Autoinduction of Bacillus subtilis phoPR operon transcription results from enhanced transcription from EsigmaA- and EsigmaE-responsive promoters by phosphorylated PhoP

Abstract

The phoPR operon encodes a response regulator, PhoP, and a histidine kinase, PhoR, which activate or repress genes of the Bacillus subtilis Pho regulon in response to an extracellular phosphate deficiency. Induction of phoPR upon phosphate starvation required activity of both PhoP and PhoR, suggesting autoregulation of the operon, a suggestion that is supported here by PhoP footprinting on the phoPR promoter. Primer extension analyses, using RNA from JH642 or isogenic sigE or sigB mutants isolated at different stages of growth and/or under different growth conditions, suggested that expression of the phoPR operon represents the sum of five promoters, each responding to a specific growth phase and environmental controls. The temporal expression of the phoPR promoters was investigated using in vitro transcription assays with RNA polymerase holoenzyme isolated at different stages of Pho induction, from JH642 or isogenic sigE or sigB mutants. In vitro transcription studies using reconstituted EsigmaA, EsigmaB, and EsigmaE holoenzymes identified PA4 and PA3 as EsigmaA promoters and PE2 as an EsigmaE promoter. Phosphorylated PhoP (PhoP approximately P) enhanced transcription from each of these promoters. EsigmaB was sufficient for in vitro transcription of the PB1 promoter. P5 was active only in a sigB mutant strain. These studies are the first to report a role for PhoP approximately P in activation of promoters that also have activity in the absence of Pho regulon induction and an activation role for PhoP approximately P at an EsigmaE promoter. Information concerning PB1 and P5 creates a basis for further exploration of the regulatory coordination or overlap of the PhoPR and SigB regulons during phosphate starvation.

FIG. 1.

DNase I footprint analysis of the phoPR promoter bound by PhoP and PhoP∼P. Various amounts of PhoP, incubated with *PhoR (1.4 μg) in the presence or absence of 4 mM ATP, were mixed with the 407-bp phoPR promoter labeled on either the coding or noncoding strand and treated with DNase I. The concentration of PhoP used in each reaction mixture was, from left to right, 0 nM, 55 nM, 275 nM, 1.38 μM, and 6.7 μM. Lanes with PhoP∼P are labeled +ATP, and those with unphosphorylated PhoP are labeled −ATP. Lanes F, PhoP-free lanes; lane G, the G-sequencing reaction lane used as a reference. The thick black vertical lines represent the PhoP and PhoP∼P binding regions, while the thin lines represent sites bound only by PhoP∼P. The hypersensitive sites are marked with a dark arrowhead. Base pairs are numbered relative to the translation start site (as +1).

FIG. 2.

Transcription start sites and PhoP binding sites on the phoPR promoter sequence and 5′ PhoP coding sequence. Gray shading identifies sequence protected by both PhoP and PhoP∼P. Stippled shading identifies sequence protected only by PhoP∼P. Transcriptional start sites for P_B1, P_E2, P_A3, P_A4, or P₅ are indicated by bold sequence base pairs that are identified by a bent arrow followed by the promoter number. The −10 consensus sequence for each promoter is underlined by a slender rectangle marked −10 followed by the promoter number. The −35 consensus sequence for each promoter is overlined by a slender rectangle marked −35 followed by the promoter number. The consensus repeats for PhoP dimer binding [TT(A/C/T)A(C/T)A] are underlined with the sequence in bold print. The translational start codon, ATG, is boxed and identified by a bent arrow marked +1. Sequence numbering is relative to the A of ATG as +1. Arrows with half arrowheads identify primers used to amplify sequences in the two promoter fusion constructs analyzed below in Fig. 3. The asterisk identifies the transcription site of P_BX1.

FIG. 3.

