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 ΔphoP 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.
Our interest concentrates on the interactions between nutritional global regulators, a subject that has emerged in the last few years as a promising field to understand the coordination of metabolism and the response to different nutritional factors in Streptomyces (Martín and Liras, 2010). The interaction between global regulators described below allows the cells to maintain an equilibrium of nutritional pathways (nutrient homeostasis), an aspect of great interest in natural conditions in the soil where feast and famine episodes are frequent. Similar coordination mechanisms have been described in other bacteria particularly in Escherichia coli and Bacillus subtilis (Kasahara et al., 1991; Commichau et al., 2006; Oh et al., 2007; Sonenshein, 2007).
The total number of genes responding to Pi limitation is considerably larger than those directly regulated by PhoP, as concluded from the transcriptome and proteome analysis of S. coelicolor wild-type and ΔphoP mutant strains after Pi depletion (Rodríguez-García et al., 2007). This is probably due to a PhoP-independent phosphate control mechanism that is still largely unknown. New interesting aspects of the interaction of PhoP with other regulators is becoming of paramount importance to understand the mechanisms of the transition from primary to secondary metabolism.
Response to phosphate starvation
Inorganic phosphate affects many reactions of primary and secondary metabolism in the cell. Therefore starvation of inorganic phosphate triggers important changes in the cell metabolism (Martín, 2004). These changes are of particular relevance in the transition from primary to secondary metabolism (Nieselt et al., 2010). Organic phosphates are abundant in plant and animal decaying materials but they are more difficult to be utilized by microorganisms than inorganic phosphate. However, most Streptomyces species have adequate phosphatase systems for efficient hydrolysis of the organic phosphates.
The primary response to Pi starvation involves the induction of extracellular enzymes to obtain Pi from organic phosphates, and the activation of genes encoding Pi transporters. Several phosphatases are activated in streptomycetes in response to phosphate starvation. The main organic phosphorus compounds in plant and animal-decaying materials in soil are phospholipids, inositol phosphates, sugar phosphates and nucleic acids. Inositol phosphates (phytates), which serve as phosphorous storage compounds in plants, frequently represent up to 80% of the total soil organic phosphorous (Quiquampoix and Mousain, 2005). The phoA and phoD genes encoding the secreted alkaline phosphatase and phospholipase D proteins, respectively, are activated by PhoP (Apel et al., 2007). The alkaline phosphatase (encoded by phoA) is a non-specific phosphomonoesterase which is commonly induced in response to Pi starvation in bacteria (Wanner, 1996). This enzyme was purified to homogeneity from Streptomyces griseus and its N-terminal amino acid sequence was used to identify the gene (Moura et al., 2001). In addition, the S. coelicolor genome contains another alkaline phosphatase gene, phoC. This gene, which shows a poor expression level, is induced by low Pi concentration in a PhoP-independent manner (Apel et al., 2007).
Another important group of enzymes that respond to phosphate starvation are phospholipases which release phosphatidic acids from phospholipids. Phospholipases are well-known virulence factors; they participate in Pi scavenging in both pathogenic and non-pathogenic bacteria (Titball, 1993; Oh et al., 2007). Several phospholipase D genes have been sequenced because of their biotechnological applications including genes from Streptomyces griseofuscus and Streptomyces antibioticus (Iwasaki et al., 1994; Masayama et al., 2008; Uesugi and Hatanaka, 2009).
Other genes encoding putative phosphate scavengers are SCO1906, SCO3790 and SCO4152. Both SCO1906 and SCO3790 encode putative secreted phosphatases (other than PhoA and PhoC) and their promoters are bound by PhoP (Rodríguez-García et al., 2007; Sola-Landa et al., 2008) (Fig. 1). SCO4152 encodes a putative secreted 5′-nucleotidase that is upregulated in the wild-type transcriptome after phosphate shift-down, but not in a ΔphoP mutant. Therefore, this gene is a candidate member of the pho regulon.
