Systems-wide analysis of the ROK-family regulatory gene rokL6 and its role in the control of glucosamine toxicity in Streptomyces coelicolor

Streptomycetes are saprophytic bacteria that grow on complex polysaccharides, such as cellulose, starch, chitin and chitosan. For the monomeric building blocks glucose, maltose and N-acetylglucosamine (GlcNAc), the metabolic pathways are well documented, but that of glucosamine (GlcN) is largely unknown. Streptomyces nagB mutants, which lack glucosamine-6-phosphate deaminase activity, fail to grow in the presence of high concentrations of GlcN. Here we report that mutations in the gene for the ROK-family transcriptional regulator RokL6 relieve the toxicity of GlcN in nagB mutants, as a result of elevated expression of the Major Facilitator Superfamily (MFS) exporter SCO1448. Systems- wide analysis using RNA sequencing, ChIP-Seq, EMSAs, 5RACE, bioinformatics and genetics revealed that RokL6 is an autoregulator that represses transcription of sco1448 by binding to overlapping promoters in the rokL6-sco1448 intergenic region. RokL6-independent expression of sco1448 fully relieved toxicity of GlcN to nagB mutants. Taken together, our data show a novel system of RokL6 as a regulator that controls the expression of the MFS transporter SCO1448, which in turn protects cells against GlcN toxicity, most likely by exporting toxic metabolites out of the cell. IMPORTANCECentral metabolism plays a key role in the control of growth and antibiotic production in streptomycetes. Specifically, aminosugars act as signaling molecules that affect development and antibiotic production, via metabolic interference with the global repressor DasR. While aminosugar metabolism directly connects to other major metabolic routes such as glycolysis and cell wall synthesis, several important aspects of their metabolism are yet unresolved. Accumulation of N-acetylglucosamine 6-phosphate (GlcNAc-6P) or glucosamine 6-phosphate (GlcN-6P) is lethal to many bacteria, a yet unresolved phenomenon referred to as "aminosugar sensitivity". We made use of this concept by selecting for suppressors in genes related to GlcN toxicity in nagB mutants, which showed that the gene pair of rok-family regulatory gene rokL6 and MFS transporter gene sco1448 forms a cryptic rescue mechanism. Inactivation of rokL6 resulted in the expression of sco1448, which then prevents toxicity of amino sugar-derived metabolites in Streptomyces. The systems biology of RokL6 and its transcriptional control of sco1448 sheds new light on aminosugar metabolism in streptomycetes and on the response of bacteria to aminosugar toxicity.