Coupling of the biosynthesis and export of the DNA gyrase inhibitor simocyclinone in Streptomyces antibioticus

Abstract

SummaryBecause most antibiotics are potentially lethal to the producing organism, there must be mechanisms to ensure that the machinery responsible for export of the mature antibiotic is in place at the time of biosynthesis. Simocyclinone D8 is a potent DNA gyrase inhibitor produced by Streptomyces antibioticus Tü 6040. Within the simocyclinone biosynthetic cluster are two divergently transcribed genes, simR and simX, encoding proteins that resemble the TetR/TetA repressor-efflux pump pair that cause widespread resistance to clinically important tetracyclines. Engineered expression of simX from a strong, heterologous promoter conferred high level simocyclinone D8 resistance on Streptomyces lividans, showing that simX encodes a simocyclinone efflux pump. Transcription of simX is controlled by SimR, which directly represses the simX and simR promoters by binding to two operator sites in the simX-simR intergenic region. Simocyclinone D8 abolishes DNA binding by SimR, providing a mechanism that couples the biosynthesis of simocyclinone to its export. In addition, an intermediate in the biosynthetic pathway, simocyclinone C4, which is essentially inactive as a DNA gyrase inhibitor, also induces simX expression in vivo and relieves simX repression by SimR in vitro.

The TetR Family of Regulators

“…This is also the case for many TFRs (e.g., SimR, CmeR, and MphR [50,56,59]), but different drug-binding stoichiometries are seen in some others. In the case of ActR, each ActR dimer is capable of binding either two molecules of actinorhodin or four molecules of (S)-2,4-dinitrophenyl acetate [(S)-DNPA] (54).…”

“…For example, ActR, QacR, SmeT, TetR, and TtgR all have a "side entry" opening distal to the dimerization interface that is believed to be the site of access for different ligands (22,(51)(52)(53)(54). Ligands appear to enter CmeR, CgmR, HrtR, LfrR, and SimR via an entry point closer to the "front" of the protein (43,46,50,55,56). Finally, DesT, EthR, and FadR exhibit a relative "top entry" (44,57,58).…”

“…This is also seen for the ligand-binding pockets of the majority of TFRs (e.g., ActR, CmeR, and QacR [53,54,56]). In contrast, the SimR ligand-binding cavity is composed of residues from both monomers such that each binds either the aminocoumarin or the angucyclinone moiety of the simocyclinone ligand (50).…”

“…This diversity supports a role for TFRs in regulating an equally diverse array of cellular processes from basic carbon and nitrogen metabolism to quorum sensing and antibiotic resistance. Structures are available for TFRs in complex with simple ligands such as citrate and resorcinol (49) to very complex molecules such as acyl-CoA derivatives (44) and antibiotics with multiple functional groups such as simocyclinone (50). There are many ways that TFRs can interact with ligands.…”

Feed-Forward Regulation of Microbisporicin Biosynthesis in Microbispora corallina

“…The primers used were LF096F and LF096R (pIJ12341), LF096F2 and LF096R (pIJ12342), LF097F and LF097R (pIJ12343), and LF097F and LF097R2 (pIJ12344). The resulting PCR fragments were cloned into EcoRI-BamHI-cut pIJ5972, an integrative Streptomyces promoter-probe plasmid carrying TTA codon-free derivatives of the luxAB reporter genes (19). The resulting constructs and pIJ5972 (negative control) were transferred by conjugation into S. coelicolor M1146 (5).…”