The roles of PhoP or PhoP binding regions in phoPR transcription differ during phosphate-limited and phosphate-replete growth. (A) Growth and phoPR expression in LPDM. An arrow marks the induction of APase in phoP⁺ strains MH5562 and MH5559. (B) Growth and phoPR expression in SSG. Filled symbols represent growth; open symbols represent expression of various phoP-lacZ promoter fusions. Circle, MH5562 (JH642 strain; phoP-lacZ); square, MH5565 (phoP strain; phoP-lacZ); triangle, MH5559 (JH642 strain; phoP-lacZ fusion containing a deletion of bp +25 to +92 that removed the 3′ PhoP binding site, phoP_Δ25-92-lacZ); inverted triangle, MH5567 (Δ phoP; phoP_Δ25-92-lacZ); diamond, MH5580 (sigE phoP-lacZ).

FIG. 4.

Primer extension identified four mRNA 5′ ends in the phoPR promoter region. (A) Primer extension analysis of the phoPR promoter region. The end-labeled primer FMH079 was annealed to RNA from transition or post-exponential-stage cultures. (A) Lane 1, RNA isolated from LPDM-grown cells during early induction (T₁); lanes 2 and 3, RNA isolated from LPDM-grown cells during later Pho induction (T₃ and T₄); lanes T, C, G, and A, sequencing ladders generated by annealing the same end-labeled primer to a plasmid (pSB5) containing the full-length promoter region of the phoPR operon and extending it with Sequenase (U.S. Biochemical Corp.). Arrowheads labeled P_B1, P_E2, P_A3, and P_A4 identify the mRNA 5′ ends. (B) Comparison of phoP 5′ ends in RNA isolated from postexponential cells grown under phosphate starvation or phosphate-replete sporulation conditions. Lane 1, RNA isolated from LPDM-grown cells during late Pho induction, T₄; lane 2, DNA isolated from SSG-grown cells at sporulation stage T₄. (Labeling is as in panel A.) (C) Putative promoter −10 and −35 consensus regions for P_B1, P_E2, P_A3, and P_A4 compared to sigma factor consensus sequences (10). Bold letters in the P_B1, P_E2, P_A3, and P_A4 sequences represent matches to the sigma binding consensus sequence. In the consensus sequence, capital letters indicate highly conserved positions and lowercase letters indicate less-conserved positions. R = A or G; W = A or T.

FIG. 5.

Growth stage-specific RNAP shows temporal expression of in vitro phoPR promoter transcripts and the absence of P_E2 with RNAP from a sigE mutant strain. (A) In vitro transcription of the phoPR promoter with RNAP isolated from stage T₀, T₃, and T₄ cells grown in LPDM. The in vitro transcription reactions were carried out as described in Materials and Methods. M, RNA marker. In vitro transcripts were generated using RNAP from LPDM-grown MH5636 cells harvested at T₀ (lane 1), T₃ (lane 2), or T₄ (lane 3) or from MH5654 (sigE) cells at T₄ (lane 4). All reaction mixtures contained 5 pmol each of PhoP and *PhoR plus 1 mM ATP. (B) In vitro transcription products identified by primer extension. Markings and procedures were the same as for Fig. 4A, except the mRNA was generated by in vitro transcription (panel A, lanes 3 and 4).

FIG. 6.

PhoP∼P enhances transcription from P_E2 and P_A4. (A) PhoP phosphorylation affects RNAP T₄ phoPR promoter transcript P_E2. Symbols are the same as for Fig. 5. Lanes 1 and 5 contain no PhoP; lanes 2 to 4 and lanes 6 to 8 contain increasing concentrations of PhoP (1, 2.5, and 5 pmol). For phosphorylation of PhoP, equal molar concentrations of PhoP and *PhoR were in reaction mixtures applied to lanes 6 to 8. (B) T₄ RNAP from a sigE strain yielded P_B1, P_A3, and P_A4 transcripts but no P_E2 transcript; PhoP∼P enhanced P_A4 transcription. Lane 1 contains an in vitro transcription reaction identical to lane 8 in panel A for a direct comparison between reactions using T₄ RNAP from JH642 and from a mutant strain. Lanes 2 and 5 contain no PhoP; lanes 3 and 4 and lanes 6 and 7 contain increasing concentrations of PhoP (2.5 and 5 pmol, respectively). Lanes 6 and 7 each contain equal molar amounts of PhoP and *PhoR plus ATP for phosphorylation of PhoP.

