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

Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK.
Molecular Microbiology (Impact Factor: 4.42). 06/2009; 72(6):1462-74. DOI: 10.1111/j.1365-2958.2009.06735.x
Source: PubMed


Because 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.

Download full-text


Available from: Hans-Peter Fiedler
  • Source
    • "The biosynthetic gene cluster for simocyclinone D8, encoded in Streptomyces antibioticus, includes a TFR/ exporter gene pair (simR/simX) similar to actR/actAB. The mature antibiotic and its biosynthetic intermediate (simocyclinone D4) can both relieve repression of simX by SimR; however, simocyclinone D8 is a more potent inducer compared to simocyclinone D4 (Le et al., 2009). Furthermore, structural information concerning the recognition of the antibiotics by SimR strongly argues that this mature molecule plays the critical role in the induction of resistance (Le et al., 2011). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Antibiotic-producing bacteria encode antibiotic resistance genes that protect them from the biologically active molecules that they produce. The expression of these genes needs to occur in a timely manner: either in advance of or concomitantly with biosynthesis. It appears that there have been at least two general solutions to this problem. In many cases, the expression of resistance genes is tightly linked to that of antibiotic biosynthetic genes. In others, the resistance genes can be induced by their cognate antibiotics or by intermediate molecules from their biosynthetic pathways. The regulatory mechanisms that couple resistance to antibiotic biosynthesis are mechanistically diverse and potentially relevant to the origins of clinical antibiotic resistance.
    Preview · Article · Jun 2014 · Molecular Microbiology
  • Source
    • "PCR amplification of the rhiR promoter from genomic DNA using the rhiR-F/R primers produced multiple bands; the appropriate fragment was purified from an agarose gel using a gel purification kit (Qiagen), which involves a heat treatment. The DNA fragments were endlabelled using [g- 32 P]-ATP and T4 polynucleotide kinase (New England Biolabs), purified using a PCR purification kit (Qiagen) and then used for electrophoretic mobility shift assays (EMSA) (Le et al., 2009). MBP-PraR was bound to DNA in 20 ml of EMSA buffer [20 mM Tris-HCl pH 8.0, 200 ng of salmon sperm DNA, 1 mM EDTA, 100 mM NaCl, 0.5 mM DTT, 5 mM MgCl2, 8% (v/v) glycerol] containing 0.1 nM radiolabelled DNA (approximately 8000 c.p.m.) and varying amounts of MBP-PraR and CinS-His6 [diluted in 50 mM NaCl, 10% glycerol (v/v), 25 mM Tris-HCl, pH 8.0]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Analysis of quorum-sensing (QS) regulation in Rhizobium leguminosarum revealed an unusual type of gene regulation that relies on the population density-dependent accumulation of an anti-repressor. The cinS gene, which is co-transcribed with the N-acyl-homoserine-lactone synthase gene cinI, is required to fully induce rhiR and raiR, whose products, together with their partner AHL synthases, regulate other genes in a QS-regulated hierarchy. Purified CinS bound to the R. leguminosarum transcriptional regulator PraR, which repressed rhiR and raiR expression. PraR bound to the rhiR and raiR promoters and CinS displaced PraR from these promoters, thereby inducing their expression. Although induction of cinS required CinI-made AHL, it appears CinS does not require the AHL for its anti-repressor function. The LuxR-type regulator ExpR was also required for normal induction of rhiR and raiR and it appears that this occurs by ExpR repressing the transcription of praR. Therefore ExpR and CinS act independently to attenuate PraR action, ExpR by repressing its transcription and CinS by attenuating its repressive activity. Thus, as CinS accumulates in a population density-dependent manner it induces the QS hierarchy by relieving PraR-mediated repression of rhiR and raiR.
    Full-text · Article · Aug 2011 · Molecular Microbiology
  • [Show abstract] [Hide abstract]
    ABSTRACT: Deals with simultaneous global external as well as global internal stabilization of linear systems with saturating actuators. The paper proposes a new family of scheduled low-and-high gain state feedback laws that yields a closed-loop system which is both globally finite gain L <sub>p</sub> stable and globally asymptotically stable. Moreover, the controller has an explicit design parameter that can be adjusted to make the L<sub>p</sub> gain of the closed-loop system arbitrarily small
    No preview · Conference Paper · Jul 1998
Show more