天蓝色链霉菌cvnA、sarA及rrdA基因功能的初步研究

【作者】 欧西军；

【导师】 赵国屏；

【作者基本信息】 复旦大学 ， 微生物学， 2009， 博士

【摘要】 第一部分天蓝色链霉菌A3（2）全基因组序列于2002年完成,在其中发现了13个非常保守的操纵子——conservon（cvn）。cvn由4-5个基因组成（cvnABCD或者cvnABCDE）,cvnA和cvnD分别编码一个跟组氨酸激酶具有较弱相似性的膜蛋白和一个类似真核G蛋白的GTP水解酶。我们在天蓝色链霉菌M145中成功构建了分别缺失13个cvnA基因的突变体,并在YBP培养基（添加了不同小分子营养物）上观察它们的表型变化,发现其中3个cvnA基因的缺失影响了菌丝发育和次生代谢。cvnA1突变体比野生型菌株发育更快,产孢更丰富,随着培养基内渗透压的升高,表型差异越来越明显。相对于野生型来说,cvnA1突变体放线紫红素（Act）和十一烷基灵菌红素（Red）的产量增加。在固体培养基YBP上,添加0.2M或更高浓度的NaCl、NaNO₃、KCl或者KNO₃后,cvnA1突变体能大量产生Act。cvnA2突变体在含甘氨酸的YBP培养基上生长发育比野生型要好。cvnA8突变体在含谷氨酰胺的培养基上产Act比野生型要早。生化实验证明CvnA1蛋白没有自磷酸化活性,但能结合并水解ATP。通过遗传互补实验我们发现CvnA1蛋白C端614个氨基酸对于上面所提到的表型不是必需的。进一步的RT-PCR实验发现cvnA1突变体中ramR、ramC、chpE、bldN,redD和redZ的转录水平比野生型显著升高了。这些结果表明CvnA1蛋白通过调控表面活化分子SapB、Chplins以及抗生素合成通路转录活化因子的mRNA水平来调控气生菌丝发育和次生代谢。第二部分链霉菌在整个生活周期中会经历一个形态分化和次生代谢的复杂过程。我们在天蓝色链霉菌M145中发现了一个在链霉菌中高度保守的新基因sarA（sco4069,sporulation and antibiotic production related gene）,该基因编码的蛋白除了跨膜结构域外没有任何已知功能结构域。跟野生型相比,sarA突变体孢子发育变快,Act和Red产量显著降低。RT-PCR分析表明SarA通过调控actⅡ-orf 4和redΖ的mRNA水平来调控抗生素的合成。第三部分链霉菌除了具有复杂的形态分化外,还能产生多种次生代谢产物特别是抗生素。为了能更深入地了解Red合成的信号调控通路,我们利用一种体内转座系统在天蓝色链霉菌中寻找参与Red合成调控的新基因。我们一共得到了25个不能产生Red的突变体,这些突变体Act的产量也同时受到了不同程度的影响。其中24个突变体的插入位点位于已知的Red生物合成基因簇中,另外一个突变体的插入位点位于rrdA（regulator of redD,sco1104）基因上游40bp。rrdA基因编码一个TetR家族的转录因子蛋白,在链霉菌中高度保守。与野生型相比,rrdA突变株中Red产量提高,Act产量下降,而在过表达rrdA基因的M145菌株中Red的产生受到抑制,Act的产量得到提高。RT-PCR结果表明RrdA通过调控redD的mRNA丰度来负调控Red的合成,而对Act合成特异转录调控因子actⅡ-orf4的转录没有影响,RrdA对Act的正调控作用很可能是因为Act和Red的合成存在底物竞争关系而形成的。更多还原

【Abstract】 PartⅠIn 2002 the first complete Streptomyces species genome（Streptomyces coelicolor A3（2）） was finished,revealing the presence of 13 four or five gene clusters named conservon（cvn,conserved operon）.The cvnA and cvnD genes of the clusters （cvnABCDE） encode a membrane protein（CvnA）,showing a weak similarity to histidine kinase,and a eukaryotic G protein like GTPase（CvnD）,respectively.We constructed null mutants for each of the 13 cvnA genes in S.coelicolor M145 and screened phenotypes for aberrant development on YBP agar with different small molecule nutrient supplements.The null mutant of cvnA1 accelerated aerial mycelia development and well-sporulation at a carbon source independent manner.Comparing to the prototype,cvnA1 mutant increased the actinorhodin（Act） and undecylprodigiosin（Red） productivity.When 0.2 M or higher concentrations of NaCl, NaNO₃,KCl or KNO₃ was added into the solid YBP medium,the mutant over-produced Act dramatically.The cvnA2 null mutant grew better than the prototype on YBP agar with glycine while the cvnA8 null mutant produced more Act earlier than the prototype on media with glutamine.Biochemical analyses revealed that CvnA1 protein could bind ATP/ADP and hydrolyze ATP,but showed no detectable autophosphorylation activity.Genetic complementation assays using various truncated C terminus and N terminus of cvnA1 for cvnA1 null mutant showed that the C terminus 614 amino acids was not necessary for the function mentioned above. RT-PCR results showed that transcriptional levels of ramR,ramC,chpE,bldN,redD and redZ were upregulated significantly in the cvnA1 null mutant.Altogether,these results indicated that CvnA1 influences the aerial morphogenesis and antibiotic production by affecting the expression of surfactants,SapB and Chaplins,and pathway-specific activators in Streptornyces coelicolor respectively. The filamentous bacteria Streptomyces exhibit a complex life cycle involving morphological differentiation and secondary metabolism.A putative membrane protein gene sarA（sco4069）,sporulation and antibiotic production related gene A, was partially characterized in Streptomyces coelicolor M145.The gene product has no characterized functional domains except for the transmembrane domain and is highly conserved in Streptomyces.Compared to the wild type M145,the sarA mutant accelerated sporulation and dramatically decreased the production of Act and Red. RT-PCR analysis showed that SarA influenced the antibiotic production through controlling the mRNA abundance of the actlI-orf4 and redZ.PartⅢStreptomyces not only exhibit complex morphological differentiation but also produce a plethora of secondary metabolites,particularly antibiotics.To improve our general understanding of the complex network of Red biosynthesis regulation,we used an in vivo transposition system to identify novel regulators that influence Red production in Streptomyces coelicolor M145.Using this screening system,we obtained 25 Red-deficient mutants.Of these,24 had a transposon inserted in the previously described Red biosynthetic gene cluster and produced different amounts of another secondary metabolite Act.One mutant was shown to have an insertion in a different region of the chromosome upstream of the previously uncharacterized gene rrdA（regulator of redD,sco1104）,which encodes a putative TetR family transcription factor.In comparison with wild-type M145,the rrdA null mutant exhibited increased Red production and decreased Act production.High rrdA expression resulted in severe reduction of Red production and Act overproduction.RT-PCR analysis showed that RrdA negatively regulated Red production by controlling redD mRNA abundance,while no change was observed at the transcript level of the Act-specific activator gene actⅡ-orf4.The effects on Act biosynthesis might arise from competition for precursors that are common to both pathways.更多还原