Genome-Wide Analysis of In Vivo Binding of the Master Regulator DasR in Streptomyces coelicolor Identifies Novel Non-Canonical Targets

Abstract

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.

Multiple allosteric effectors control the affinity of DasR for its target sites

“…This is a very interesting observation that sheds new light on our recent discovery by system-wide in vivo DNA binding studies that DasR does not only bind to dre sites, but also to non-canonical target sites that do not conform to the dre consensus sequence [9]. During normal growth, DasR primarily bound to canonical (Class I) target sites that conformed to the dre consensus sequence, while during development specificity changed, with a strong increase in noncanonical binding events, whereby the latter were not bound in vitro [9]. Unfortunately, our in vitro studies suffered from the fact that not all DasR protein preparations were stable for longer periods of time, which obscured more detailed studies of the affinity of DasR for the different phosphorylated aminosugars (GlcNAc-6P or GlcN-6P).…”

“…This strongly suggests that DasR-GlcNAc-6P has a different affinity for its in vivo DNA binding sites (dre or otherwise) than DasR-GlcN-6P. This is a very interesting observation that sheds new light on our recent discovery by system-wide in vivo DNA binding studies that DasR does not only bind to dre sites, but also to non-canonical target sites that do not conform to the dre consensus sequence [9]. During normal growth, DasR primarily bound to canonical (Class I) target sites that conformed to the dre consensus sequence, while during development specificity changed, with a strong increase in noncanonical binding events, whereby the latter were not bound in vitro [9].…”

“…In contrast, the crystal structures of NagR in complex with GlcN-6-P and GlcNAc-6-P suggests that both molecules similarly displace the NagR-DNAbinding domains and therefore should similarly affect the DNAbinding ability of the regulator [19]. Recently, we have shown via genome-wide systems biology analysis that DasR can also bind to genes that do not contain a dre site in their promoter region, in particular during a later stage of its life cycle [8,9]. How DasR recognizes such alternative sites, designated Class II (as opposed to Class I for genes controlled via a canonical dre), is yet unclear but presumably requires a co-repressor and/or an effector molecule.…”

“…'Das' stands for Defect in aerial hyphae and spore formation which refers to the impaired sporulation phenotype exhibited by the dasR null mutants of Streptomyces griseus [7] and Streptomyces coelicolor [2]. The observed developmental defect of dasR mutants fits with a regulon that extends well beyond GlcNAc and chitin utilization genes [8,9], such as gene clusters involved in the production of antibiotics prodiginines and actinorhodin [10] and the desferrioxamine siderophores [11] which are essential for proper sporulation of S. coelicolor [12,13]. The identification of the DasR regulon and the functional characterization of the genes it controls allows the description of the very first complete signalling cascade from extracellular signal transport to the control of secondary metabolite production in the industrially important Streptomyces genus [10].…”

The master regulator PhoP coordinates phosphate and nitrogen metabolism, respiration, cell differentiation and antibiotic biosynthesis: comparison in Streptomyces coelicolor and Streptomyces avermitilis

“…26 Similarly, in chitin metabolism, a regulatory protein, DasR, binds to promoter regions involved in N-acetylglucosamine metabolism that are also regulated by PhoP. 41 The interaction of regulatory proteins may estabilize the binding of PhoP to weakly conserved PHO boxes and explains why there are so many PhoPbinding regions detected in the DNA by the ChIP-on-chip technique.…”