One of the most important achievements of bacteria is its capability to adapt to the changing conditions of the environment. The competition for nutrients with other microorganisms, especially in the soil, where nutritional conditions are more variable, has led bacteria to evolve a plethora of mechanisms to rapidly fine-tune the requirements of the cell. One of the essential nutrients that are normally found in low concentrations in nature is inorganic phosphate (Pi). Bacteria, as well as other organisms, have developed several systems to cope for the scarcity of this nutrient. To date, the unique mechanism responding to Pi starvation known in detail is the Pho regulon, which is normally controlled by a two component system and constitutes one of the most sensible and efficient regulatory mechanisms in bacteria. Many new members of the Pho regulon have emerged in the last years in several bacteria; however, there are still many unknown questions regarding the activation and function of the whole system. This review describes the most important findings of the last three decades in relation to Pi regulation in bacteria, including: the PHO box, the Pi signalling pathway and the Pi starvation response. The role of the Pho regulon in nutritional regulation cross-talk, secondary metabolite production and pathogenesis is discussed in detail.

Summary: The regulatory proteins AfsR and PhoP control expression of the biosynthesis of actinorhodin and undecylprodigiosin in Streptomyces coelicolor. Electrophoretic mobility shift assays showed that PhoP^DBD does not bind directly to the actII‐ORF4, redD and atrA promoters, but it binds to the afsS promoter, in a region overlapping with the AfsR operator. DNase I footprinting studies revealed a PhoP protected region of 26 nt (PHO box; two direct repeats of 11 nt) that overlaps with the AfsR binding sequence. Binding experiments indicated a competition between AfsR and PhoP; increasing concentrations of PhoP^DBD resulted in the disappearance of the AfsR–DNA complex. Expression studies using the reporter luxAB gene coupled to afsS promoter showed that PhoP downregulates afsS expression probably by a competition with the AfsR activator. Interestingly, AfsR binds to other PhoP‐regulated promoters including those of pstS (a component of the phosphate transport system) and phoRP (encoding the two component system itself). Analysis of the AfsR‐protected sequences in each of these promoters allowed us to distinguish the AfsR binding sequence from the overlapping PHO box. The reciprocal regulation of the phoRP promoter by AfsR and of afsS by PhoP suggests a fine interplay of these regulators on the control of secondary metabolism.

Bacterial growth requires equilibrated concentration of C, N and P sources. This work shows a phosphate control over the nitrogen metabolism in the model actinomycete Streptomyces coelicolor. Phosphate control of metabolism in Streptomyces is exerted by the two component system PhoR-PhoP. The response regulator PhoP binds to well-known PHO boxes composed of direct repeat units (DRus). PhoP binds to the glnR promoter, encoding the major nitrogen regulator as shown by EMSA studies, but not to the glnRII promoter under identical experimental conditions. PhoP also binds to the promoters of glnA and glnII encoding two glutamine synthetases, and to the promoter of the amtB-glnK-glnD operon, encoding an ammonium transporter and two putative nitrogen sensing/regulatory proteins. Footprinting analyses revealed that the PhoP-binding sequence overlaps the GlnR boxes in both glnA and glnII. ‘Information theory’ quantitative analyses of base conservation allowed us to establish the structure of the PhoP-binding regions in the glnR, glnA, glnII and amtB genes. Expression studies using luxAB as reporter showed that PhoP represses the above mentioned nitrogen metabolism genes. A mutant deleted in PhoP showed increased expression of the nitrogen metabolism genes. The possible conservation of phosphate control over nitrogen metabolism in other microorganisms is discussed.

Summary: Limitation of different nutrients in Streptomyces coelicolor A3(2) triggers nutrient‐stress responses, mediated by PhoP, GlnR, AfsR and other regulators, that are integrated at the molecular level and control secondary metabolite biosynthesis and differentiation. In addition, utilization of chitin or N ‐acetylglucosamine regulates secondary metabolite biosynthesis by a mechanism mediated by DasR. Phosphate control of primary and secondary metabolism in Streptomyces species is mediated by the two‐component PhoR–PhoP system. In S. coelicolor , PhoP controls secondary metabolism by binding to a PHO box in the afsS promoter overlapping with the AfsR binding site. Therefore, the afsS promoter serves to integrate the PhoP‐mediated response to phosphate limitation and AfsR‐mediated responses to other unknown environmental stimuli. Interestingly, phosphate control oversees nitrogen regulation but not vice versa. In Δ phoP mutants, expression of some nitrogen metabolism genes including glnA , glnII and glnK is increased. Phosphate control of these genes is exerted through binding of PhoP to the promoters of glnR (the global nitrogen regulator), glnA , glnII and the amtB–glnK–glnD operon. This regulation allows a ‘metabolic homeostasis’ of phosphate and nitrogen utilization pathways, preventing nutritional unbalances. Similar mechanisms of interaction between phosphate control and carbon catabolite regulation or between phosphate and DasR‐mediated N ‐acetylglucosamine regulation appear to exist. Transport of N ‐acetylglucosamine by the NagE2 permease and, therefore, regulation of secondary metabolism, is dependent upon the balance of phosphorylated/dephosphorylated proteins of the N ‐acetylglucosamine phosphotransferase system. These findings provide the bases for understanding the mechanisms underlying systems biology of Streptomyces species.

The transport of inorganic phosphate (P_i) is essential for the growth of all organisms. The metabolism of soil-dwelling Streptomyces species, and their ability to produce antibiotics and other secondary metabolites, are strongly influenced by the availability of phosphate. The transcriptional regulation of the SCO4138 and SCO1845 genes of Streptomyces coelicolor was studied. These genes encode the two putative low-affinity P_i transporters PitH1 and PitH2, respectively. Expression of these genes and that of the high-affinity transport system pstSCAB follows a sequential pattern in response to phosphate deprivation, as shown by coupling their promoters to a luciferase reporter gene. Expression of pitH2, but not that of pap-pitH1 (a bicistronic transcript), is dependent upon the response regulator PhoP. PhoP binds to specific sequences consisting of direct repeats of 11 nt in the promoter of pitH2, but does not bind to the pap-pitH1 promoter, which lacks these direct repeats for PhoP recognition. The transcription start point of the pitH2 promoter was identified by primer extension analyses, and the structure of the regulatory sequences in the PhoP-protected DNA region was established. It consists of four central direct repeats flanked by two other less conserved repeats. A model for PhoP regulation of this promoter is proposed based on the four promoter DNA–PhoP complexes detected by electrophoretic mobility shift assays and footprinting studies.