The released Pi from organic sources can enter the cell by means of (at least) three Pi transporters including the high-affinity Pst system, and the low-affinity high-velocity PitH1 and PitH2 systems (Díaz et al., 2005; Sola-Landa et al., 2005; Santos-Beneit et al., 2008). The pitH1 promoter is active in the ΔphoP mutant, whereas the promoters of the pstS gene (the first gene of the pstSCAB cluster) and of the pitH2 gene are totally dependent upon PhoP binding to become active. Following Pi depletion the highest activation of the pitH2 promoter precedes that of the pstS promoter. The high-affinity transporter (Pst) is energetically more expensive to the cell (because it consumes ATP) than the low-affinity Pit systems which is driven by the proton motive force. Nevertheless, the Pst system is effective in conditions of very low Pi concentrations due to its high affinity for Pi. These factors explain the sequential pattern of expression of these genes observed in flask cultures of S. coelicolor (Santos-Beneit et al., 2008) and in fermenters (Nieselt et al., 2010).
In addition to phosphate scavenging and release of Pi from organic phosphates, starvation of the cultures for phosphate triggers several other responses to phosphate starvation stress (Table 1).
Table 1. Metabolic responses to phosphate starvation.
All these genes are upregulated after Pi depletion and are PhoP-dependent.
Phosphate starvation appears to produce a PhoP-dependent switch in energy metabolism, downregulating the aerobic metabolism genes and upregulating the nitrate reductase.
Most genes encoding ribosomal proteins are downregulated after Pi depletion.
Production of undecylprodigiosin and actinorhodin takes place after Pi depletion (undecylprodigiosin is switched on a few hours earlier than actinorhodin).
Carbon and nitrogen sources, in addition to phosphate, play an important role in the regulation of primary and secondary metabolism in almost all bacteria including streptomycetes (Martín and Demain, 1980; Hobbs et al., 1990; Merrick and Edwards, 1995). These different mechanisms of control have been widely studied in an individual form, but the connections existing between them are still poorly known. A few examples of genes regulated simultaneously by different nutritional sources in Streptomyces species have been described up to now. The glpQ1 and glpQ2 genes of S. coelicolor (see previous section) are directly regulated by PhoP (Rodríguez-García et al., 2007; Santos-Beneit et al., 2009b) and their expression is also affected, in a different manner, by the carbon source (Santos-Beneit et al., 2009b).
An interaction of phosphate and carbon source regulation has been described for the pstS gene in Streptomyces lividans and S. coelicolor. PstS is the phosphate-binding protein belonging to the high-affinity phosphate-specific transport system (pst), a member of the pho regulon (Díaz et al., 2005; Sola-Landa et al., 2005; Rodríguez-García et al., 2007). The extracellular PstS protein is accumulated in high concentrations in liquid cultures when the bacteria are grown in the presence of different sugars under phosphate limitation conditions (Díaz et al., 2005; Esteban et al., 2008). Phosphorylated intermediates of the glycolysis may serve as integrator molecules of the phosphate and carbon metabolism. An interaction between phosphate and carbon sources has been shown also in other microorganisms; in Vibrio vulnificus, the expression of alkaline phosphatase and phospholipase genes is affected by both phosphate concentration and the carbon source (Oh et al., 2007). In B. subtilis it is known that the carbon catabolite protein-A (CcpA) exerts a control over the expression of phoRP genes, although there is a discrepancy about whether such regulation is exerted through the cre sequence (Puri-Taneja et al., 2006) or indirectly (Choi and Saier, 2005). The cross-talk between CcpA and the PhoRP system is a good example of interaction between mechanisms controlling metabolic fluxes in bacteria. In S. coelicolor the integration of the carbon and phosphate signalling pathways may be mediated by phosphorylated glycolytic intermediates since the phosphofructokinase gene (pfkA2) has been reported to play an important role in the control of secondary metabolite biosynthesis (Borodina et al., 2008).
