Analysis and validation of the pho regulon in the tacrolimus-producer strain Streptomyces tsukubaensis: differences with the model organism Streptomyces coelicolor

Abstract

Inorganic and organic phosphate controls both primary and secondary metabolism in Streptomyces genus. Metabolism regulation by phosphate in Streptomyces species is mediated by the PhoR-PhoP two-component system. Response regulator PhoP binds to conserved sequences of 11 nucleotides called direct repeat units (DRus), whose organization and conservation determine the binding of PhoP to distinct promoters. Streptomyces tsukubaensis is the industrial producer of the clinical immunosuppressant tacrolimus (FK506). A bioinformatic genome analysis detected several genes with conserved PHO boxes involved in phosphate scavenging and transport, nitrogen regulation, and secondary metabolite production. In this article, the PhoP regulation has been confirmed by electrophoretic mobility shift assays (EMSA) of the most relevant members of the traditional pho regulon such as the two-component system PhoR-P or genes involved in high-affinity phosphate transport (pstSCAB) and low-affinity phosphate transport (pit). However, the PhoP control over phosphatase genes in S. tsukubaensis is significantly different from the pattern reported in the model bacteria Streptomyces coelicolor. Thus, neither the alkaline phosphatase PhoA nor PhoD is regulated by PhoP. On the contrary, the binding of PhoP to the promoter of a novel putative phosphatase PhoX was confirmed. A crosstalk of the PhoP and GlnR regulators, which balances phosphate and nitrogen utilization, also occurs in S. tsukubaensis but slightly modified. Finally, PhoP regulates genes, like afsS, that link phosphate control and secondary metabolite production in S. tsukubaensis. In summary, there are notable differences between the regulation of specific genes of the pho regulon in S. tsukubaensis and the model organism S. coelicolor.

SenX3-RegX3, an Important Two-Component System, Regulates Strain Growth and Butenyl-spinosyn Biosynthesis in Saccharopolyspora pogona

“…Initially, the phosphate effect was attributed to a decrease or inhibition of natural product production as a result of a phosphate increase in the culture medium ( Curdová et al., 1976 ). Further studies on phosphate metabolism-related genes identified several of these genes, such as alkaline phosphatase, polyphosphatase, and low- and high-affinity transporter genes, and the pho regulon ( Moura et al., 2001 ; Vuppada et al., 2018 ; Santos-Beneit et al., 2008 ; Martínez-Castro et al., 2018 ). Additionally, it was found that the molecular mechanism of phosphate control is mainly regulated by a two-component system ( Martín et al., 2017 ).…”

Regulation of Geldanamycin Biosynthesis by Cluster-Situated Transcription Factors and the Master Regulator PhoP

“…These two Streptomyces species grow in the absence of inorganic phosphate because they were able to hydrolyze organic phosphate present in the soy-peptone medium, whereas S. hygroscopicus seems unable to do so. This might be due to the absence of some extracellular phosphatases in S. hygroscopicus , as described also in S. tsukubaensis [62]. Although S. hygroscopicus phoP mutant grows well at 5 mM phosphate, the production of geldanamycin was higher at 15 mM which is a phosphate concentration normally inhibitory for antibiotic biosynthesis, as is the case in S. natalensis for pimaricin biosynthesis [55], suggesting that the phoP mutant is deregulated in PhoP mediated control of geldanamycin biosynthesis.…”

“…The response regulator PhoP has 223 amino acids and is 98% identical to the homologous protein of S. coelicolor and S. avermitilis . PhoP is extremely well conserved in all Streptomyces species [62] supporting its important role on the PhoR-PhoP mediated response [63]. The PhoP protein has the conserved aminoacids Asp 6 and Asp 49 in the amino terminal region, lysine K 98 for phosphorylation, as part of the receiver domain, and a DNA binding domain (DBD) in the carboxyl terminal end (amino acids 190-201).…”

Evaluation of Apple Root-Associated Endophytic Streptomyces pulveraceus Strain ES16 by an OSMAC-Assisted Metabolomics Approach

“…In particular, by limiting the nitrogen availability, Streptomyces species can be starved of essential amino acids leading to a stringent response that restricts transcription of numerous genes involved in vegetative growth (i.e., induces sporulation) and stimulates the expression of stress response genes (Bibb, 2005;Tiffert et al, 2008Tiffert et al, , 2011Dalebroux and Swanson, 2012;Martín and Liras, 2020). Additionally, phosphate starvation influences the two-component phosphate control of the metabolism system, PhoR-PhoP (Hutchings et al, 2004;Martín et al, 2012), slows down primary metabolism and triggers the production of secondary metabolites (Sola-Landa et al, 2003, 2005Martínez-Castro et al, 2018). Therefore, we successfully employed PDA limited in both nitrogen and phosphate ( Supplementary Table 2) as a high stress-inducing and sporulating medium for endophytic S. pulveraceus ES16.…”