Haufschild Tom, Hammer Jonathan, Rabold Nico, Plut Veronika, Jogler Christian, Kallscheuer Nicolai
Department of Microbial Interactions, Institute for Microbiology, Friedrich Schiller University, 07743, Jena, Germany.
Cluster of Excellence Balance of the Microverse, Friedrich Schiller University, 07743, Jena, Germany.
Appl Microbiol Biotechnol. 2025 Mar 31;109(1):79. doi: 10.1007/s00253-025-13462-w.
Members of the phylum Planctomycetota possess a plethora of intriguing and hitherto underexplored features including an enlarged periplasmic space, asymmetric cell division ("budding"), and a mostly undiscovered small molecule portfolio. Due to the large phylogenetic distance to frequently used and easily genetically accessible model bacteria, most of the established genetic tools are not readily applicable for the here-investigated bacterial phylum. However, techniques for targeted gene inactivation and the introduction of heterologous genes are crucial to investigate the cell biology in the phylum in greater detail. In this study, the targeted genomic modification of model planctomycetes was achieved by enforcing two types of homologous recombination events: simultaneous double homologous recombination for the deletion of coding regions and insertion-duplication mutagenesis for the introduction of foreign DNA into the chromosome. Upon testing the expression of commonly used fluorescent protein-encoding genes, many of the tested native promoters could not be harnessed for variation of the expression strength. Since also four commonly used inducible gene expression systems did not work in the tested model strain Planctopirus limnophila, a native rhamnose-dependent transcriptional regulator/promoter pair was established as an inducible expression system. The expanded molecular toolbox will allow the future characterization of genome-encoded features in the understudied phylum. KEY POINTS: • Two recombination methods were used for the genetic modification of planctomycetes • Commonly used fluorescent proteins are functional in model planctomycetes • A rhamnose-dependent regulator was turned into an inducible expression system.
浮霉菌门的成员具有大量有趣且迄今未被充分探索的特征，包括扩大的周质空间、不对称细胞分裂（“出芽”）以及大多未被发现的小分子组合。由于与常用且易于进行基因操作的模式细菌在系统发育上距离较远，大多数已建立的遗传工具并不容易应用于本文所研究的细菌门。然而，靶向基因失活和异源基因导入技术对于更详细地研究该门的细胞生物学至关重要。在本研究中，通过实施两种同源重组事件实现了模式浮霉菌的靶向基因组修饰：用于缺失编码区的同时双同源重组以及用于将外源DNA引入染色体的插入 - 重复诱变。在测试常用荧光蛋白编码基因的表达时，许多测试的天然启动子无法用于调节表达强度。由于四种常用的诱导型基因表达系统在测试的模式菌株嗜泥平皮球菌中也不起作用，因此建立了一对天然的鼠李糖依赖性转录调节因子/启动子作为诱导型表达系统。这个扩展的分子工具箱将有助于未来对这个研究较少的门类中基因组编码特征的表征。要点：• 使用两种重组方法对浮霉菌进行基因改造 • 常用荧光蛋白在模式浮霉菌中具有功能 • 鼠李糖依赖性调节因子被转化为诱导型表达系统。
