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Regulators of the WhiB-like (wbl) family are playing important role in the complex regulation of metabolic and morphological differentiation in Streptomyces. In this study, we investigated the role of wblI, a member of this family, in the regulation of secondary metabolite production in Streptomyces lividans. The over-expression of wblI was correlated with an enhanced biosynthesis of undecylprodigiosin and actinorhodin and with a reduction of the biosynthesis of yCPK and of the grey spore pigment encoded by the whiE locus. Five regulatory targets of WblI were identified using in vitro formaldehyde crosslinking and confirmed by EMSA and qRT-PCR. These included the promoter regions of wblI itself, two genes of the ACT cluster (actVA3 and the intergenic region between the divergently orientated genes actII-1 and actII-2) and that of wblA, another member of the Wbl family. Quantitative RT-PCR analysis indicated that the expression of actVA3 encoding a protein of unknown function as well as that of actII-1, a TetR regulator repressing the expression of actII-2, encoding the ACT transporter, were down regulated in the WblI over-expressing strain. Consistently the expression of the transporter actII-2 was up-regulated. The expression of WblA, that is known to have a negative impact on ACT biosynthesis, was strongly down regulated in the WblI over-expressing strain. These data are consistent with the positive impact that WblI over-expression has on ACT biosynthesis. The latter might result from direct activation of ACT biosynthesis and export and from repression of the expression of WblA, a likely indirect, repressor of ACT biosynthesis.

The Streptomyces genus is well known for its ability to produce bio-active secondary metabolites of great medical interest. However, the metabolic features accompanying these bio-productions remain to be defined. In this study, the comparison of related model strains producing differing levels of actinorhoddin (ACT), showed that S. lividans, a weak producer, had high TriAcylGlycerol (TAG) content indicative of a glycolytic metabolism. In contrast, the strong producer, S. coelicolor, was characterized by low TAG content, active consumption of its polyphosphate (PolyP) stores and extremely high ATP/ADP ratios. This indicated highly active oxidative metabolism that was correlated with induction of ACT biosynthesis. Interestingly, in conditions of phosphate limitation, the ppk mutant had TAG content and ACT production levels intermediary between those of S. lividans and S. coelicolor. This strain was characterized by high ADP levels indicating that Ppk was acting as an Adenosine Di Phosphate Kinase. Its absence resulted in energetic stress that is proposed to trigger an activation of oxidative metabolism to restore its energetic balance. This process, which is correlated with ACT biosynthesis, requires acetylCoA to fuel the Krebs cycle and phosphate for ATP generation by the ATP synthase coupled to the respiratory chain, resulting in low TAG and polyP content of the ACT producing strains.

Polyphosphate kinases (PPK) from different bacteria, including that of Streptomyces lividans, were shown to contain the typical HKD motif present in phospholipase D (PLD) and showed structural similarities to the latter. This observation prompted us to investigate the PLD activity of PPK of S. lividans, in vitro. The ability of PPK to catalyze the hydrolysis of phosphatidylcholine (PC), the PLD substrate, was assessed by the quantification of [ 3 H]phosphatidic acid (PA) released from [ 3 H]PC-labeled ELT3 cell membranes. Basal cell membrane PLD activity as well as GTPγS-activated PLD activity was higher in the presence than in absence of PPK. After abolition of the basal PLD activity of the membranes by heat or tryptic treatment, the addition of PPK to cell membranes was still accompanied by an increased production of PA demonstrating that PPK also bears a PLD activity. PLD activity of PPK was also assessed by the production of choline from hydrolysis of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the presence of the Amplex Red reagent and compared to two commercial PLD enzymes. These data demonstrated that PPK is endowed with a weak but clearly detectable PLD activity. The question of the biological signification, if any, of this enzymatic promiscuity is discussed.

