Dataset Information

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

ABSTRACT: Lincomycin is one of the most important antibiotics in clinical practice. To further understand the regulatory mechanism on lincomycin biosynthesis, we investigated a pleiotropic transcriptional regulator AdpAlin in the lincomycin producer Streptomyces lincolnensis NRRL 2936. Deletion of adpA lin (which generated ?adpA lin ) interrupted lincomycin biosynthesis and impaired the morphological differentiation. We also found that putative AdpA binding sites were unusually scattered in the promoters of all the 8 putative operons in the lincomycin biosynthetic gene cluster (BGC). In ?adpA lin , transcript levels of structural genes in 8 putative operons were decreased with varying degrees, and electrophoretic mobility shift assays (EMSAs) confirmed that AdpAlin activated the overall putative operons via directly binding to their promoter regions. Thus, we speculated that the entire lincomycin biosynthesis is under the control of AdpAlin. Besides, AdpAlin participated in lincomycin biosynthesis by binding to the promoter of lmbU which encoded a cluster sited regulator (CSR) LmbU of lincomycin biosynthesis. Results of qRT-PCR and catechol dioxygenase activity assay showed that AdpAlin activated the transcription of lmbU. In addition, AdpAlin activated the transcription of the bldA by binding to its promoter, suggesting that AdpAlin indirectly participated in lincomycin biosynthesis and morphological differentiation. Uncommon but understandable, AdpAlin auto-activated its own transcription via binding to its own promoter region. In conclusion, we provided a molecular mechanism around the effect of AdpAlin on lincomycin biosynthesis in S. lincolnensis, and revealed a cascade regulation of lincomycin biosynthesis by AdpAlin, LmbU, and BldA.

SUBMITTER: Kang Y

PROVIDER: S-EPMC6819324 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Kang Yajing Y Wang Yingying Y Hou Bingbing B Wang Ruida R Ye Jiang J Zhu Xiaoyu X Wu Haizhen H Zhang Huizhan H

Frontiers in microbiology 20191023

Lincomycin is one of the most important antibiotics in clinical practice. To further understand the regulatory mechanism on lincomycin biosynthesis, we investigated a pleiotropic transcriptional regulator AdpAlin in the lincomycin producer Streptomyces lincolnensis NRRL 2936. Deletion of adpA lin (which generated ΔadpA lin ) interrupted lincomycin biosynthesis and impaired the morphological differentiation. We also found that pu ...[more]

PMID: 31708899

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Project description: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.

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.

Dataset Information

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

Publications

AdpA<sub>lin</sub>, a Pleiotropic Transcriptional Regulator, Is Involved in the Cascade Regulation of Lincomycin Biosynthesis in <i>Streptomyces lincolnensis</i>.

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