Phosphate control oversees nitrogen regulation
The relationship between the phosphate and nitrogen regulatory pathways in S. coelicolor is complex because it affects not only the expression of genes involved in transport and assimilation of the nitrogen sources but also the direct nitrogen regulators. GlnR is the main nitrogen regulator and controls the expression of several genes involved in nitrogen metabolism at the transcriptional level (Wray and Fisher, 1993; Reuther and Wohlleben, 2007). In response to nitrogen limitation GlnR activates the transcription of numerous genes (Tiffert et al., 2008), including that of glnA and glnII (encoding two different types of glutamine synthetases) and the operon amtB–glnK–glnD, involved in ammonium transport and metabolism (Reuther and Wohlleben, 2007). GlnRII is a second regulator similar to GlnR that also binds to the promoters of glnA, glnII and amtB genes (Fink et al., 2002). However, GlnRII is not totally homologous to GlnR and its role in nitrogen regulation is not yet clear (Reuther and Wohlleben, 2007).
Recently, it was discovered that the regulation of nitrogen metabolism in S. coelicolor is under PhoP control (Rodríguez-García et al., 2007). Under phosphate limitation the expression of some nitrogen metabolism genes like glnA, glnII, amtB and glnK is increased in a S. coelicolorΔphoP mutant (Fig. 2). The transcriptomic data clearly indicate that these genes are overexpressed in the ΔphoP mutant. The control of PhoP over these genes is exerted indirectly through binding of this regulator to the promoter of glnR, responsible for their activation. In addition, there is also a direct control exerted by the binding of PhoP to the promoters of glnA, glnII and the amtB–glnK–glnD operon (Rodríguez-García et al., 2009). In the regulation of glnA there is a direct competition between the PhoP and GlnR proteins, which recognize overlapping sequences in its promoter (Rodríguez-García et al., 2009) (Fig. 3). GlnR has a higher affinity in vitro for this region than PhoP (A. Sola-Landa, unpublished). Overlapping binding sequences occur also in the glnII promoter. In the amtB promoter the PhoP- and GlnR-binding sequences are not overlapping, but PhoP appears to act as a ‘road-block’, preventing the correct formation of the RNA polymerase complex needed for the transcription (Fig. 3). This double mechanism (repression of glnR and also of the amtB–glnD–glnK operon) ensures a fine control of glnA, glnII and amtB–glnK–glnD by PhoP. A possible explanation for the phosphate–nitrogen control is the need for overall coordination of metabolism. Under phosphate limitation, the cells need to channel their energy resources to obtain phosphate and decrease the expression of the genes for nitrogen assimilation in order to equilibrate the P/N ratio in the hyphae, avoiding the energy expenses related to the utilization of unnecessary nitrogen sources.
Interestingly, PhoP binds to the promoter of glnR gene but not to the promoter of glnRII, suggesting that there is a ‘second’ nitrogen regulatory system independent of PhoP (Rodríguez-García et al., 2009). The cross-regulation between phosphate and nitrogen metabolism is not reciprocal since GlnR does not bind to promoters of genes of the pho regulon such as pstS and phoRP (A. Sola-Landa, unpubl. results).
Similar coordination mechanisms occur also in other microorganisms. A negative effect of phosphate starvation over the expression of nitrogen metabolism genes has been described in E. coli and Sinorhizobium meliloti (VanBogelen et al., 1996; Krol and Becker, 2004). It is very likely that similar phosphate–nitrogen interaction mechanisms will be discovered when systems biology studies are applied to other bacteria.
Interaction of global regulators PhoP and AfsR in the control of secondary metabolite biosynthesis
The complex regulation of secondary metabolites production in Streptomyces species and other bacteria is under the control of both global and pathway-specific regulators, which in turn respond to a variety of signals. As indicated above, phosphate limitation triggers the biosynthesis of hundreds of secondary metabolites in different Streptomyces species. The PhoR–PhoP system was shown to be involved in the regulation of actinorhodin and undecylprodigiosin production in S. lividans and S. coelicolor (Sola-Landa et al., 2003; Santos-Beneit et al., 2009a) and also in pimaricin production in Streptomyces natalensis (Mendes et al., 2007). The absence of PHO boxes and the lack of direct binding of PhoP to the genes encoding the antibiotic biosynthetic enzymes or the pathway-specific regulators, actII-ORF4 and redD, suggests that the PhoP regulatory effect may be exerted through signalling cascades involving intermediate regulatory genes or through interaction of PhoP with other regulators.
