【作者】 罗毅；

【导师】 孙明；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2012， 博士

【摘要】 Zwittermicin A(ZmA)是一种聚酮-非核糖体肽杂合的物质,它由一些芽胞杆菌所产生,包括蜡状芽胞杆菌菌株UW85和苏云金芽胞杆菌菌株YBT-1520。ZmA一方面具有抗生素广谱的抑菌活性,一方面对苏云金芽胞杆菌的晶体蛋白有杀虫增效活性。其特殊的分子结构和多样的生物活性,使得我们有兴趣对其开展合成基因簇的鉴定及调控机制的研究。国外的研究团队通过对菌株UW85中与ZmA合成相关的非核糖体肽酶(nonribosomal peptide synthetase, NRPS)和聚酮合酶(polyketide synthase, PKS)功能域的分析,推测了ZmA的合成途径,但是并没有给出直接的实验证据,也没有表明基因簇的完整性。在本研究中,我们期望通过异源表达的方式来鉴定ZmA完整的合成基因簇。为此,我们构建了菌株YBT-1520的全基因组细菌人工染色体(BAC)文库。文库包含1200个克隆子,平均插入片段大小为45Kb,文库约覆盖总基因组10倍。以已知与ZmA合成相关的基因设计引物为探针,通过聚合酶链反应(PCR)到文库中经行筛选,得到了9个阳性克隆。将这9个阳性克隆末端测序,结合其限制性酶切物理图谱和菌株YBT-1520基因组测序,构建了覆盖ZmA合成基因簇的重叠联合群,该重叠联合群共覆盖了103Kb区域。通过本研究所构建的在芽胞杆菌中相容的、可克隆大片段的穿梭载体pEMB0557、pEMB0603和pEMB0606,将这9个阳性克隆的插入片段组合式的导入不产ZmA的苏云金芽胞杆菌菌株BMB171中,检测ZmA的表达。有2个BAC克隆子的插入片段(总共覆盖约60Kb的DNA区域)共同导入菌株BMB171后,可以产生ZmA,表明该60Kb对于ZmA的合成是充分的。在这60Kb区域下游,有一个之前未经发现的基因簇,含有三个开放读码框,命名为zmaWXY,将包含该基因簇和60Kb基因簇(共约70Kb)的2个BAC克隆子组合在BMB171中表达发现,ZmA的产量有所增加。实验证实,ZmaWXY为ZmA抗性转运蛋白,其可以阻止ZmA分子进入胞内,是不同于乙酰化酶ZmaR(早先报道的ZmA抗性基因表达蛋白)的另一种抗性机制；同时,增加抗性基因zmaWXY或zmaR的拷贝数,可以提高ZmA的产量。对于抗生素的产生菌,为了防止其被自身所产生的抗生素所抑制,一定存在相应的抗性机制。早前就有研究报道指出,对于ZmA产生菌,将抗性基因zmaR突变后,其仍然可以产生ZmA,但对高浓度的ZmA变得敏感,说明一定存在着其他抗性机制,但一直未知。结合我们在本研究中的发现,我们提出了完整的ZmA产生菌对ZmA抗性免疫的模式：ZmaWXY首先阻挡胞外一定浓度的ZmA进入胞内,当胞外ZmA的浓度超过ZmaWXY所能发挥的功能阈值时,ZmA分子仍然可以进入胞内,此时就由胞内的ZmaR发挥乙酰化功能,使ZmA分子失去抗生素活性,从而保证产生菌的安全。此外,我们在60Kb基因簇内部还发现了一个之前未发现的基因orf123,对其进行基因缺失突变后发现ZmA的产量明显提高,推测其为潜在的ZmA合成的负调控基因。在这两个基因簇的两端(70Kb),存在推测的转座酶基因,暗示这两个基因簇是可移动的区域并且涵盖ZmA完整的合成基因簇。通过本研究,我们利用异源表达手段确认了ZmA完整的合成基因簇,指出完整的基因簇由合成、抗性免疫和调控三个部分组成；发现并证实了与zmaR互补的、新的ZmA抗性基因zmaWXY,并提出了ZmA产生菌对ZmA完整的抗性免疫模式；揭示了潜在的ZmA合成的负调控基因orf123。本研究还建立了通过异源表达方式,从芽胞杆菌、甚至其他细菌中直接克隆、鉴定大片段功能基因簇的平台。更多还原

