Interplay of CodY and ScoC in the Regulation of Major Extracellular Protease Genes of Bacillus subtilis

Abstract

AprE and NprE are two major extracellular proteases in Bacillus subtilis whose expression is directly regulated by several pleiotropic transcriptional factors, including AbrB, DegU, ScoC, and SinR. In cells growing in a rich, complex medium, the aprE and nprE genes are strongly expressed only during the post-exponential growth phase; mutations in genes encoding the known regulators affect the level of post-exponential-phase gene expression but do not permit high-level expression during the exponential growth phase. Using DNA-binding assays and expression and mutational analyses, we have shown that the genes for both exoproteases are also under strong, direct, negative control by the global transcriptional regulator CodY. However, because CodY also represses scoC, little or no derepression of aprE and nprE was seen in a codY null mutant due to overexpression of scoC. Thus, CodY is also an indirect positive regulator of these genes by limiting the synthesis of a second repressor. In addition, in cells growing under conditions that activate CodY, a scoC null mutation had little effect on aprE or nprE expression; full effects of scoC or codY null mutations could be seen only in the absence of the other regulator. However, even the codY scoC double mutant did not show high levels of aprE and nprE gene expression during exponential growth phase in a rich, complex medium. Only a third mutation, in abrB, allowed such expression. Thus, three repressors can contribute to reducing exoprotease gene expression during growth in the presence of excess nutrients.

Importance: The major Bacillus subtilis exoproteases, AprE and NprE, are important metabolic enzymes whose genes are subject to complex regulation by multiple transcription factors. We show here that expression of the aprE and nprE genes is also controlled, both directly and indirectly, by CodY, a global transcriptional regulator that responds to the intracellular pools of amino acids. Direct CodY-mediated repression explains a long-standing puzzle, that is, why exoproteases are not produced when cells are growing exponentially in a medium containing abundant quantities of proteins or their degradation products. Indirect regulation of aprE and nprE through CodY-mediated repression of the scoC gene, encoding another pleiotropic repressor, serves to maintain a significant level of repression of exoprotease genes when CodY loses activity.

FIG 1

Binding of CodY to the aprE regulatory region. (A) Sequence (5′ to 3′) of the coding (nontemplate) strand of the aprE regulatory region within the aprE640p⁺-lacZ fusion. Coordinates are reported with respect to the transcription start point (10). The upstream endpoints of inserts within the aprE640 and aprE334 fusions are at positions −557 and −251, respectively; the latter junction is indicated by a vertical arrow above the sequence. The downstream endpoints of both inserts coincide with the 3′ end of the presented sequence. The likely translation initiation codon, the −10 promoter region, and the apparent transcription start point are shown in bold. The directions of transcription and translation are indicated by horizontal arrows. The sequences that were protected by CodY (this work), ScoC (12), or AbrB (11) in DNase I footprinting experiments are shown by bracketed lines. The sequences of the four CodY-binding motifs, with three or four mismatches each, are italicized and underlined. The mutated nucleotides are shown in lowercase above the sequence. (B and C) Gel shift assays of CodY binding to aprE fragments. The aprE640p⁺ (B) and aprE640p2 (C) PCR fragments obtained with oligonucleotides oBB67 and oBB102, using pGB1 and pGB13, respectively, as templates, and labeled on the template strand were incubated with increasing amounts of purified CodY in the presence of 10 mM ILV. CodY concentrations used (monomers) are reported below the lanes; concentrations corresponding to the apparent K_D for binding are underlined. (D) DNase I footprinting analysis of CodY binding to the aprE regulatory region. The aprE640p⁺ PCR fragment used for panel B was incubated with increasing amounts of purified CodY in the presence of 10 mM ILV and then with DNase I. The protected area is indicated by a vertical line, and the corresponding sequence is reported; the protected nucleotides are italicized. The apparent transcription start point and direction of transcription are shown by a bent arrow. CodY concentrations used (nanomolar [monomers]) are indicated below the lanes. The A+G sequencing ladder of the template DNA strand is shown in the right lane.

FIG 2

Expression of the aprE640p⁺-lacZ and nprE396p⁺-lacZ fusions in DS nutrient broth medium. Cells were grown in DS medium, and samples for β-galactosidase determination were taken at the indicated times. Times are shown with respect to T₀, i.e., the transition point between the exponential and stationary growth phases. At least two experiments were performed for each strain, and the results of a representative experiment are shown. Other biological replicates of each experiment gave very similar patterns of gene expression.

FIG 3

Binding of CodY to the nprE regulatory region. (A) Sequence (5′ to 3′) of the coding (nontemplate) strand of the nprE regulatory region within the nprE396-lacZ fusion. The 5′ and 3′ nucleotides of the sequence presented correspond to the first and last nucleotides of the nprE insert within the fusion. Coordinates are reported with respect to the transcription start point (60). The upstream boundary of the nprE153-lacZ fusion, at position −95, is indicated by a vertical arrow above the sequence. The likely translation initiation codon, the −10 and −35 promoter regions, and the transcription start point are shown in bold. The directions of transcription and translation are indicated by horizontal arrows. The sequences that were protected by CodY or ScoC (12) in DNase I footprinting experiments are shown by bracketed lines. The sequences of the two CodY-binding motifs, with three or four mismatches each, are italicized and underlined. The mutated nucleotides are shown in lowercase above the sequence. (B and C) Gel shift assays of CodY binding to nprE fragments. The nprE396p⁺ (B) and nprE396p1 (C) DNA fragments obtained with oligonucleotides oBB67 and oBB102, using pGB2 and pGB6, respectively, as templates, and labeled on the template strand were incubated with increasing amounts of purified CodY in the presence of 10 mM ILV. CodY concentrations used (nanomolar [monomers]) are reported below the lanes; concentrations corresponding to the apparent K_D for binding are underlined. (D) DNase I footprinting analysis of CodY binding to the nprE regulatory region. The nprE396p⁺ DNA fragment used for panel B was incubated with increasing amounts of purified CodY in the presence of 10 mM ILV and then with DNase I. The protected area is indicated by a vertical line, and the corresponding sequence is reported; the protected nucleotides are italicized. The apparent transcription start point and direction of transcription are shown by a bent arrow. CodY concentrations used (monomers) are indicated below the lanes. The A+G sequencing ladder of the template DNA strand is shown in the right lane.

FIG 4

Model of regulation of the aprE and nprE promoters by the combined actions of CodY, ScoC, and AbrB. The sizes of the circles reflect the relative amounts of the active forms of the proteins. 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 strengths of transcription of the target genes, and the numbers show activities of the corresponding lacZ fusions during exponential growth in TSS+16 aa medium.