The AfsR action was initially studied in S. griseus and in S. coelicolor (Horinouchi et al., 1983). Recently we have described a cross-talk between the PhoP and AfsR proteins (Santos-Beneit et al., 2009a). AfsR is a positive regulator of actinorhodin and undecylprodigiosin in S. coelicolor and S. lividans. Floriano and Bibb (1996) found that the stimulatory effect of AfsR on antibiotic production is exerted through the activation of the pathway-specific regulatory genes actII-ORF4 and redD. The only known target of the AfsR regulator is a small gene located downstream of afsR, named afsS in S. coelicolor and afsR2 in S. lividans. A similar gene has been identified in the genomes of S. griseus and other Streptomyces species. Amplification of afsR2/afsS on a high-copy-number plasmid confers overproduction of actinorhodin and undecylprodigiosin in both S. coelicolor and S. lividans (Vögtli et al., 1994; Matsumoto et al., 1995; Floriano and Bibb, 1996). The binding sequence of AfsR to the afsS promoter region was identified by Lee and colleagues (2002); our analysis of this sequence identified a putative PHO box overlapping with the AfsR-binding sequence, which suggests that both PhoP and AfsR might interact in the regulation of afsS. DNase I footprinting studies revealed a PhoP protected region of 26 nucleotides that overlaps with the AfsR recognition sequence. Binding experiments indicated a competition between AfsR and PhoP; increasing concentrations of PhoP prevented formation of the AfsR–DNA complex (Santos-Beneit et al., 2009a).
Expression studies with S. coelicolor wild-type and ΔphoP strains using the reporter luxAB gene coupled to the afsS promoter showed that PhoP downregulates afsS expression probably by a competition with the AfsR activator. The binding site of PhoP in the afsS promoter overlaps the −35 region. The exact role of PhoP in the absence of AfsR is being investigated but we cannot exclude the possibility that in response to phosphate starvation PhoP itself activates afsS when AfsR is not present.
Interestingly, AfsR binds to other PhoP-regulated promoters including those of pstS (a member of the phosphate transport system) and phoRP (encoding the two-component system itself). Analysis of the AfsR- and PhoP-protected sequences in each of these promoters together with mutagenesis analyses of the overlapping sequences allowed us to distinguish some nucleotides in the AfsR-binding sequence different from the consensus PHO box (Santos-Beneit et al., 2009a; F. Santos-Beneit, unpublished).
The regulation of afsS by AfsR and PhoP allows the bacteria to integrate different signals (i.e. nutrients starvation) in the regulation of primary and secondary metabolism; in other words, the afsS promoter region integrates different signals, e.g. phosphate starvation through PhoP or the still unknown stress signal sensed by AfsR-associated sensor kinases. The same argument is true for the phoRP and pstS promoters that bind both PhoP and AfsR.
Regulation of secondary metabolite biosynthesis by N-acetylglucosamine and the global regulator DasR
An additional mechanism of nutritional control by N-acetylglucosamine occurs in S. coelicolor and probably in some other Streptomyces species, although it is unclear if this mechanism is universal in all streptomycetes. N-acetylglucosamine is the monomeric component of chitin, a constituent of cell wall of many filamentous fungi that is also present in the cuticle of insects and crustaceous. This polymer is utilized as carbon and nitrogen source by chitinases that have been characterized in different Streptomyces species (Schrempf, 2001; Kezuka et al., 2006).
