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 Streptomyces lincolnensis. 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 S. lincolnensis LCGL. SLCG_2919 was found to specifically bind to promoter regions of the lincomycin biosynthetic gene cluster (lin 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, SLCG_2920, which encodes an ATP/GTP binding protein whose overexpression increased resistance against lincomycin and Lin-A yields in S. lincolnensis. The precise SLCG_2919 binding site within the promoter region of SLCG_2920 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 S. lincolnensis. The present study improves our understanding of molecular regulation for lincomycin biosynthesis. IMPORTANCE TetR family transcriptional regulators (TFRs) are generally found to regulate diverse cellular processes in bacteria, especially antibiotic biosynthesis in Streptomyces species. However, knowledge of their function in lincomycin biosynthesis in S. lincolnensis 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, SLCG_2919 and its adjoining gene, SLCG_2920, which encodes an ATP/GTP binding protein, were extensively distributed in diverse Streptomyces species. In addition, we revealed a new TFR binding motif, in which SLCG_2919 binds to the promoter region of SLCG_2920, 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 Streptomyces species to improve antibiotic production.

AdpAlin, a Pleiotropic Transcriptional Regulator, Is Involved in the Cascade Regulation of Lincomycin Biosynthesis in Streptomyces lincolnensis

“…Meng et al (2017) revealed the regulatory network between nitrate metabolism and lincomycin biosynthesis where GlnR activates the transcription of lmrA , the lincomycin exporter gene. Lately, a TetR-type regulator SLCG_2919 has been identified as a repressor of lincomycin biosynthesis that controls the transcription of lmbA , lmbC , lmbE , lmbG , lmbK , lmbR , lmbV , and lmbW (Xu et al, 2019). However, to complete the regulatory network of lincomycin biosynthesis, there are lots of gaps remained.…”

LmbU, a Cluster-Situated Regulator for Lincomycin, Consists of a DNA-Binding Domain, an Auto-Inhibitory Domain, and Forms Homodimer

“…In our previous study, we have demonstrated that a global regulator BldA (Hou et al, 2018) and a CSR LmbU (Hou et al, 2017) are involved in lincomycin biosynthesis. Recently, a TetR-type regulator SLCG_2919 has been proved to negatively regulate lincomycin biosynthesis (Xu et al, 2018). However, the regulatory mechanism of lincomycin biosynthesis is still unclear at present.…”

Uncovering and Engineering a Mini-Regulatory Network of the TetR-Family Regulator SACE_0303 for Yield Improvement of Erythromycin in Saccharopolyspora erythraea

“…The DNase I footprinting assay was performed as previously described (Xu et al, 2019a). To precisely determine the DNA binding site of SACE_0303, 100 ng FAM/HEX-labeled P 0303−0304 was successively incubated with 0, 70, and 490 nM His6-SACE_0303 in a total 50 µL of binding buffer at 20 • C for 20 min, and then 2 µL DNase I (1 U/µg; Promega) was performed at 20 • C for 30 s, 10 µL DNase I stop solution was added to the mixture and reacted at 65 • C for 10 min.…”