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A Molecular Biology Tool Kit for the Phototrophic Firmicute Heliobacterium modesticaldum

机译：用于光养性顽固性HeliobacteriumModesticaldum的分子生物学工具套件

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The heliobacteria are members of the bacterial order Clostridiales and form the only group of phototrophs in the phylum Firmicutes. Several physiological and metabolic characteristics make them an interesting subject of investigation, including their minimalist photosynthetic system, nitrogen fixation abilities, and ability to reduce toxic metals. While the species Heliobacterium modesticaldum is an excellent candidate as a model system for the family Heliobacteriaceae, since an annotated genome and transcriptomes are available, studies in this organism have been hampered by the lack of genetic tools. We adapted techniques for genetic manipulation of related clostridial species for use with H. modesticaldum. Five heliobacterial DNA methyltransferase genes were expressed in an Escherichia coli strain engineered as a conjugative plasmid donor for broad-host-range plasmids. Premethylation of the shuttle vectors before conjugation into H. modesticaldum is absolutely required for production of transconjugant colonies. The introduced shuttle vectors are maintained stably and can be recovered using a modified minipreparation procedure developed to inhibit endogenous DNase activity. Furthermore, we describe the formulation of various growth media, including a defined medium for metabolic studies and isolation of auxotrophic mutants.IMPORTANCE Heliobacteria are anoxygenic phototrophic bacteria with the simplest known photosynthetic apparatus. They are unique in using bacteriochlorophyll g as their main pigment and lacking a peripheral antenna system. Until now, research on this organism has been hampered by the lack of a genetic transformation system. Without such a system, gene knockouts, site-directed mutations, and gene expression studies cannot be performed to help us further understand or manipulate the organism. Here we report the genetic transformation of a heliobacterium, which should enable future genetic studies in this unique phototrophic organism.
机译：Heliobacteria是梭状梭菌的成员，并且是Firmicutes门中唯一的一组营养养分。几种生理和代谢特征使其成为一个有趣的研究主题，包括其极简的光合作用系统，固氮能力和还原有毒金属的能力。尽管HeliosbacteriumModeticaldum物种是Heliobacteriaceae家族模式系统的极佳候选者，但由于可获得带注释的基因组和转录组，该生物的研究因缺乏遗传工具而受到阻碍。我们调整了与梭状芽胞杆菌一起使用的相关梭菌物种的遗传操作技术。在作为广泛宿主范围质粒的缀合质粒供体而工程改造的大肠杆菌菌株中表达了五个Heliobacterial DNA甲基转移酶基因。穿梭载体在结合入莫德氏链霉菌之前必须进行预甲基化，以产生反结合结合菌落。所引入的穿梭载体被稳定地维持，并且可以使用开发用于抑制内源性DNA酶活性的改良的微量制备方法来回收。此外，我们描述了各种生长培养基的配方，包括用于代谢研究和营养缺陷型突变体分离的特定培养基。重要事项嗜盐细菌是具有最简单的已知光合作用的产氧性光养细菌。它们以细菌叶绿素g为主要色素而独特，并且缺乏外围天线系统。迄今为止，由于缺乏基因转化系统，对该生物的研究一直受到阻碍。没有这样的系统，就无法进行基因敲除，定点突变和基因表达研究来帮助我们进一步了解或操纵该生物。在这里，我们报告了一种日光细菌的遗传转化，这应该能够在这种独特的光养生物中进行未来的遗传研究。

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