N-acetylglucosamine induces the enzymes involved in its utilization, and also, increases the biosynthesis of actinorhodin and undecylprodigiosin in S. coelicolor (Rigali et al., 2008). This control is mediated by the global regulator DasR, a member of the GntR family (Colson et al., 2007; Rigali et al., 2008). A dasR mutant showed increased production of both actinorhodin and undecylprodigiosin suggesting that DasR regulates negatively the biosynthesis of these antibiotics. A DasR-responsive element was found in the upstream region of the crr–pts1 operon encoding the phosphotransferase system (PTS) enzyme, IIA (IIACrr), which partially matched the consensus DasR-binding sequence (Colson et al., 2007). DasR-responsive elements were found in the upstream regions of actII-ORF4 and redZ, the transcriptional activators of the act and red gene clusters.
It is intriguing why chitin (or N-acetylglucosamine) utilization is so important in the biosynthesis of actinorhodin and undecylprodigiosin. It seems that detection and utilization of these carbon sources may serve to sense the presence of fungi in soil, triggering a ‘competition stress’ mechanism that will lead to production of secondary metabolites by S. coelicolor, some of which may have antifungal activity, or may serve as communication signals triggering differentiation.
The DasR-mediated regulation is triggered during N-acetylglucosamine transport into the cells
Transport of N-acetylglucosamine is the first step that triggers the DasR-mediated transcriptional response. The NagE2 permease of the S. coelicolor phosphotransferase system is the key protein acting as N-acetylglucosamine receptor (Nothaft et al., 2010). Uptake of N-acetylglucosamine requires a phosphoryl group transfer from phosphoenolpyruvate via the phosphotransferases EI, HPr and IIACrr to NagF, which in turn phosphorylates N-acetylglucosamine during transport by the NagE2 permease, as described previously in Streptomyces olivaceoviridis (Wang et al., 2002; Saito and Schrempf, 2004). Transcription of the nagF and nagE2 genes is induced by N-acetylglucosamine.
The need of sequential phosphorylation (phosphorelay) of these enzymes and the supply of phosphoenolpyruvate connect the phosphorylation status of the cell (i.e. its energy charge) with the transport of N-acetylglucosamine and the induction of secondary metabolite biosynthesis. Further research is still needed on the role of DasR at a global regulator of secondary metabolism and its connection with the mechanism of classical carbon catabolite regulation exerted by glucose.
Conclusions and future outlook
Several examples of the interactions between the global nutritional regulators PhoP, GlnR, DasR, AfsR and other poorly known nutritional regulators occur in S. coelicolor. The interaction or competition between these global regulators allows the cell to reach a ‘metabolic homeostasis’ in response to nutritional unbalances in the soil. We have limited the scope of this minireview to those well-known examples but several other global regulators related to nutrition, are known in Streptomyces species (e.g. DmdR, SoxR), which probably respond to other nutritional stresses or environmental stimuli. Other regulators of antibiotic production like AbsA2, CutS, AbaB, AbsB, RapA2 and AfsQ2 have been described in Streptomyces species (Horinouchi et al., 1983; Champness et al., 1992; Scheu et al., 1997; Price et al., 1999; Chang et al., 2005; Lu et al., 2007; Shu et al., 2009) and further research is needed on the possible interactions with the global regulators described in detail in this article. A detailed knowledge of the molecular interactions and the integration of signals on the promoters of specific ‘integrator’ regulatory genes (e.g. afsS) will allow us to get a better picture of the key regulatory interactions that serve as the basis of systems biology.
The evidence available suggests that the PhoR–PhoP system is present in all sequenced Streptomyces genomes (e.g. Streptomyces ambofaciens, Streptomyces avermitilis, S. griseus, Streptomyces clavuligerus, etc.); however, given the great diversity of Streptomyces species, it is likely that modifications of the interactions between global regulators occur in some Streptomyces.
This work was supported by grants of the CICYT Consolider Project Bio2006-14853-O2-1 (Ministry of Science and Innovation, Spain) and the ERA Sysmo STREAM PROJECT (GEN2006-27745-E/SYS). Carlos Prieto was supported by a Juan de la Cierva programme (JCI-2009-05444) of the Ministry of Science and Innovation (Spain).