Abstract
Streptomycetes are important biotechnological bacteria with complex differentiation. Copper is a well-known positive regulator of differentiation and antibiotic production. However, the specific mechanisms buffering cytosolic copper and the biochemical pathways modulated by copper remain poorly understood. Here, we developed a new methodology to quantify cytosolic copper in single spores which allowed us to propose that cytosolic copper modulates asynchrony of germination. We also characterised the SCO2730/2731 copper chaperone/P-type ATPase export system. A Streptomyces coelicolor strain mutated in SCO2730/2731 shows an important delay in germination, growth and sporulation. Secondary metabolism is heavily enhanced in the mutant which is activating the production of some specific secondary metabolites during its whole developmental cycle, including germination, the exponential growth phase and the stationary stage. Forty per cent of the S. coelicolor secondary metabolite pathways, are activated in the mutant, including several predicted pathways never observed in the lab (cryptic pathways). Cytosolic copper is precisely regulated and has a pleiotropic effect in gene expression. The only way that we know to achieve the optimal concentration for secondary metabolism activation, is the mutagenesis of SCO2730/2731. The SCO2730/2731 genes are highly conserved. Their inactivation in industrial streptomycetes may contribute to enhance bioactive compound discovery and production.
Highlights
Streptomycetes are important biotechnological bacteria from which two thirds of the bioactive secondary metabolites used in clinic were discovered[1,2]
There are some proteins known to be involved in Streptomyces spore germination: NepA, a structural cell wall protein involved in the maintenance of spore dormancy in S. coelicolor[8]; SsgA, a protein marking cell-wall sites where germination takes place[9]; resuscitation-promoting factors (Rpfs), cell wall hydrolases[10] controlling germination; OsdR, a Streptomyces orthologue to the M. tuberculosis DevR dormancy regulator, which was demonstrated to be functional in Mycobacterium[11]; and SCO4439, a D-alanyl-D-alanine carboxypeptidase that controls spore
Cytosolic copper reaches its minimum concentration during germination (11.8 ± 0.3 ng Cu/mg protein) which suggests that the SCO2730/2731 secretion system has a higher affinity for copper than does the SCO1045/1046 chaperone/transporter that is activated at the higher cytosolic copper levels reached in the mycelium (Fig. 3A,B)
Summary
Streptomycetes are important biotechnological bacteria from which two thirds of the bioactive secondary metabolites used in clinic (mainly antibiotics, and antitumorals, immunosupressors, etc.) were discovered[1,2] They have a complex developmental cycle that makes this bacterium a multicellular prokaryotic model including programmed cell death (PCD) and hyphae differentiation, which leads to aerial mycelium formation and sporulation[3,4]. Worrall and Vijgenboom[16] predicted the existence of two copper chaperone/P-type ATPases (CopZ/ CopA) modulating copper export (SCO1045/1046, SCO2730/2731). They demonstrated that the expression of the genes encoding these transporters is regulated by CsoR, the master transcriptional repressor modulating copper effects in gene transcription[16]. The existence of a membrane-bound periplasmic cupric reductase and a P-type ATPase importing Cu(I) has been postulated[18]
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