NovG, a DNA-binding protein acting as a positive regulator of novobiocin biosynthesis
NovG, a DNA-binding protein acting as a positive regulator of novobiocin biosynthesis
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DOI：
10.1099/mic.0.27669-0
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发表时间：
2005-06-01
期刊：
影响因子：
2.8
通讯作者：
Heide, L
中科院分区：
文献类型：
--
作者：
Eustáquio, AS;Li, SM;Heide, L
氨基香豆素类抗生素novobiocin的生物合成基因簇包含两个可能的调控基因，即novE和novG。预测的novG基因产物显示了一个假定的螺旋-螺旋-螺旋dna结合基序，并且与StrR（一种经过充分研究的链霉素生物合成通路特异性转录激活因子）具有序列相似性。在这里，通过遗传和生化方法，为NovG作为新生物素生物合成的积极调节剂的作用提供了功能证明。球链霉菌（Streptomyces spheroides）的整个novobiocin簇在异源宿主coelicolstreptomyces M512中表达，并在异源表达的簇中产生了缺乏novG基因的菌株。这些Delta novG菌株产生的新生物素仅为携带完整新生物素簇的S. coelicolor M512菌株形成的2%。通过将novG的完整拷贝导入突变体，可以恢复生产。在含有完整簇的菌株中，novG在多拷贝质粒上的存在导致抗生素的生产几乎增加了三倍，这表明novG蛋白的可用性限制了新生物素的生物合成。此外，纯化的n端His(6)标记的NovG分别对NovG - novh和cloG-cloY基因簇的NovG - novh和cloG-cloY基因簇表现出特异性的dna结合活性。通过比较结合NovG的片段的DNA序列，在两个片段中都发现了保守的反向重复序列，与鉴定为StrR结合位点的重复序列相似。StrR和推测的NovG结合位点的一致序列为GTTCRACTG(N)(11)CRGTYGAAC。因此，NovG和StrR显然属于同一家族的dna结合调节蛋白。
The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin contains two putative regulatory genes, i.e. novE and novG. The predicted gene product of novG shows a putative helix-turn-helix DNA-binding motif and shares sequence similarity with StrR, a well-studied pathway-specific transcriptional activator of streptomycin biosynthesis. Here functional proof is provided, by genetic and biochemical approaches, for the role of NovG as a positive regulator of novobiocin biosynthesis. The entire novobiocin cluster of the producer organism Streptomyces spheroides was expressed in the heterologous host Streptomyces coelicolor M512, and additional strains were produced which lacked the novG gene within the heterologously expressed cluster. These Delta novG strains produced only 2% of the novobiocin formed by the S. coelicolor M512 strains carrying the intact novobiocin cluster. The production could be restored by introducing an intact copy of novG into the mutant. The presence of novG on a multicopy plasmid in the strain containing the intact cluster led to almost threefold overproduction of the antibiotic, suggesting that novobiocin biosynthesis is limited by the availability of NovG protein. Furthermore, purified N-terminal His(6)-tagged NovG showed specific DNA-binding activity for the novG-novH and the cloG-cloY intergenic regions of the novobiocin and clorobiocin biosynthetic gene clusters, respectively. By comparing the DNA sequences of the fragments binding NovG, conserved inverted repeats were identified in both fragments, similar to those identified as the binding sites for StrR. The consensus sequence for the StrR and the putative NovG binding sites was GTTCRACTG(N)(11)CRGTYGAAC. Therefore, NovG and StrR apparently belong to the same family of DNA-binding regulatory proteins.