Inaoka Takashi, Takahashi Kosaku, Ohnishi-Kameyama Mayumi, Yoshida Mitsuru, Ochi Kozo
Microbial Function Laboratory, National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan.
J Biol Chem. 2003 Jan 24;278(4):2169-76. doi: 10.1074/jbc.M208722200. Epub 2002 Oct 7.
We found that a polycistronic operon (ywfBCDEFG) and a monocistronic gene (ywfH) are required for the biosynthesis of bacilysin in Bacillus subtilis. The disruption of these genes by plasmid integration caused loss of the ability to produce bacilysin, accompanied by a lack of bacilysin synthetase activity in the crude extract. We investigated the regulatory mechanism for bacilysin biosynthesis using the transcriptional lacZ fusion system. The transcription of these genes was found to be induced at the transition from exponential to stationary phase. Induction of transcription was accelerated by depleting a required amino acid, which was done by transferring the wild-type (rel(+)) cells to an amino acid-limited medium. In contrast, no enhancement of the gene expression was detected in relA mutant cells. In wild-type (rel(+)) cells, a forced reduction of intracellular GTP, brought about by addition of decoyinine, which is a GMP synthetase inhibitor, enhanced the expression of both the ywfBCDEFG operon and the ywfH gene, resulting in a 2.5-fold increase in bacilysin production. Disruption of the codY gene, which regulates stationary phase genes by detecting the level of GTP, also induced transcription of these genes. In contrast, the expression of ywfBCDEFG in relA cells was not activated either by decoyinine addition or codY disruption, although the expression of ywfH was induced. Moreover, the codY disruption resulted in an increase of bacilysin production only in rel(+) cells. These results indicate that guanosine 5'-diphosphate 3'-diphosphate (ppGpp) plays a crucial role in transcription of the ywfBCDEFG operon and that the transcription of these genes are dependent upon the level of intracellular GTP which is transmitted as a signal via the CodY-mediated repression system. We propose that, unlike antibiotic production in Streptomyces spp., bacilysin production in B. subtilis is controlled by a dual regulation system composed of the guanine nucleotides ppGpp and GTP.
我们发现，枯草芽孢杆菌中杆菌溶素的生物合成需要一个多顺反子操纵子（ywfBCDEFG）和一个单顺反子基因（ywfH）。通过质粒整合破坏这些基因会导致产生杆菌溶素的能力丧失，同时粗提物中缺乏杆菌溶素合成酶活性。我们使用转录lacZ融合系统研究了杆菌溶素生物合成的调控机制。发现这些基因的转录在从指数期到稳定期的转变过程中被诱导。通过消耗必需氨基酸来加速转录诱导，这是通过将野生型（rel(+)）细胞转移到氨基酸限制培养基中来实现的。相反，在relA突变体细胞中未检测到基因表达的增强。在野生型（rel(+)）细胞中，添加作为GMP合成酶抑制剂的脱氧助间型霉素导致细胞内GTP的强制减少，增强了ywfBCDEFG操纵子和ywfH基因的表达，导致杆菌溶素产量增加2.5倍。通过检测GTP水平来调节稳定期基因的codY基因的破坏也诱导了这些基因的转录。相反，尽管ywfH的表达被诱导，但在relA细胞中，添加脱氧助间型霉素或破坏codY均未激活ywfBCDEFG的表达。此外，codY破坏仅在rel(+)细胞中导致杆菌溶素产量增加。这些结果表明，鸟苷5'-二磷酸3'-二磷酸（ppGpp）在ywfBCDEFG操纵子的转录中起关键作用，并且这些基因的转录依赖于细胞内GTP的水平，GTP作为信号通过CodY介导的阻遏系统传递。我们提出，与链霉菌属中抗生素的产生不同，枯草芽孢杆菌中杆菌溶素的产生受由鸟嘌呤核苷酸ppGpp和GTP组成的双重调节系统控制。
