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. 2010 Jun;192(12):3103-13.
doi: 10.1128/JB.00089-10. Epub 2010 Apr 9.

Direct regulation of Bacillus subtilis phoPR transcription by transition state regulator ScoC

Bindiya Kaushal  1 , Salbi Paul, F Marion Hulett
Affiliations

Direct regulation of Bacillus subtilis phoPR transcription by transition state regulator ScoC

Bindiya Kaushal et al. J Bacteriol. 2010 Jun.

Abstract

Induction of the Pho response in Bacillus subtilis occurs when the P(i) concentrations in the growth medium fall below 0.1 mM, a condition which results in slowed cellular growth followed by entry into stationary phase. The phoPR promoter region contains three sigma(A)-responsive promoters; only promoter P(A4) is PhoP autoregulated. Expression of the phoPR operon is postexponential, suggesting the possibility of a repressor role for a transition-state-regulatory protein(s). Expression of a phoPR promoter-lacZ fusion in a scoC loss-of-function mutant strain grown in low-phosphate defined medium was significantly higher than expression in the wild-type strain during exponential growth or stationary phase. Derepression in the scoC strain from a phoP promoter fusion containing a mutation in the CcpA binding site (cre1) was further elevated approximately 1.4-fold, indicating that the repressor effects of ScoC and CcpA on phoP expression were cumulative. DNase I footprinting showed protection of putative binding sites by ScoC, which included the -10 and/or -35 elements of five (P(B1), P(E2), P(A3), P(A4), and P(A6)) of the six promoters within the phoPR promoter region. P(A6) was expressed in vivo from the phoP cre1 promoter fusion in both wild-type and scoC strains. Evidence for ScoC repression in vivo was shown by primer extension for P(A4) and P(A3) from the wild-type promoter and for P(A4) and P(E2) from the phoP cre1 promoter. The latter may reflect ScoC repression of sporulation that indirectly affects phoPR transcription. ScoC was shown to repress P(A6), P(A4), P(E2), and P(B1) in vitro.

