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Abstract：
Streptomyces γ-butyrolactones (GBLs) are quorum sensing communication signals triggering antibiotic production. The GBL system of Streptomyces filipinensis, the producer of the antifungal agent filipin, has been investigated. Inactivation of sfbR (for S. filipinensis γ-butyrolactone receptor), a GBL receptor, resulted in a strong decrease in production of filipin, and deletion of sfbR2, a pseudo-receptor, boosted it, in agreement with lower and higher levels of transcription of filipin biosynthetic genes, respectively. It is noteworthy that none of the mutations affected growth or morphological development. While no ARE (autoregulatory element)-like sequences were found in the promoters of filipin genes, suggesting indirect control of production, five ARE sequences were found in five genes of the GBL cluster, whose transcription has been shown to be controlled by both S. filipinensis SfbR and SfbR2. In vitro binding of recombinant SfbR and SfbR2 to such sequences indicated that such control is direct. Transcription start points were identified by 5\' rapid amplification of cDNA ends, and precise binding regions were investigated by the use of DNase I protection studies. Binding of both regulators took place in the promoter of target genes and at the same sites. Information content analysis of protected sequences in target promoters yielded an 18-nucleotide consensus ARE sequence. Quantitative transcriptional analyses revealed that both regulators are self-regulated and that each represses the transcription of the other as well as that of the remaining target genes. Unlike other GBL receptor homologues, SfbR activates its own transcription whereas SfbR2 has a canonical autorepression profile. Additionally, SfbR2 was found here to bind the antifungal antimycin A as a way to modulate its DNA-binding activity.IMPORTANCE Streptomyces GBLs are important signaling molecules that trigger antibiotic production in a quorum sensing-dependent manner. We have characterized the GBL system from S. filipinensis, finding that two key players of this system, the GBL receptor and the pseudo-receptor, each counteracts the transcription of the other for the modulation of filipin production and that such control over antifungal production involves an indirect effect on the transcription of filipin biosynthetic genes. Additionally, the two regulators bind the same sites, are self-regulated, and repress the transcription of three other genes of the GBL cluster, including that encoding the GBL synthase. In contrast to all the GBL receptors known, SfbR activates its own synthesis. Moreover, the pseudo-receptor was identified as the receptor of antimycin A, thus extending the range of examples supporting the idea of signaling effects of antibiotics in Streptomyces The intricate regulatory network depicted here should provide important clues for understanding the regulatory mechanism governing secondary metabolism.
摘要：
链霉菌γ-丁内酯（GBLs）是触发抗生素生产的群体感应通信信号。丝状链霉菌的GBL系统，抗真菌剂filipin的生产商，已被调查。sfbR的失活（对于菲律宾沙门氏菌γ-丁内酯受体），GBL受体，导致filipin的产量大幅下降，和假受体sfbR2的缺失，增强了它，与filipin生物合成基因的较低和较高水平的转录一致，分别。值得注意的是,没有突变影响生长或形态发育。虽然在filipin基因的启动子中没有发现ARE（自动调节元件）样序列，建议间接控制生产，在GBL簇的五个基因中发现了五个ARE序列，其转录已被证明由S.filipinensisSfbR和SfbR2控制。重组SfbR和SfbR2与此类序列的体外结合表明此类控制是直接的。通过5个cDNA末端的快速扩增鉴定转录起始点，通过使用DNaseI保护研究，研究了精确的结合区域。两种调节子的结合发生在靶基因的启动子和相同位点。靶启动子中受保护序列的信息含量分析产生了18个核苷酸的共有ARE序列。定量转录分析表明，两个调节因子都是自我调节的，并且每个调节因子都抑制另一个以及其余靶基因的转录。与其他GBL受体同源物不同，SfbR激活其自身的转录,而SfbR2具有典型的自抑制谱。此外,此处发现SfbR2结合抗真菌抗霉素A作为调节其DNA结合活性的方式。重要性链霉菌GBLs是以群体感应依赖性方式触发抗生素产生的重要信号分子。我们已经描述了菲律宾沙门氏菌的GBL系统，发现这个系统的两个关键角色，GBL受体和假受体，每个都抵消另一个的转录以调节filipin的产生，并且这种对抗真菌产生的控制涉及对filipin生物合成基因转录的间接影响。此外,这两个调节器绑定了相同的位点，是自我调节的，并抑制GBL簇的其他三个基因的转录，包括编码GBL合酶的。与所有已知的GBL受体相反，SfbR激活其自身的合成。此外，假受体被鉴定为抗霉素A的受体，因此，扩展了支持抗生素在链霉菌中的信号传导效应思想的示例范围。此处描述的复杂的调节网络应为理解控制次级代谢的调节机制提供重要线索。
