Transcriptional studies and regulatory interactions between the phoR-phoP operon and the phoU, mtpA, and ppk genes of Streptomyces lividans TK24

Abstract

The PhoR/PhoP two-component system of Streptomyces lividans was previously shown to allow the growth of the bacteria at low Pi concentrations and to negatively control antibiotic production. The present study focuses on the transcriptional analysis of phoR and phoP, along with the phoU and mtpA genes that are transcribed divergently from the phoRP operon in S. lividans. The effect of phoR, phoP, phoU, and ppk mutations on transcription of these genes was examined under phosphate-replete and phosphate-limited conditions. We demonstrated that phoR and phoP were cotranscribed as a leaderless bicistronic transcript cleaved at discrete sites toward the 3' end of phoR. In addition, phoP could also be transcribed alone from a promoter located at the 3' end of phoR. The phoU and mtpA genes, predicted to encode metal binding proteins, were shown to be transcribed as monocistronic transcripts. The expression of phoR-phoP, phoP, and phoU was found to be induced under conditions of Pi limitation in S. lividans TK24. This induction, requiring both PhoR and PhoP, was significantly weaker in the phoU mutant but much stronger in the ppk mutant than in the parental strain. The expression of mtpA was also shown to be up-regulated when Pi was limiting but independently of PhoR/PhoP. The induction of mtpA expression was much stronger in the phoU mutant strain than in the other strains. This study revealed interesting regulatory interactions between the different genes and allowed us to propose putative roles for PhoU and MtpA in the adaptation to phosphate scarcity.

FIG. 1.

Isolated colonies of S. lividans TK24, S. lividans TK24 phoU::Ωaac (phoU), S. lividans TK24 phoP::Ωaac (phoP), and S. lividans TK24 phoR::Ωaac (phoR) grown on the solid modified minimal medium containing 0.44 mM KH₂PO₄ (condition of P_i limitation).

FIG. 2.

(A) Organization and transcripts of the genes mtpA, phoU, phoR, and phoP from S. lividans TK24. Probes P4, P3, P2, and P1 are indicated as a line under the corresponding genes, the transcripts are indicated as dotted lines, and the putative processing sites are indicated as vertical arrows. (B) Northern blot analysis of the expression of phoR, phoP, phoU, and mtpA from S. lividans TK24. A total of 20 μg of total RNA isolated from cultures of S. lividans TK24 (wt), S. lividans TK24 ppk::Ωhygro (ppk), S. lividans TK24 phoP::Ωaac (phoP), S. lividans TK24 phoR::Ωaac (phoR), and S. lividans TK24 phoU::Ωaac (phoU), grown for 48 h on plates of modified minimal medium containing 2.2 mM K₂HPO₄ (Pi sufficiency, +) or 0.44 mM K₂HPO₄ (Pi limitation, −), were loaded in each well and hybridized with the probes P1, P2, P3, and P4 corresponding, respectively, to phoP, phoR, phoU, and mptA. MW stands for molecular weight and corresponds to a DNA ladder from BRL.

FIG. 3.

High-resolution S1 nuclease mapping of the 5′ ends of the phoR, phoP, and phoU transcripts from S. lividans TK24. A total of 40 μg of total RNA isolated from cultures of S. lividans TK24 (wt), S. lividans TK24 ppk::Ωhygro (ppk), and S. lividans TK24 phoP::Ωaac (phoP) grown for 48 h on plates of modified minimal medium containing either 2.2 mM K₂HPO₄ (P_i sufficiency, +) or 0.44 mM K₂HPO₄ (P_i limitation, −) was hybridized with the PCR-labeled DNA fragments P5 and P6 (Table 1). The RNA-DNA hybrid was then treated with 100 U of S1 nuclease. E. coli tRNA hybridized with the same probes under the same conditions constituted an internal control for DNA/DNA reannealing. The RNA-DNA hybrids were then run on a polyacrylamide gel together with GATC sequencing ladders. All S1 nuclease mapping experiments were performed twice with independent sets of RNA and gave similar results. The positions of the putative transcriptional start site (Tsp) and of the major processing sites (Ps) within phoR are indicated by arrows. The sequences of the regions of the Ps and the Tsp are written vertically. On the sequences, the putative Tsp and the direction of transcription is indicated by an arrow; the Ps are indicated by asterisks.

FIG. 4.

(A) Nucleotide sequence of the phoU and phoR-phoP intergenic region of S. lividans TK24. (B) Nucleotide sequence of the 5′ region of phoP of S. lividans TK24. The translational start and stop codons are boxed. Putative RBSs are underlined by dots. The Tsp of the promoters are indicated by arrows above the sequence. The −10 and −35 promoters sequences are in boldface letters and are underlined. The gray box corresponds to a putative stem-loop. The asterisks correspond to the different processing sites detected by S1 mapping. The likely translational start codon of PhoP is GTG¹, as it is preceded by a Shine-Dalgarno-like sequence. It overlaps the TGA stop codon of the kinase PhoR and is located two codons upstream of the GTG² proposed by the Sanger Centre. Arrows above or below the sequence represent some of the primers listed in Table 1. PCR region I was amplified using the primers PhoP1-EcoRI/PhoP1 HindIII, and the PCR region II was amplified using the primers PhoP2-EcoRI/PhoP2-HindIII.

FIG. 5.

A total of 10⁶ spores of S. lividans TK24 and of the phoP, ppk, and phoU mutants were spread on the rich medium R2YE (8), in which no K₂HPO₄ (−, 1 mM final concentration) or 3.7 mM K₂HPO₄ (+, 4.7 mM final concentration) was added. A 5-mm-diameter cellulose disk impregnated with 20 μl of 100 mM H₂O₂ was deposited in the center of the plate just after spreading. Plates were incubated for 60 h at 30°C. The diameters of the zones of growth inhibition are indicated on the picture.