The ROK-family regulator RokL6 is involved in GlcN toxicity by controlling the transcription of SCO1448 in Streptomyces coelicolor [RNA-seq]

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 the metabolic pathway is well documented, but that of glucosamine (GlcN) is currently unknown. Importantly, GlcN is lethal to Streptomyces coelicolor nagB mutants, which lack glucosamine-6-phosphate deaminase activity. Here we report that spontaneous and directed mutations in the gene for the ROK-family protein RokL6 (SCO1447) relieve GlcN toxicity in nagB mutants of S. coelicolor. RNA sequencing, ChIP-Seq and over-expression studies revealed that RokL6 acts by directly repressing SCO1448, which encodes a sugar exporter; RokL6 thereby only binds to the rokL6-SCO1448 intergenic region in vivo, with consensus binding site C(T)TATCAGG - 7 nt - CCTGATAG(A). The exact transcriptional start sites for rokL6 and SCO1448 were determined using 5'RACE. RokL6 represses the transcription of both rokL6 and SCO1448 by binding to overlapping promoter sequences. Taken together, our data show that RokL6 and SCO1448 are novel GlcN-related genes, whereby RokL6 directly controls the transcription of SCO1448. The latter is a key protein in the defense of S. coelicolor against the toxicity of GlcN in a nagB-mutant background, most likely via the export of GlcN-derived toxic intermediates. Overall design: To investigare the transcriptional control by RokL6, RNAseq analysis was performed using RNA extracted from Streptomyces coelicolor M145 and its rokL6 null mutant grown on minimal medium agar plates with mannitol or mannitol plus GlcN.