Dataset Information

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.

SUBMITTER: Xu Y

PROVIDER: S-EPMC6293104 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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

Xu Yurong Y Ke Meilan M Li Jie J Tang Yaqian Y Wang Nian N Tan Guoqing G Wang Yansheng Y Liu Ruihua R Bai Linquan L Zhang Lixin L Wu Hang H Zhang Buchang B

Applied and environmental microbiology 20181213 1

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 (< ...[more]

PMID: 30341075

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Project description:Lincomycin A is a clinically important antimicrobial agent produced by Streptomyces lincolnensis In this study, a new regulator designated LmbU (GenBank accession no. ABX00623.1) was identified and characterized to regulate lincomycin biosynthesis in S. lincolnensis wild-type strain NRRL 2936. Both inactivation and overexpression of lmbU resulted in significant influences on lincomycin production. Transcriptional analysis and in vivo neomycin resistance (Neor) reporter assays demonstrated that LmbU activates expression of the lmbA, lmbC, lmbJ, and lmbW genes and represses expression of the lmbK and lmbU genes. Electrophoretic mobility shift assays (EMSAs) demonstrated that LmbU can bind to the regions upstream of the lmbA and lmbW genes through the consensus and palindromic sequence 5'-CGCCGGCG-3'. However, LmbU cannot bind to the regions upstream of the lmbC, lmbJ, lmbK, and lmbU genes as they lack this motif. These data indicate a complex transcriptional regulatory mechanism of LmbU. LmbU homologues are present in the biosynthetic gene clusters of secondary metabolites of many other actinomycetes. Furthermore, the LmbU homologue from Saccharopolyspora erythraea (GenBank accession no. WP_009944629.1) also binds to the regions upstream of lmbA and lmbW, which suggests widespread activity for this regulator. LmbU homologues have no significant structural similarities to other known cluster-situated regulators (CSRs), which indicates that they belong to a new family of regulatory proteins. In conclusion, the present report identifies LmbU as a novel transcriptional regulator and provides new insights into regulation of lincomycin biosynthesis in S. lincolnensisIMPORTANCE Although lincomycin biosynthesis has been extensively studied, its regulatory mechanism remains elusive. Here, a novel regulator, LmbU, which regulates transcription of its target genes in the lincomycin biosynthetic gene cluster (lmb gene cluster) and therefore promotes lincomycin biosynthesis, was identified in S. lincolnensis strain NRRL 2936. Importantly, we show that this new regulatory element is relatively widespread across diverse actinomycetes species. In addition, our findings provide a new strategy for improvement of yield of lincomycin through manipulation of LmbU, and this approach could also be evaluated in other secondary metabolite gene clusters containing this regulatory protein.

Project description:UnlabelledPristinamycin I (PI), produced by Streptomyces pristinaespiralis, is a streptogramin type B antibiotic, which contains two proteinogenic and five aproteinogenic amino acid precursors. PI is coproduced with pristinamycin II (PII), a member of streptogramin type A antibiotics. The PI biosynthetic gene cluster has been cloned and characterized. However, thus far little is understood about the regulation of PI biosynthesis. In this study, a TetR family regulator (encoded by SSDG_03033) was identified as playing a positive role in PI biosynthesis. Its homologue, PaaR, from Corynebacterium glutamicum serves as a transcriptional repressor of the paa genes involved in phenylacetic acid (PAA) catabolism. Herein, we also designated the identified regulator as PaaR. Deletion of paaR led to an approximately 70% decrease in PI production but had little effect on PII biosynthesis. Identical to the function of its homologue from C. glutamicum, PaaR is also involved in the suppression of paa expression. Given that phenylacetyl coenzyme A (PA-CoA) is the common intermediate of the PAA catabolic pathway and the biosynthetic pathway of L-phenylglycine (L-Phg), the last amino acid precursor for PI biosynthesis, we proposed that derepression of the transcription of paa genes in a ?paaR mutant possibly diverts more PA-CoA to the PAA catabolic pathway, thereby with less PA-CoA metabolic flux toward L-Phg formation, thus resulting in lower PI titers. This hypothesis was verified by the observations that PI production of a ?paaR mutant was restored by L-Phg supplementation as well as by deletion of the paaABCDE operon in the ?paaR mutant. Altogether, this study provides new insights into the regulation of PI biosynthesis by S. pristinaespiralis.ImportanceA better understanding of the regulation mechanisms for antibiotic biosynthesis will provide valuable clues for Streptomyces strain improvement. Herein, a TetR family regulator PaaR, which serves as the repressor of the transcription of paa genes involved in phenylacetic acid (PAA) catabolism, was identified as playing a positive role in the regulation of pristinamycin I (PI) by affecting the supply of one of seven amino acid precursors, L-phenylglycine, in Streptomyces pristinaespiralis. To our knowledge, this is the first report describing the interplay between PAA catabolism and antibiotic biosynthesis in Streptomyces strains. Considering that the PAA catabolic pathway and its regulation by PaaR are widespread in antibiotic-producing actinomycetes, it could be suggested that PaaR-dependent regulation of antibiotic biosynthesis might commonly exist.

Dataset Information

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

Publications

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

Similar Datasets

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