【Abstract】 Zwittermicin A (ZmA) is a hybrid polyketide-nonribosomal peptide that is produced by some Bacillus strains, including Bacillus cereus strain UW85and Bacillus thuringiensis strain YBT-1520. It displays broad-spectrum antimicrobial activity and synergistically activity with the crystal proteins from B. thuringiensis. Its unusual structure and diverse biological activities make us interested in identifying its gene cluster and regulation.The biosynthetic pathway of ZmA in B. cereus strain UW85by analyzing the nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules involved in ZmA biosynthesis has been proposed, but there is no any experimental evidence to show that the ZmA biosynthetic gene gluster is complete and sufficient for ZmA biosynthesis.In this study, we constructed a bacterial artificial chromosome (BAC) library from genomic DNA of B. thuringiensis subsp. kurstaki strain YBT-1520and screened for the presence of genes known to be involved in the biosynthesis of ZmA. The library consists of1200clones, with an average insert size around45kb, and would have a10-fold coverage of the strain YBT-1520genome. Nine positive clones were identified. By the BAC clones terminal sequence result and the strain YBT-1520genome sequence result, these nine clones can overlap a103-kb CONTIG.Two of the nine clones (totally covering approximately60-kb region) are found to be able to confer ZmA biosynthetic capability upon B. thuringiensis strain BMB171after simultaneously transferring them into this surrogate host by a series of compatible shuttle BAC vectors constructed in this study (pEMB0557, pEMB0603or pEMB0606). Another gene cluster containing three previously unidentified ORFs (named zmaWXY), downstream the60-kb region, were found contribute to the yield of ZmA after transferring another two BAC clone inserts (totally covering approximately70-kb region, including the60-kb region and zmaWXY) into the host BMB171. They are experimentally defined function as ZmA resistance transporter which expels ZmA from the cells.As we known, bacteria which produces antibiotic always has a corresponding resistance mechanism to protect itself from the antibiotic. According to the earlier reports, the zmaR (the ZmA self-resistance gene which has been identified before) mutant was sensitive to high concentrations of ZmA, but could still produce ZmA at near-wild-type levels without compromising growth compared to the parent strain, suggesting that another mechanism of self-resistance must be present. This additional mechanism of self-resistance has remained undiscovered until now. In this study, the whole mechanism for ZmA self-resistance of the producer strain has been proposed: resistance transporter ZmaWXY recognise ZmA and expel it from the cytoplasmic membrane, thus keeping ZmA molecules out of the cell; if the concentration of ZmA in the culture supernatants is higher than the concentration ZmaWXY can cope with, ZmA molecules could diffuse into the cell; once the ZmA molecules get into the cell, ZmaR acetylates ZmA, thereby inactivating it and rendering the cell resistant to ZmA.Gene knockout experiment showed that a previously unidentified gene orfl23which locates in the60-kb region maybe act as a potential negative regulatory gene of ZmA biosynthesis. On the flanking regions of the two gene clusters are putative transposase genes which implies the mobile nature of these two gene clusters.The ZmA intact gene cluster was validated by heterologous expression and is proposed to comprise three parts:biosynthesis, resistance, and regulation. A resistance mechanism of ZmA complementary to zmaR was revealed and the whole mechanism for ZmA self-resistance of the producer strain has been proposed. This study also develops a straightforward strategy to isolate and identify a huge gene cluster from Bacillus, even from bacteria.更多还原