Phosphate control of phoA, phoC and phoD gene expression in Streptomyces coelicolor reveals significant differences in binding of PhoP to their promoter regions

Abstract

Three putative alkaline phosphatase genes, phoA, phoC and phoD, were identified in the genome of Streptomyces coelicolor by homology with the amino acid sequence obtained from the PhoA protein of Streptomyces griseus. PhoA and PhoC correspond to broad-spectrum alkaline phosphatases whereas PhoD is similar to a Ca 2+ -dependent phospholipase D of Streptomyces chromofuscus. The phoA and phoD genes were efficiently expressed in R5 medium under phosphate-limited conditions, as shown by studies using the xylE reporter gene, whereas phoC was poorly transcribed under the same conditions. Expression of phoA was clearly PhoPdependent since it was not transcribed in the S. coelicolor DphoP mutant and was strongly activated under low phosphate concentrations. Similarly, expression of phoD was PhoPdependent and highly sensitive to phosphate availability. By contrast, expression of phoC was not PhoP-dependent. Electrophoretic mobility shift assays showed that PhoP binds to the phoA and phoD promoters, but not to that of phoC. Footprinting studies with GST-PhoP revealed the presence of a PHO box (two direct 11 nt repeats) in the phoA promoter and two PHO boxes in the promoter of phoD. The transcription start points of the three promoters were identified by primer extension. The transcription start point of phoD coincides with the G of its translation start codon, indicating that this gene is transcribed as a leaderless mRNA. The deduced "10 and "35 regions of phoD (but not those of phoA) overlapped with the PHO boxes in this promoter, suggesting that an excess of PhoP interferes with binding of the RNA polymerase to this promoter. In summary, the three promoters showed clear differences in the modulation of their expression by PhoP.

The master regulator PhoP coordinates phosphate and nitrogen metabolism, respiration, cell differentiation and antibiotic biosynthesis: comparison in Streptomyces coelicolor and Streptomyces avermitilis

“…[19][20][21] THE pho REGULON: PhoP-REGULATED GENES Transcriptomic studies, footprinting assays and in vivo expression studies with reporter genes allowed the initial identification of about 50 genes, which are regulated by PhoP and form the core of the pho regulon. 17 A list of relevant genes regulated by PhoP is shown in Table 1 and a summary of downregulated and upregulated genes is shown in Figure 2. New approaches using chromatin immunoprecipitation (ChIP-on-chip) have enlarged the scope of these studies.…”

“…15,16 In S. coelicolor, the most conserved sequence is GTTCACC, obtained from the alignment of the PhoP-binding sites of the best studied PhoP-regulated genes. 5,16,17 The sequence logo representing the PhoP-binding DRus is shown in Figure 1. Two DRus are required for binding of PhoP to DNA; one DRu is not sufficient.…”

Analysis of the Peltigera membranacea metagenome indicates that lichen-associated bacteria are involved in phosphate solubilization

“…The process of mineral phosphate solubilization (MPS) is complex and involves several gene products (Chhabra et al, 2013;Rodríguez et al, 2006;Zhao et al, 2013;Apel et al, 2007). Acidification is a key step in the dissolution of many poorly soluble mineral phosphates, and hence, as proposed by Goldstein (1996), the direct oxidation of glucose to gluconic acid is a key step in some of the major mechanisms for MPS in Gram-negative bacteria, which are suggested to be more efficient at MPS than Gram-positive bacteria owing to secretion of organic acids from sugar metabolism (Sashidhar & Podile, 2009).…”

Extracellular sugar phosphates are assimilated by Streptomyces in a PhoP-dependent manner

“…In the R2YE medium limited in P i , where the expression of phoR/phoP was shown to be very high and constitutive (Darbon et al 2012), the deletion of phoP in S. lividans suppresses the 'exSPs effect' on development but not that of exogenous addition of P i . In order to rationalize these observations, we propose that the exSPs would be first dephosphorylated by PhoP-dependent secreted phosphatases (such as PhoA and PhoD) (Apel et al 2007) and the resulting inorganic phosphate would enter the cell by classical medium or high affinity phosphate transporters (PitH2 or PstSCAB; Fig. 4).…”

“…P i , inorganic phosphate, PhoR phosphate limitation two-component histidine kinase, PhoP phosphate limitation response regulator associated to PhoR, PitH1 low-affinity P i transporter under inorganic phosphate excess, PitH2 P i low-affinity transporter under phosphate limitation conditions, PstABC P i high-affinity ABC-transporter. The model is deduced from the compilation of previous works (Sola-Landa et al 2003Diaz et al 2005;Ghorbel et al 2006;Apel et al 2007;Esteban et al 2008;Santos-Beneit et al 2008) Fig. 5 Concentration dependence of the sugar-phosphates effect on Streptomyces development.…”