Validation of the Intact Zwittermicin A Biosynthetic Gene Cluster and Discovery of a Complementary Resistance Mechanism in Bacillus thuringiensis
Validation of the Intact Zwittermicin A Biosynthetic Gene Cluster and Discovery of a Complementary Resistance Mechanism in Bacillus thuringiensis
复制标题
苏云金芽孢杆菌完整两性霉素 A 生物合成基因簇的验证和互补耐药机制的发现
DOI：
10.1128/aac.00111-11
复制
发表时间：
2011-09-01
影响因子：
4.9
通讯作者：
Sun, Ming
中科院分区：
文献类型：
--
作者：
Luo, Yi;Ruan, Li-Fang;Sun, Ming
摘要两性霉素A(Zwittermicin A，ZMA)是由某些蜡状芽孢杆菌群菌株产生的一种聚酮-非核糖体杂合肽。它表现出广谱的抗菌活性。通过分析参与ZMA生物合成的非核糖体肽合成酶(NRPS)和聚酮合成酶(PKS)模块，提出了ZMA在蜡样芽孢杆菌中的生物合成途径。在本研究中，我们构建了苏云金芽孢杆菌亚种细菌人工染色体文库。库尔斯塔克菌株YBT-1520基因组DNA。用聚合酶链式反应技术研究了该文库中与ZMA生物合成相关的已知基因的存在。鉴定出9个阳性克隆，其中2个(覆盖约60kb区域)通过两个相容的穿梭BAC载体同时转入苏云金芽孢杆菌BMB171，具有合成ZMA的能力。另一个以前未知的基因簇zmaWXY被发现可以提高ZMA的产量，并被实验定义为ZMA抗性转运蛋白，将ZMA从细胞中驱逐出去。在两个基因簇(zma合成簇和zmaWXY)的侧翼区域检测到了可能的转座酶基因，这表明这两个基因簇具有移动性。对完整的ZMA基因簇进行了验证，揭示了与已鉴定的ZMA自抗基因zmaR互补的抗性机制。这项研究也为从芽孢杆菌中分离和鉴定一个巨大的基因簇提供了一种简单的策略。
ABSTRACT Zwittermicin A (ZmA) is a hybrid polyketide-nonribosomal peptide produced by certain Bacillus cereus group strains. It displays broad-spectrum antimicrobial activity. Its biosynthetic pathway in B. cereus has been proposed through analysis of the nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules involved in ZmA biosynthesis. In this study, we constructed a bacterial artificial chromosome (BAC) library from Bacillus thuringiensis subsp. kurstaki strain YBT-1520 genomic DNA. The presence of known genes involved in the biosynthesis of ZmA in this BAC library was investigated by PCR techniques. Nine positive clones were identified, two of which (covering an approximately 60-kb region) could confer ZmA biosynthesis ability upon B. thuringiensis BMB171 after simultaneous transfer into this host by two compatible shuttle BAC vectors. Another previously unidentified gene cluster, named zmaWXY, was found to improve the yield of ZmA and was experimentally defined to function as a ZmA resistance transporter which expels ZmA from the cells. Putative transposase genes were detected on the flanking regions of the two gene clusters (the ZmA synthetic cluster and zmaWXY), which suggests a mobile nature of these two gene clusters. The intact ZmA gene cluster was validated, and a resistance mechanism complementary to that for zmaR (the previously identified ZmA self-resistance gene) was revealed. This study also provided a straightforward strategy to isolate and identify a huge gene cluster from Bacillus.