Scarless Genome Editing and Stable Inducible Expression Vectors for Geobacter sulfurreducens.
Scarless Genome Editing and Stable Inducible Expression Vectors for Geobacter sulfurreducens.
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DOI：
10.1128/aem.01967-15
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发表时间：
2015-10
影响因子：
4.4
通讯作者：
Bond DR
中科院分区：
文献类型：
--
作者：
Chan CH;Levar CE;Zacharoff L;Badalamenti JP;Bond DR
地菌科成员的金属还原是由多个基因簇编码的，细胞外电子转移的研究通常需要在表面形成生物膜。利用极性抗生素盒插入的基因工具限制了突变体的构建和互补。此外，不稳定的质粒会造成新陈代谢负担，从而减缓生长，而卡那霉素等抗生素的存在会干扰地杆菌生物膜的生长速度和程度。我们在这里报告了模型厌氧金属还原细菌硫化还原地杆菌的遗传系统改进。用SACB/蔗糖反选择法精确去除中断fgrM鞭毛调节基因的转座子，构建了一株可移动的硫还原型革兰氏菌，并通过缺失编码内膜细胞色素imcH的基因消除了Fe(III)的还原。我们还表明，与不稳定的pBBR1质粒相比，在没有抗生素选择的情况下，RK2基质粒在硫还原菌中保持了15代以上。因此，我们设计了一系列新的含有天然结构性地杆菌启动子的RK2载体，并对其中一个启动子进行了修饰，使之能够被小分子芳香羧酸香草酸盐诱导成依赖VanR的基因。对于Fe(III)还原、Mn(IV)氧化物还原和在平衡电极上的生长，可诱导质粒完全互补ΔimcH突变体。介绍了一种实时、高通量的柠檬酸还原铁离子检测方法，该方法可以同时筛选多个硫还原革兰氏菌的构建，并通过香草酸盐系统显示imcH表达的敏感性。这些工具将使硫还原革兰氏菌进行更复杂的基因研究，而不会产生极性插入效应，也不需要多种抗生素。
Metal reduction by members of the Geobacteraceae is encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent of Geobacter biofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacterium Geobacter sulfurreducens. A motile strain of G. sulfurreducens was constructed by precise removal of a transposon interrupting the fgrM flagellar regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochrome imcH. We also show that RK2-based plasmids were maintained in G. sulfurreducens for over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutive Geobacter promoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented ΔimcH mutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerous G. sulfurreducens strain constructs simultaneously and shows the sensitivity of imcH expression by the vanillate system. These tools will enable more sophisticated genetic studies in G. sulfurreducens without polar insertion effects or need for multiple antibiotics.