【作者】 朱冬青；

【导师】 邓子新；

【作者基本信息】 上海交通大学 ， 微生物学， 2008， 博士

【摘要】 来自抗生链霉菌的酪氨酸酶基因melC以高拷贝形式(pIJ702)进入不同的链霉菌菌株中后,菌落周围就会产生棕黑色的黑色素圈,这是一种非常明显的表型。转化子中有大约1 %的自发突变的白色的突变株,其中有一部分是因为melC本身发生突变,另一部分是因为宿主染色体发生突变影响到melC的表达。ZX66(衍生于变铅青链霉菌野生型JT46)就是染色体发生突变的白色转化子。来自于JT46染色体的一个3.3 kb的DNA片段含有一个可以回补ZX66黑色素的表达的基因。这个基因编码的蛋白属于转录调节因子AraC家族的一员,高度同源于天蓝色链霉菌中的AdpAc,因此命名为AdpAl。ZX66染色体上的这个基因发生了一个碱基的颠换,引起一个关键的氨基酸,苏氨酸,变成苯丙氨酸。将天蓝色链霉菌、变铅青链霉菌和抗生链霉菌中的adpA进行缺失,所得突变株均不能合成黑色素。在adpA的突变株中,melC的转录水平明显降低。蛋白AdpAl能够结合在melC的启动子区域激活melC基因的转录。我们可以得出结论:在链霉菌中AdpA家族蛋白正调控melC操纵子。阿维链霉菌是能够产生阿维菌素和寡霉素的重要工业微生物。阿维链霉菌AdpAa是灰色链霉菌AdpAg的同源蛋白。阿维链霉菌adpAa突变株(ZD7)不能合成黑色素,也不能产生气生菌丝。而且HPLC检测突变株不再产生阿维菌素、寡霉素。CAS平板检测突变株不再分泌铁载体。反转录PCR检测野生型菌株和adpAa突变株ZD7中基因的转录水平。可能参与形态分化的基因的转录水平在adpAa突变株有不同程度的下降。黑色素、阿维菌素和寡霉素的生物合成基因簇在adpAa突变株检测不到转录。阿维链霉菌基因组中32簇次生代谢基因簇超过一半的转录水平明显下降,还有一簇基因的转录水平升高。我们可以得出结论:在阿维链霉菌中AdpA正调控melC操纵子和形态分化,以及阿维菌素、寡霉素和铁载体的生物合成,并且影响超过一半次生代谢基因簇的的转录。aveR是阿维链霉菌阿维菌素生物合成基因簇中唯一的可能调节基因。为了验证aveR是否参与阿维菌素生物合成基因的转录调节,我们中断aveR基因获得突变株(ZD10)。高压液相色谱检测表明,与野生型菌株相比,突变株不再产生阿维菌素,并且寡霉素的产量明显高于野生型。进一步的反转录PCR分析显示,与野生型菌株相比,突变株ZD10的聚酮合酶基因aveA3转录水平有明显下降。结果显示AveR是阿维菌素生物合成的正调节因子,通过调节结构基因的转录表达来影响阿维菌素的产生。更多还原

【Abstract】 Dark brown haloes of melanin around colonies are an easily visualized phenotype displayed by many Streptomyces strains harboring plasmid pIJ702 carrying the melC operon of Streptomyces antibioticus IMRU3270. Spontaneous melanin-negative mutants of pIJ702 occur with a frequency of ca. 1%, and often mutation occurs in the melC operon, which removes the BglII site as part of an inverted repeat. Other melanin-negative mutations seem to occur spontaneously in S. lividans, resulting in white colonies from which intact, melanin-producing pIJ702 can be isolated by introduction into a new host. S. lividans ZX66 was found to be such a mutant and to have a secondary mutation influencing expression of the melC operon on the chromosome. A 3.3 kb DNA fragment was isolated from its progenitor strain, JT46, and a gene able to restore melC operon expression was found to encode a member of an AraC family of transcriptional regulators, which was equivalent to AdpAc in S. coelicolor and therefore was designated AdpAl. Lack of melC operon expression was correlated with a single A-to-C transversion, which altered a single key amino acid residue from Thr to Pro. The transcription of the melC operon was found to be greatly reduced in the adpA mutant background. The counterpart gene in the S. antibioticus strain in which the melC operon carried on pIJ702 originated was also isolated and was found to have an identical regulatory role. Thus, we concluded that the melC operon is under general direct positive control by AdpA family proteins, perhaps at the transcriptional level and certainly at the translational level via bldA, in Streptomyces.S. avermitilis is a soil bacterium that carries out not only a complex morphological differentiation but also the production of secondary metabolites such as avermectin and oligomycin. AdpAa, a regulator of the AvaC family encoded by SAV5261 of S. avermitilis genome, is highly similar to AdpAg, a central transcriptional regulator in the A-factor regulatory cascade that leads to morphological development and secondary metabolism in S. griseus. adpAa was interrupted and the mutant ZD7 fails to produce melanin and to form aerial mycelium. Furthermore adpAa is also involved in production of avermectin and oligomycin. HPLC analysis indicates that AdpA regulates avermectin and oligomycin biosynthesis positively. S. avermitilis can produce siderophore and siderophore production of the mutant of adpAa was blocked. Reverse transcription PCR for transcript analysis also showed the difference of transcription of genes involved in morphological differentiation and secondary metabolites between wild type strain and ZD7. the transcriptional levels of some genes involved in morphological differentiation were lower in ZD7 compared with those in the wild type strain. There are 32 secondary metabolite clusters in S. avermitilis, and the transcriptional levels of 15 clusters of them were lower in ZD7 compared with those in the wild type strain.Gene aveR is the only putative regulatory gene in avermectin biosynthetic gene cluster from S. avermitilis. In order to test if aveR is involved in the regulation of transcription of avermectin biosynthetic genes, aveR mutant ZD10 was obtained. Analyzed by LC-MS, avermectin productivity was abolished, whereas the yield of oligomycin significantly increased in ZD10. Reverse transcriptase PCR analysis indicated that the transcription level of aveA3, an essential PKS gene, was drastically reduced in ZD10. Therefore, AveR was proved as a positive regulator for avermectin biosynthesis by promoting the transcription of structural genes.更多还原