Conserved <i>cis</i>-Acting Elements Upstream of Genes Composing the Chitinolytic System of Streptomycetes Are DasR-Responsive Elements

Abstract

For soil-dwelling bacteria that usually live in a carbon-rich and nitrogen-poor environment, the ability to utilize chitin – the second most abundant polysaccharide on earth – is a decisive evolving advantage as it is a source for both elements. Streptomycetes are high-GC Gram-positive soil bacteria that are equipped with a broad arsenal of chitinase-degrading genes. These genes are induced when the streptomycetes sense the presence of chitooligosaccharides. Their expression is repressed as soon as more readily assimilated carbon sources become available. This includes for example glucose or N-acetylglucosamine, the monomer subunit of chitin. Historically, the first cis-acting elements involved in carbon regulation in streptomycetes were found more than a decade ago upstream of chitinase genes, but the transcriptional regulator had so far remained undiscovered. In this work, we show that these cis-acting elements consist of inverted repeats with multiple occurrences and are bound by the HutC/GntR type regulator DasR. We have therefore designated these sites as DasR-responsive elements (dre). DasR, which is also the repressor of the genes for the N-acetylglucosamine-specific phosphotransferase transport system, should therefore play a critical role in sensing the balance between the monomeric and polymeric forms of N-acetylglucosamine.

Taxonomy, Physiology, and Natural Products of Actinobacteria

“…For bacteria, GlcNAc is a favorable C and N source and a major constituent of the cell wall peptidoglycan. Some 13 chitinases and chitosanases have been identified in S. coelicolor (353)(354)(355).…”

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

“…This principle has been applied successfully to enhance the expression of antibiotic biosynthetic gene clusters, including the clusters for Act, Red, and the cryptic polyketide Cpk (34). The GntR regulator DasR acts as a responsive nutrient sensor which has a pleiotropic regulon and represses, among others, nag metabolic genes, chitinolytic genes, and the pathway-specific activator genes for antibiotic production (9,26,32,34). Since the repressing activity of DasR is relieved allosterically by the effector molecule GlcN-6P (32), it follows that controlling the flux of GlcNAc may be a promising approach for metabolic engineering relating to improved screening of novel antimicrobial compounds.…”

“…Production and purification of His 6 -tagged DasR were based on a previously described protocol (9,23), with several modifications. Escherichia coli BL21 Rosetta (DE3) cells carrying pFT240 (33) were grown at 37°C in 250 ml LB medium containing 100 g/ml of ampicillin until the culture reached an optical density at 600 nm (OD 600 ) of 0.6.…”

“…Prior to assessing the effect of inactivation of the nag genes on the GlcNAc-mediated signaling pathway in S. coelicolor, we first analyzed the GlcNAc-dependent control of the nagA, nagB, and nagK genes. DasR is a global regulator of primary metabolism and antibiotic production and is known to control, among others, the chitinolytic system in Streptomyces coelicolor (9,26), as well as nagB (32). There is a DasR-responsive element (dre) at position Ϫ68 upstream of nagB (tGTGGTtTAGACCAaT; dre nagB ) with a 13-nt match to the dre consensus sequence (AGT GGTCTAGACCACT; dre cons ) (32).…”

Properties of CsnR, the Transcriptional Repressor of the Chitosanase Gene, csnA , of Streptomyces lividans

“…Reg1, the negative regulator of ␣-amylase genes in S. lividans, seems also to be involved in the genetic regulation of chitinase genes (36,37). Finally DasR, a member of the HutC/GntR subfamily, regulates the expression of some chitinase genes through interaction with the dre motif in S. coelicolor (10). DasR also has a more global effect on other genes involved in GlcNAc metabolism (10,41).…”