Comprehensive Set of Integrative Plasmid Vectors for Copper-Inducible Gene Expression in Myxococcus xanthus
Comprehensive Set of Integrative Plasmid Vectors for Copper-Inducible Gene Expression in Myxococcus xanthus
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DOI：
10.1128/aem.07502-11
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发表时间：
2012-04-01
影响因子：
4.4
通讯作者：
Munoz-Dorado, Jose
中科院分区：
文献类型：
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作者：
Gomez-Santos, Nuria;Treuner-Lange, Anke;Munoz-Dorado, Jose
黄色粘球菌被广泛用作研究滑动运动、多细胞发育和细胞分化的模型系统。此外，M. xanthus 是新型次生代谢物的丰富来源。对这些过程的分析受到了有限的诱导基因表达工具的阻碍。在这里，我们报告了一组质粒载体的构建，以允许铜诱导基因在 M. xanthus 中表达。铜对菌株DK1622影响的分析表明，生长期间铜浓度高达500μM，发育期间铜浓度高达60μM，不会影响细胞活力、运动性或聚集成子实体等生理过程。迄今为止报道的 M. xanthus 中的铜响应启动子中，多铜氧化酶 cuoA 启动子用于构建表达载体，因为在不存在铜的情况下观察不到基础表达，并且诱导线性依赖于培养基中的铜浓度。已经构建了四种不同的质粒载体，其具有不同的标记选择基因和M.xanthus染色体中的整合位点。载体已经过测试，并使用 lacZ 基因对基因表达进行了定量。此外，我们证明了铜诱导的 pilB 基因表达可以对因 PilB 缺乏而引起的运动缺陷进行功能补充。这些多功能载体可能会加深我们对 M. xanthus 生物学的理解，并且也可能具有生物技术应用。
Myxococcus xanthus is widely used as a model system for studying gliding motility, multicellular development, and cellular differentiation. Moreover, M. xanthus is a rich source of novel secondary metabolites. The analysis of these processes has been hampered by the limited set of tools for inducible gene expression. Here we report the construction of a set of plasmid vectors to allow copper-inducible gene expression in M. xanthus. Analysis of the effect of copper on strain DK1622 revealed that copper concentrations of up to 500 mu M during growth and 60 mu M during development do not affect physiological processes such as cell viability, motility, or aggregation into fruiting bodies. Of the copper-responsive promoters in M. xanthus reported so far, the multicopper oxidase cuoA promoter was used to construct expression vectors, because no basal expression is observed in the absence of copper and induction linearly depends on the copper concentration in the culture medium. Four different plasmid vectors have been constructed, with different marker selection genes and sites of integration in the M. xanthus chromosome. The vectors have been tested and gene expression quantified using the lacZ gene. Moreover, we demonstrate the functional complementation of the motility defect caused by lack of PilB by the copper-induced expression of the pilB gene. These versatile vectors are likely to deepen our understanding of the biology of M. xanthus and may also have biotechnological applications.