TetR-Type Regulator SLCG_2919 Is a Negative Regulator of Lincomycin Biosynthesis in Streptomyces lincolnensis

Abstract

Lincomycin A (Lin-A) is a widely used antibacterial antibiotic fermented by <i>Streptomyces lincolnensis</i> However, the transcriptional regulatory mechanisms underlying lincomycin biosynthesis have seldom been investigated. Here, we first identified a TetR family transcriptional regulator (TFR), SLCG_2919, which negatively modulates lincomycin biosynthesis in <i>S. lincolnensis</i> LCGL. SLCG_2919 was found to specifically bind to promoter regions of the lincomycin biosynthetic gene cluster (<i>lin</i> cluster), including 25 structural genes, three resistance genes, and one regulatory gene, and to inhibit the transcription of these genes, demonstrating a directly regulatory role in lincomycin biosynthesis. Furthermore, we found that SLCG_2919 was not autoregulated, but directly repressed its adjacent gene, <i>SLCG_2920</i>, which encodes an ATP/GTP binding protein whose overexpression increased resistance against lincomycin and Lin-A yields in <i>S. lincolnensis</i> The precise SLCG_2919 binding site within the promoter region of <i>SLCG_2920</i> was determined by a DNase I footprinting assay and by electrophoretic mobility shift assays (EMSAs) based on base substitution mutagenesis, with the internal 10-nucleotide (nt) AT-rich sequence (AAATTATTTA) shown to be essential for SLCG_2919 binding. Our findings indicate that SLCG_2919 is a negative regulator for controlling lincomycin biosynthesis in <i>S. lincolnensis</i> The present study improves our understanding of molecular regulation for lincomycin biosynthesis.<b>IMPORTANCE</b> TetR family transcriptional regulators (TFRs) are generally found to regulate diverse cellular processes in bacteria, especially antibiotic biosynthesis in <i>Streptomyces</i> species. However, knowledge of their function in lincomycin biosynthesis in <i>S. lincolnensis</i> remains unknown. The present study provides a new insight into the regulation of lincomycin biosynthesis through a TFR, SLCG_2919, that directly modulates lincomycin production and resistance. Intriguingly, <i>SLCG_2919</i> and its adjoining gene, <i>SLCG_2920</i>, which encodes an ATP/GTP binding protein, were extensively distributed in diverse <i>Streptomyces</i> species. In addition, we revealed a new TFR binding motif, in which SLCG_2919 binds to the promoter region of <i>SLCG_2920</i>, dependent on the intervening AT-rich sequence rather than on the flanking inverted repeats found in the binding sites of other TFRs. These insights into transcriptional regulation of lincomycin biosynthesis by SLCG_2919 will be valuable in paving the way for genetic engineering of regulatory elements in <i>Streptomyces</i> species to improve antibiotic production.