Complex regulation of the Bacillus subtilis aconitase gene

Abstract

The roles of the CcpC, CodY, and AbrB proteins in regulation of the Bacillus subtilis aconitase (citB) gene were found to be distinct and to vary with the conditions and phase of growth. CcpC, a citrate-inhibited repressor that is the primary factor regulating citB expression in minimal-glucose-glutamine medium, also contributed to repression of citB during exponential-phase growth in broth medium. A null mutation in codY had no effect on citB expression during growth in minimal medium even when combined with ccpC and abrB mutations. However, a codY mutation slightly relieved repression during exponential growth in broth medium and completely derepressed citB expression when combined with a ccpC mutation. An abrB mutation led to decreased expression of citB during stationary phase in both broth and minimal medium. All three proteins bound in vitro to specific and partially overlapping sites within the citB regulatory region. Interaction of CcpC and CodY with the citB promoter region was partially competitive.

FIG. 1.

Growth phase-dependent expression of a citB-lacZ fusion in wild-type and codY, ccpC, and abrB mutant strains. β-Galactosidase activity was measured in wild-type and mutant strains sampled at various times during growth in DS medium. Arrows indicate the end of the exponential growth phase. (A) Strains: AF21 (wild type [wt]), HKB95 (codY), CJB9 (ccpC), HKB98 (codY ccpC), and HKB99 (codY ccpC abrB). (B) Strains: AF21 (wild-type), HKB94 (abrB), HKB95 (codY), HKB96 (abrB codY), and HKB99 (codY ccpC abrB). (C) Strains: AF21 (wild-type), CJB9 (ccpC), HKB94 (abrB), and HKB97 (ccpC abrB).

FIG. 2.

Effect of glucose on citB expression. Strains AF21 (wild type [wt]), CJB9 (ccpC), HKB98 (codY ccpC), and HKB99 (abrB codY ccpC) were grown in DS medium supplemented with 2% glucose, and β-galactosidase activity was measured in samples harvested at various time points during growth. Arrows indicate the end of the exponential growth phase.

FIG. 3.

Effect on citB expression of deletion of the CcpC binding site in the citB promoter region. Strains AF23 (citBp23-lacZ), AF24 (citBp24-lacZ), HKB125 (citBp23-lacZ codY), and HKB126 (citBp24-lacZ codY) were grown in DS medium, and β-galactosidase activity was measured in samples harvested at various time points during growth. Arrows indicate the end of the exponential growth phase. The citBp23-lacZ fusion contains the citB promoter region from positions −84 to +36 with respect to the transcriptional start site. The citBp24-lacZ fusion contains the citB promoter region from positions −67 to +36 and is thus missing the left arm of the dyad symmetry element (9).

FIG. 4.

Effects on citB expression of citrate synthase, aconitase, and spo0A mutations. Cultures were grown in DS medium, and β-galactosidase activity was measured in samples harvested at various time points during growth. Arrows indicate the end of the exponential growth phase. (A) Strains: AF21 (wild type [wt]), HKB76 (citA citZ), HKB165 (citB), and HKB166 (citA citZ citB). (B) Strains: AF21 (wild type), HKB168 (spo0A), HKB165 (citB), HKB169 (citB spo0A), and HKB170 (citA citZ citB spo0A).

FIG. 5.

DNase I footprint assay of CodY interaction with the citB regulatory region. A 523-bp PCR product, corresponding to positions −320 to +202 with respect to the citB transcriptional start site and labeled at the 5′ end of the template (noncoding) strand, was incubated with increasing amounts of purified CodY-His₆ in the presence or absence of 2 mM GTP. After treatment with DNase I, the DNA was denatured and subjected to electrophoresis. A set of Sanger sequencing reactions (not shown) were primed with the same oligonucleotide used for synthesis of the template strand of the PCR product and used to establish the positions of the protected bands. Vertical lines on the left indicate regions protected by CodY.

FIG. 6.

Simultaneous binding of CcpC and CodY to the citB regulatory region. A PCR product encompassing positions −166 to +123 with respect to the citB transcription start site was labeled at the 5′ end on the nontemplate (coding) strand. A set of Sanger sequencing reactions (A, C, G, and T) were primed with the same oligonucleotide used for synthesis of the nontemplate strand of the PCR product and used to establish the positions of the protected bands. The vertical bars indicate regions protected by CcpC or CodY, and the arrowhead points to a DNase I-hypersensitive site typically created by binding of CcpC to the citB promoter region (22). (A) The PCR product was incubated with increasing amounts of CcpC protein in the presence or absence of 900 nM CodY-His₆. All reaction mixes also contained 2 mM GTP. After DNase I treatment, the DNA fragments were purified as described in Materials and Methods, denatured, and subjected to electrophoresis. CcpC concentrations (nanomolar) were as follows: lanes 1 and 11, 0; lanes 2 and 12, 12; lanes 3 and 13, 25; lanes 4 and 14, 50; lanes 5 and 15, 100; lanes 6 and 16, 200; lanes 7 and 17, 500; and lanes 8 and 18, 1,000. (B) The PCR product was incubated with 50 nM CcpC and increasing amounts of CodY with or without added GTP (2 mM). CodY concentrations (nanomolar) were as follows: lane 2, 0; lane 3, 300; lane 4, 600; lane 5, 1,000; lane 6, 1,800; and lane 7, 3,000. The sample in lane 1 contained neither CcpC nor CodY.

FIG. 7.

DNase I footprint of AbrB binding to the citB regulatory region. (A) The template strand was labeled at its 3′ end. (B) The nontemplate strand was labeled at its 3′ end. Lanes: 1, 10 μM AbrB; 2, 5 μM AbrB; 3, 1 μM AbrB; 4 and 5, no AbrB. Maxam-Gilbert purine (U) and pyrimidine (Y) sequencing ladders for each strand are shown for reference. The positions of the +1 and −35 nucleotides of the promoter are indicated.