Functional Analysis of the N-Acetylglucosamine Metabolic Genes of Streptomyces coelicolor and Role in Control of Development and Antibiotic Production

Abstract

N-Acetylglucosamine, the monomer of chitin, is a favored carbon and nitrogen source for streptomycetes. Its intracellular catabolism requires the combined actions of the N-acetylglucosamine-6-phosphate (GlcNAc-6P) deacetylase NagA and the glucosamine-6-phosphate (GlcN-6P) deaminase/isomerase NagB. GlcNAc acts as a signaling molecule in the DasR-mediated nutrient sensing system, activating development and antibiotic production under poor growth conditions (famine) and blocking these processes under rich conditions (feast). In order to understand how a single nutrient can deliver opposite information according to the nutritional context, we carried out a mutational analysis of the nag metabolic genes nagA, nagB, and nagK. Here we show that the nag genes are part of the DasR regulon in Streptomyces coelicolor, which explains their transcriptional induction by GlcNAc. Most likely as the result of the intracellular accumulation of GlcN-6P, nagB deletion mutants fail to grow in the presence of GlcNAc. This toxicity can be alleviated by the additional deletion of nagA. We recently showed that in S. coelicolor, GlcNAc is internalized as GlcNAc-6P via the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS). Considering the relevance of GlcNAc for the control of antibiotic production, improved insight into GlcNAc metabolism in Streptomyces may provide new leads toward biotechnological applications.

Taxonomy, Physiology, and Natural Products of Actinobacteria

“…GlcNAc-derived GlcN-6-P acts as an allosteric effector of the GntR family regulator DasR (186), a global regulator that controls the GlcNAc regulon (186,360,361), and also the production of antibiotics (187) and siderophores (362). GlcNAc-dependent nutritional signaling is most likely mediated through changes in the intracellular level of GlcN-6-P, which binds as a ligand to the GntR family regulator DasR, leading to derepression of DasR-mediated control of antibiotic production (187).…”

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

“…The first indirect evidence of a role of GlcNAc-6P in the GlcNAc-mediated response was provided by the examination of the phenotype of the S. coelicolor nagA null mutant (DnagA IFD (GAM4), [23,24]) in which GlcNAc-6P accumulates inside the cytoplasm. As described previously [2], GlcNAc blocks morphological differentiation (sporulation) and antibiotic production of the parental strain S. coelicolor M145 grown on rich R2YE agar plates (see also strains M145 and ET001 in Fig.…”

“…DasR-6His was produced in E. coli BL21(DE3) and purified onto a Ni 2þ -nitrilotriacetic acid-agarose column as previously described [23]. Pooled fractions with the pure protein were dialyzed overnight at 4 C against EMSA buffer (10 mM Tris-Cl, pH 7.5, 1 mM dithiothreitol, 0.25 mM CaCl 2 , 0.5 mM MgCl 2 , 50 mM KCl, and 2% glycerol).…”

“…Oligonucleotide used to generate Cy5-fluorescent 40-bp double-stranded DNA probes (dre nagKA , dre dasA , and dre nagB ) are described in Ref. [23].…”

Extracellular sugar phosphates are assimilated by Streptomyces in a PhoP-dependent manner

“…Although the role of ubiquist molecules in signalisation has been poorly investigated, several studies confirmed that these molecules are likely to signal specific metabolic state and thus also play important regulatory roles (Viollier et al 2001a, b;Li et al 2008;Rigali et al 2008;Luti and Mavituna 2011). For instance, we have previously demonstrated that the peptidoglycan aminosugar building block N-acetylglucosamine (GlcNAc) has a different impact on development depending on the nutritional context and its concentration (Rigali et al 2006(Rigali et al , 2008Nothaft et al 2010;Craig et al 2012;Swiatek et al 2012).…”