6, 8](http://www.genome.jp/kegg/ expression/)
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