Intermediate Levels of Bacillus subtilis CodY Activity Are Required for Derepression of the Branched-Chain Amino Acid Permease, BraB

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.

Fig 1. 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.

Fig 2. 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.

Fig 3. Role of braB CodY-binding sites in CodY binding.

The braB156p ⁺ (A) or braB144p ⁺, braB144p2, and braB144p3 (B) DNA fragments obtained by PCR with oligonucleotides oBB67 and oBB102 and labelled on the template strand were incubated with increasing amounts of purified CodY in the presence of 10 mM ILV and then with DNase I. The protected areas are indicated by vertical lines. 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.

Fig 4. Gel shift assays of CodY binding to braB fragments.

The braB242p ⁺ (A), braB156p ⁺ (B), braB156p1 (C), braB144p ⁺ (D), braB144p3 (E), braB144p2 (F), and braB144p2/3 (G) DNA fragments labeled on the template strand were incubated with increasing amounts of purified CodY in the presence of 10 mM ILV. The fragments were obtained by PCR with oligonucleotides oBB67 and oBB102 (A), oBB358 and oBB102 (B, C), and oBB422 and oBB102 (D-G) and their positions in the gel are indicated by right-pointing arrows. The unspecific DNA fragment, present in some panels, is indicated by a left-pointing arrow. CodY concentrations used (nM of monomer) are reported below each lane; concentrations corresponding to the apparent K_D for binding are underlined.

Fig 5. 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 null 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.

Fig 6. Expression of the braB242-gfp fusion in individual cells.

Cells were grown in TSS + 16 aa medium. GFP fluorescence was detected using Zeiss Axio Observer.Z1 microscope. A. Strain BB4082 (wild type). B. Strain BB4083 (codY::spc). C. Strain BB4084 [codY(R61K)].

Fig 7. 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.

Fig 8. Competition between CodY and ScoC for braB binding.

A and B. The braB64 DNA fragment, obtained by PCR with oligonucleotides oBB730 and oBB731 and labelled on the template strand, was incubated for 32 min with increasing amounts of purified ScoC (A) or CodY (B) in the presence of 10 mM ILV. C and D. The braB64 DNA fragment was preincubated for 16 min with 12.5 nM CodY (C) or 800 nM ScoC (D), and then increasing concentrations of either ScoC (C) or CodY (D) were added for an additional 16 min. The positions of the free DNA fragment and protein-DNA complexes are indicated by right-pointing and left-pointing arrows, respectively. Protein concentrations used (nM of monomer) are reported below each lane; concentrations corresponding to the apparent K_D for binding are underlined.

Fig 9. A model of regulation of the braB promoter by the combined actions of CodY and ScoC.

The sizes of the circles reflect the relative amount of the active form of each protein. The solid vertical lines indicate relatively strong effects on transcription. Dotted lines indicate relatively weak effects on transcription. The boldness of the horizontal arrows indicates the relative strength of transcription of the target genes.