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 adpAlin (which generated ΔadpAlin) 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 ΔadpAlin, 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.
Highlights
Lincomycin is a naturally occurring antibiotic isolated from soil sample, and it was first introduced into clinical practice in 1963 (Macleod et al, 1964)
S. lincolnensis NRRL 2936 which served as wild type (WT) and its mutants were incubated at 28◦C on mannitol soya flour (MS) medium (Kieser et al, 2000) for 3–5 days for routine cultivation, phenotype observation, and strain preservation, and cultivated at 28◦C in YEME liquid medium [10 g/L yeast extract (OXOID, United States), 5 g/L polypeptone (Nihon Pharmaceutical, Japan), 10 g/L glucose (Lingfeng, China), 3 g/L maltose (Generay, China), 5 mM MgCl2·2H2O (Lingfeng, China), 340 g/L sucrose (Titan, China), dissolved in dH2O] with shaking (210 rpm) for 3–5 days for routine cultivation, total DNA extraction, and sporeless strain preservation
We investigated the effects of AdpAlin on lincomycin biosynthesis and attempted to propose some innovative idea on this classic regulator
Summary
Lincomycin is a naturally occurring antibiotic isolated from soil sample, and it was first introduced into clinical practice in 1963 (Macleod et al, 1964). Hou et al (2018a, 2019) demonstrated that LmbU, as a CSR, positively regulates lincomycin biosynthesis by controlling the transcription of lmbA, lmbC, lmbJ, lmbK, lmbW, and lmbU itself, and subsequently solved the subtle mechanism of LmbU regulon. For the biosynthesis of grixazone (Higashi et al, 2007), nikkomycin (Pan et al, 2009), and natamycin (Yu et al, 2018), AdpA activates the transcription of CSRs in their BGCs and indirectly regulates antibiotic biosynthesis. It was reported that AdpA from S. xiamenensis 318 negatively regulates morphological differentiation as well as polycyclic tetramate macrolactams (PTMs) production, while positively regulates xiamenmycin production by activating the transcription of two of the structural genes ximA and ximB (Bu et al, 2019). ANS65440.1) and attempted to investigate its regulatory mechanism of lincomycin biosynthesis in Streptomyces lincolnensis in this study
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