FIG. 7.

PhoP∼P activates transcription from P_A4 and P_E2 by using different forms of RNAP. (A) Expression of P_A3 and P_A4 requires Eσ^A. Lane M contains the 200-nucleotide marker. Lanes 1 to 5 and 7 to 13 contain reconstituted Eσ^A. Lane 6 contains core RNAP with no sigma factor added. Lanes 1 and 7 contain no PhoP or PhoP∼P. Lanes 2 to 5 contain increasing amounts of PhoP (0.1 to 5.0 pmol). Lane 6 contains 5.0 pmol of PhoP∼P. Lanes 8 to 13 contain increasing amounts of PhoP∼P (0.1 to 5 pmol). Lanes 8 to 13 contain equal molar amounts of PhoP and *PhoR plus ATP for phosphorylation of PhoP. (B) PhoP∼P specifically activates the P_E2 Eσ^E promoter of the phoPR operon. The 100-nucleotide marker is in the lane marked M. Lanes 1 to 8 contain reconstituted Eσ^E plus the phoPR template. Lanes 2 to 4 contain increasing amounts (0.5, 1, and 5 pmol) of PhoP. Lanes 5 to 8 contain increasing amounts (0.25, 0.5, 1, and 5 pmol) of PhoP∼P. Lanes 9 to 13 contain reconstituted Eσ^E plus the spoIIID template. Lanes 10 and 11 contain 1 and 5 pmol of PhoP, respectively. Lanes 12 and 13 contain 1 and 5 pmol of PhoP∼P, respectively.

FIG. 8.

Core plus SigB is sufficient to transcribe from P_B1 or P_(x1); RNAP from a sigB mutant strain cannot transcribe from P_B1 or P_(x1). (A) Eσ^B transcription from the P_B1 or P_(x1) promoter does not require PhoP or PhoP∼P. Lane M contains the 100-nucleotide marker. Reaction mixtures in lanes 1 to 9 contained the phoPR promoter template and reconstituted Eσ^B. Lanes 2 to 5 and 6 to 9 contained increasing amounts (0.1, 0.5, 1, and 5 pmol) of PhoP or PhoP∼P, respectively. (B) Neither T₀ RNAP nor T₄ RNAP from a sigB mutant strain can transcribe from P_B1. Lanes 1 to 7 contain the phoPR template. The reaction mixture in lane 1 contained T₀ sigE RNAP plus PhoP∼P and identified the migration positions of P_(BX1), P_B1, P_A3, and P_A4. Lane 2 contains core RNAP plus σ^B as in lane 1 of panel A. Reaction mixtures in lanes 3 and 4 contained T₀ RNAP, and those in lanes 5 to 7 contained T₄ RNAP from a sigB mutant strain. Reaction mixtures in lanes 3 and 5 contained PhoP, and lanes 4, 6, and 7 contained PhoP∼P.

FIG. 9.

RNA from a sigE mutant strain contains no P_E2 transcript, and RNA from a sigB mutant strain shows temporal regulation of phoPR promoter transcripts and identifies a 5′ mRNA terminus upstream of P_A4. Primer extension and generation of sequencing ladders were the same as described in the legend for Fig. 4. WT lanes used RNA for primer extension studies that was isolated from the parental strain JH642 at T₀, T₂, or T₄. σ^E lanes used RNA for primer extension studies that was isolated from the sigE mutant strain EU8701 at T₀, T₂, or T₄. σ^B lanes used RNA for primer extension studies that was isolated from sigB mutant strain PB344 at T₀, T₁, T₂, T₃, and T₄.