Department of Microbiology, University of Massachusetts, Amherst, MA, USA.
Environ Microbiol Rep. 2012 Feb;4(1):82-8. doi: 10.1111/j.1758-2229.2011.00305.x. Epub 2011 Nov 27.
Geobacter metallireducens is an important model organism for many novel aspects of extracellular electron exchange and the anaerobic degradation of aromatic compounds, but studies of its physiology have been limited by a lack of techniques for gene deletion and replacement. Therefore, a genetic system was developed for G. metallireducens by making a number of modifications in the previously described approach for homologous recombination in Geobacter sulfurreducens. Critical modifications included, among others, a 3.5-fold increased in the quantity of electrotransformed linear DNA and the harvesting of cells at early-log. The Cre-lox recombination system was used to remove an antibiotic resistance cassette from the G. metallireducens chromosome permitting the generation of multiple mutations in the same strain. Deletion of the gene fliC, which encodes the flagellin protein, resulted in a strain that did not produce flagella, was non-motile, and was defective for the reduction of insoluble Fe(III). Deletion of pilA, which encodes the structural protein of the type IV pili, inhibited the production of lateral pili as well as Fe(III) oxide reduction and electron transfer to an electrode. These results demonstrate the importance of flagella and pili in the reduction of insoluble Fe(III) by G. metallireducens and provide methods for additional genetic-based approaches for the study of G. metallireducens.
产亚铁还原菌是研究细胞外电子传递和芳香族化合物厌氧降解等诸多新领域的重要模式生物，但由于缺乏基因缺失和替换技术，其生理学研究受到限制。因此，通过对先前描述的脱硫肠状菌同源重组方法进行多项修改，为产亚铁还原菌开发了一种遗传系统。关键的修改包括，将电转化线性 DNA 的量增加了 3.5 倍，并在早期对数期收获细胞。使用 Cre-lox 重组系统从产亚铁还原菌染色体中去除抗生素抗性盒，从而允许在同一菌株中产生多个突变。删除编码鞭毛蛋白的 fliC 基因导致不产生鞭毛、不运动且不能还原不溶性 Fe(III)的菌株。删除编码 IV 型菌毛结构蛋白的 pilA 基因，抑制了侧菌毛的产生以及 Fe(III)氧化物的还原和电子向电极的转移。这些结果表明鞭毛和菌毛在产亚铁还原菌还原不溶性 Fe(III)中的重要性，并为进一步基于遗传的研究方法提供了产亚铁还原菌。