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Figures

FIG. 1.
FIG. 1.
The phoPR promoter sequence and 5′ PhoP coding sequence showing six transcription start sites along with the putative ScoC binding sites. Transcription start sites for PB1, PE2, PA3, PA4, P5, and PA6 are indicated by boldface sequence and are identified by a bent arrow followed by the promoter number and a letter identifying the form of RNAP (where known) required for the transcription. The −10 region is marked below, and the −35 is marked above the sequence for each promoter based on published consensus sequences (16). The region of +1 to +92 is the 5′ PhoP coding sequence followed by the lacZ fusion. The translation start site, ATG, is boxed and identified by a bent arrow marked +1. Sequence numbering is relative to the A of ATG as +1. A single putative ScoC binding site in the 5′ region of the phoPR promoter, 5′-−227GAAAGTATT−219-3′ (site A), is located on the coding strand. Three conserved putative ScoC binding sites in the 3′ region of the phoPR promoter are 5′-78AATAAAATC−71-3′ (site B), 5′-−51CATAAAATA−43-3′ (site C), and 5′-−23AATTATAAT−15-3′ (site D) located on the coding strand. The ScoC binding consensus sequence is RATANTATY, where R is A or G, Y is C or T, and N is A, G, C, or T. The putative ScoC binding sites are indicated with nine stars above the sequences. The CcpA binding site (cre box) is shown in bold, marked above with plus signs. Primers used are shown as underlined or overlined sequences with an arrow in the direction of synthesis. FMH1025 is the primer used for primer extension analysis specific to the lacZ fusion. The ScoC DNase I-protected regions are shown as gray boxes on both strands of the phoPR promoter. The white boxes show the weaker ScoC protection. The consensus repeats for PhoP dimer binding, TT(A/C/T)A(C/T)A, are underlined, and the sequence is in boldface.
FIG. 2.
FIG. 2.
Effect of scoC mutation on phoPR transcription throughout growth. Cells were grown in LPDMG over a period of 12 h to monitor the growth and level of phoPR transcription. The first hour when APase activity was induced as a reporter for Pho induction is identified as T0. Solid symbols represent the growth, and open symbols represent the β-galactosidase specific activity of the full-length phoPR-lacZ fusion in each strain. Circle, WT (MH5562); square, scoC (MH7400).
FIG. 3.
FIG. 3.
DNase I footprint analysis of ScoC binding to the phoPR promoter. The plasmid pBK1 was used as a template for a PCR probe. The ScoC concentration (μM) is shown at the top of each lane. F, free of ScoC; G, Maxam-Gilbert G sequencing reaction, used as a marker. Base pairs are numbered on coding and noncoding strands relative to the translation start site (as +1). Solid lines identify the DNase I protection. Dotted lines identify the weaker protection by ScoC. (A) DNase I protection by on the coding strand in the 5′ region of the phoPR promoter. End-labeled FMH880 and nonlabeled FMH881 were used to create the PCR probe. (B) DNase I protection on the noncoding strand in the 5′ region of the phoPR promoter. End-labeled FMH881 and nonlabeled FMH880 were used to create the PCR probe. (C) DNase I protection on the coding strand of the 3′ promoter region. End-labeled FMH1018 and nonlabeled FMH1019 were used to create the PCR probe. (D) DNase I protection on the noncoding strand of the 3′ promoter region. End-labeled FMH1019 and nonlabeled FMH1018 were used to create the PCR probe.
FIG. 4.
FIG. 4.
Primer extension analysis of the phoPR promoter region from the total RNA isolated from a WT (MH5562) or a scoC strain (MH7400) grown in LPDMG. The end-labeled primer FMH1025 was annealed to RNA isolated from exponential phase, transition stage, or postexponential phase cultures. (A) Lanes 3 to 10 show the primer extension reactions of RNA samples taken from a WT strain (MH5562) growing in LPDMG at the times indicated. The expression from four transcriptional start sites (PB1, PE2, PA3, and PA4) is indicated with arrows. T0 is the time of Pho induction and T1, T2, T3, T4, and T5 are 1, 2, 3, 4, and 5 h of growth, respectively, in LPDMG after Pho induction. T−2 and T−1 samples were taken at 2 h and 1 h before Pho induction, respectively. (B) Lanes 3 to 10 show the primer extension of RNA samples taken from the scoC (MH7400) strain at the times indicated. (C) Quantification of individual transcripts. Radioactivity was determined in arbitrary units for each transcript by phosphorimaging. The sum of all transcripts from a reaction was considered 100%. The percent contribution of each transcript to the total transcription from each primer extension reaction was calculated and plotted in a set of bar graphs. The time of RNA sampling was indicated relative to Pho induction, T0. Bars correspond to PA4, PA3, PE2, and PB1, respectively.
FIG. 5.
FIG. 5.
Combined effect of scoC and phoP cre1 promoter mutation on phoPR transcription during growth and stationary phase. Cells were grown in LPDMG for 10 h to monitor growth and level of transcription. Solid symbols represent the growth, and open symbols represent the β-galactosidase specific activity of the full-length phoPR-lacZ fusion in each strain. Circle, WT phoPR-lacZ (MH6024); square, phoPR-lacZ cre1-lacZ strain (MH6040); triangle, scoC phoPR-lacZ cre1-lacZ strain (MH7417).
FIG. 6.
FIG. 6.
Primer extension analysis of the phoPR promoter region from the total RNA isolated from the phoP cre1-lacZ (MH6040) strain or scoC phoP cre1-lacZ (MH7417) strain grown in LPDMG. The end-labeled primer FMH1025 was annealed to RNA isolated from exponential phase, transition stage, and postexponential phase cultures. (A) Lanes 3 to 9 show the primer extension reactions of RNA samples taken at 3 to 9 h from the phoP cre1-lacZ (MH6040) strain grown in LPDMG (Fig. 5) at the times indicated. T0 is the time of Pho induction, and T1, T2, T3, T4, and T5 are 1, 2, 3, 4, and 5 h of growth, respectively, after Pho induction in LPDMG. T−1 is 1 h before Pho induction (growth hour 3). Promoter expression from six transcriptional start sites, PB1, PE2, PA3, PA4, P5, and PA6, is indicated with arrows. (B) Lanes 3 to 9 show the primer extension of RNA samples taken at the indicated times from the scoC phoP cre1-lacZ (MH7417) strain. (C) Quantification of individual transcripts as described for Fig. 4. Bars correspond to PA6, PA4, PA3, PE2, and PB1, respectively.
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