Streptomycetes produce a wealth of natural products, including over half of all known antibiotics. It was previously demonstrated that N-acetylglucosamine and secondary metabolism are closely entwined in streptomycetes. Here we show that DNA recognition by the N-acetylglucosamine-responsive regulator DasR is growth-phase dependent, and that DasR can bind to sites in the S. coelicolor genome that have no obvious resemblance to previously identified DasR-responsive elements. Thus, the regulon of DasR extends well beyond what was previously predicted and includes a large number of genes with functions far removed from N-acetylglucosamine metabolism, such as genes for small RNAs and DNA transposases. Conversely, the DasR regulon during vegetative growth largely correlates to the presence of canonical DasR-responsive elements. The changes in DasR binding in vivo following N-acetylglucosamine induction were studied in detail and a possible molecular mechanism by which the influence of DasR is extended is discussed. Discussion of DasR binding was further informed by a parallel transcriptome analysis of the respective cultures. Evidence is provided that DasR binds directly to the promoters of all genes encoding pathway-specific regulators of antibiotic production in S. coelicolor, thereby providing an exquisitely simple link between nutritional control and secondary metabolism.

Summary Actinobacteria are producers of a plethora of natural products of agricultural, biotechnological and clinical importance. In an era where mankind has to deal with rapidly spreading antimicrobial resistance, streptomycetes are of particular importance as producers of half of all antibiotics used in the clinic. Genome sequencing efforts revealed that their capacity as antibiotic producers has been underestimated, in particular as many biosynthetic pathways are silent under standard laboratory conditions. Here we review the global regulatory networks that control antibiotic production in streptomycetes, with emphasis on carbon‐ and aminosugar‐related nutrient sensory pathways. Recent research has revealed intriguing connections between these regulons, and overlap and antagonism between the activities of among others the global regulatory proteins AtrA, DasR and Rok7B7 as well as GlnR (nitrogen control) and PhoP (phosphate control), are discussed. Finally, we provide ideas as to how these novel insights might help us to find ways to activate the transcription of silent biosynthetic gene clusters.

TonB-dependent transporters (TBDTs) are ubiquitous outer membrane β-barrel proteins that import nutrients and bacteriocins across the outer membrane in a proton motive force-dependent manner, by directly connecting to the ExbB/ExbD/TonB system in the inner membrane. Here, we show that the TBDT Oar in Myxococcus xanthus is required for secretion of a protein, protease PopC, to the extracellular milieu. PopC accumulates in the periplasm before secretion across the outer membrane, and the proton motive force has a role in secretion to the extracellular milieu. Reconstitution experiments in Escherichia coli demonstrate that secretion of PopC across the outer membrane not only depends on Oar but also on the ExbB/ExbD/TonB system. Our results indicate that TBDTs and the ExbB/ExbD/TonB system may have roles not only in import processes but also in secretion of proteins.

a b s t r a c tThe global transcriptional regulator DasR connects N-acetylglucosamine (GlcNAc) utilization to the onset of morphological and chemical differentiation in the model actinomycete Streptomyces coelicolor. Previous work revealed that glucosamine-6-phosphate (GlcN-6P) acts as an allosteric effector which disables binding by DasR to its operator sites (called dre, for DasR responsive element) and allows derepression of DasR-controlled/GlcNAc-dependent genes. To unveil the mechanism by which DasR controls S. coelicolor development, we performed a series of electromobility shift assays with histidinetagged DasR protein, which suggested that N-acetylglucosamine-6-phosphate (GlcNAc-6P) could also inhibit the formation of DasR-dre complexes and perhaps even more efficiently than GlcN-6P. The possibility that GlcNAc-6P is indeed an efficient allosteric effector of DasR was further confirmed by the high and constitutive activity of the DasR-repressed nagKA promoter in the nagA mutant, which lacks GlcNAc-6P deaminase activity and therefore accumulates GlcNAc-6P. In addition, we also observed that high concentrations of organic or inorganic phosphate enhanced binding of DasR to its recognition site, suggesting that the metabolic status of the cell could determine the selectivity of DasR in vivo, and hence its effect on the expression of its regulon.

In the model species Streptomyces coelicolor A3(2), the uptake of chitin-degradation byproducts, mainly N,N′- diacetylchitobiose ([GlcNAc]2) and N-acetylglucosamine (GlcNAc), is performed by the ATP-binding cassette (ABC) transporter DasABC-MsiK and the sugar-phosphotransferase system (PTS), respectively. Studies on the S. coelicolor chromosome have suggested the occurrence of additional uptake systems of GlcNAc-related compounds, including the SCO6005–7 cluster, which is orthologous to the ABC transporter NgcEFG of S. olivaceoviridis. However, despite conserved synteny between the clusters in S. coelicolor and S. olivaceoviridis, homology between them is low, with only 35% of residues being identical between NgcE proteins, suggesting different binding specificities. Isothermal titration calorimetry experiments revealed that recombinant NgcESco interacts with GlcNAc and (GlcNAc)2, with Kd values (1.15 and 1.53 μM, respectively) that were higher than those of NgcE of S. olivaceoviridis (8.3 and 29 nM, respectively). The disruption of ngcESco delayed (GlcNAc)2 consumption, but did not affect GlcNAc consumption ability. The ngcESco-dasA double mutation severely decreased the ability to consume (GlcNAc)2 and abolished the induction of chitinase production in the presence of (GlcNAc)2, but did not affect the GlcNAc consumption rate. The results of these biochemical and reverse genetic analyses indicate that NgcESco acts as a (GlcNAc)2- binding protein of the ABC transporter NgcEFGSco-MsiK. Transcriptional and biochemical analyses of gene regulation demonstrated that the ngcESco gene was slightly induced by GlcNAc, (GlcNAc)2, and chitin, but repressed by DasR. Therefore, a model was proposed for the induction of the chitinolytic system and import of (GlcNAc)2, in which (GlcNAc)2 generated from chitin by chitinase produced leakily, is mainly transported via NgcEFG-MsiK and induces the expression of chitinase genes and dasABCD.