Phosphate, as a constituent of the high energy molecules, ATP/GTP and polyphosphate, plays a crucial role in most of the metabolic processes of living organisms. Therefore, the adaptation to low Pi availability is a major challenge for bacteria. In Streptomyces, this adaptation is tightly controlled by the two component PhoR/PhoP system. In this study, the free intracellular Pi, ATP, ADP and polyP content of the wild type and the phoP mutant strain of S. lividans TK24 were analyzed at discrete time points throughout growth in Pi replete and limited media. PolyP length and content was shown to be directly related to the Pi content of the growth medium. In Pi repletion, ATP and high molecular weight (HMW) polyP contents were higher in the phoP mutant than in the WT strain. This supports the recently proposed repressive effect of PhoP on oxidative phosphorylation. High oxidative phosphorylation activity might also have a direct or indirect positive impact on HMW polyP synthesis. In Pi sufficiency as in Pi limitation, the degradation of these polymers was shown to be clearly delayed in the phoP mutant, indicating PhoP dependent expression of the enzymes involved in this degradation. The efficient storage of Pi as polyphosphate and/or its inefficient degradation in Pi in the phoP mutant resulted in low levels of free Pi and ATP that are likely to be, at least in part, responsible for the very poor growth of this mutant in Pi limitation. Furthermore, short polyP was shown to be present outside the cell, tightly bound to the mycelium via electrostatic interactions involving divalent cations. Less short polyP was found to be associated with the mycelium of the phoP mutant than with that of the WT strain, indicating that generation and externalization of these short polyP molecules was directly or indirectly dependent on PhoP.

Streptomyces lividans TK24 is a strain that naturally produces antibiotics at low levels, but dramatic overproduction of antibiotics occurs upon interruption of the ppk gene. However, the role of the Ppk enzyme in relation to the regulation of antibiotic biosynthesis remains poorly understood. In order to gain a better understanding of the phenotype of the ppk mutant, the proteomes of the wild-type (wt) and ppk mutant strains, grown for 96 h on R2YE medium limited in phosphate, were analyzed. Intracellular proteins were separated on two-dimensional (2D) gels, spots were quantified, and those showing a 3-fold variation or more were identified by mass spectrometry. The expression of 12 proteins increased and that of 29 decreased in the ppk mutant strain. Our results suggested that storage lipid degradation rather than hexose catabolism was taking place in the mutant. In order to validate this hypothesis, the triacylglycerol contents of the wt and ppk mutant strains of S. lividans as well as that of Streptomyces coelicolor M145, a strain that produces antibiotics at high levels and is closely related to S. lividans, were assessed using electron microscopy and thin-layer chromatography. These studies highlighted the large difference in triacylglycerol contents of the three strains and confirmed the hypothetical link between storage lipid metabolism and antibiotic biosynthesis in Streptomyces.

The ppk gene of Streptomyces lividans encodes an enzyme catalyzing, in vitro, the reversible polymerization of the gamma phosphate of ATP into polyphosphate and was previously shown to play a negative role in the control of antibiotic biosynthesis (H. Chouayekh and M. J. Virolle, Mol. Microbiol. 43:919-930, 2002). In the present work, some regulatory features of the expression of ppk were established and the polyphosphate content of S. lividans TK24 and the ppk mutant was determined. In Pi sufficiency, the expression of ppk was shown to be low but detectable. DNA gel shift experiments suggested that ppk expression might be controlled by a repressor using ATP as a corepressor. Under these conditions, short acid-soluble polyphosphates accumulated upon entry into the stationary phase in the wild-type strain but not in the ppk mutant strain. The expression of ppk under Pi-limiting conditions was shown to be much higher than that under Pi-sufficient conditions and was under positive control of the two-component system PhoR/PhoP. Under these conditions, the polyphosphate content of the cell was low and polyphosphates were reproducibly found to be longer and more abundant in the ppk mutant strain than in the wild-type strain, suggesting that Ppk might act as a nucleoside diphosphate kinase. In light of our results, a novel view of the role of this enzyme in the regulation of antibiotic biosynthesis in S. lividans TK24 is proposed.

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.