Abstract：
The sulfate-reducing microbe Desulfovibrio ferrophilus is of interest due to its relatively rare ability to also grow with Fe(III) oxide as an electron acceptor and its rapid corrosion of metallic iron. Previous studies have suggested multiple agents for D. ferrophilus extracellular electron exchange including a soluble electron shuttle, electrically conductive pili, and outer surface multiheme c-type cytochromes. However, the previous lack of a strategy for genetic manipulation of D. ferrophilus limited mechanistic investigations. We developed an electroporation-mediated transformation method that enabled replacement of D. ferrophilus genes of interest with an antibiotic resistance gene via double-crossover homologous recombination. Genes were identified that are essential for flagellum-based motility and the expression of the two types of D. ferrophilus pili. Disrupting flagellum-based motility or expression of either of the two pili did not inhibit Fe(III) oxide reduction, nor did deleting genes for multiheme c-type cytochromes predicted to be associated with the outer membrane. Although redundancies in cytochrome or pilus function might explain some of these phenotypes, overall, the results are consistent with D. ferrophilus primarily reducing Fe(III) oxide via an electron shuttle. The finding that D. ferrophilus is genetically tractable not only will aid in elucidating further details of its mechanisms for Fe(III) oxide reduction but also provides a new experimental approach for developing a better understanding of some of its other unique features, such as the ability to corrode metallic iron at high rates and accept electrons from negatively poised electrodes. IMPORTANCE Desulfovibrio ferrophilus is an important pure culture model for Fe(III) oxide reduction and the corrosion of iron-containing metals in anaerobic marine environments. This study demonstrates that D. ferrophilus is genetically tractable, an important advance for elucidating the mechanisms by which it interacts with extracellular electron acceptors and donors. The results demonstrate that there is not one specific outer surface multiheme D. ferrophilus c-type cytochrome that is essential for Fe(III) oxide reduction. This finding, coupled with the lack of apparent porin-cytochrome conduits encoded in the D. ferrophilus genome and the finding that deleting genes for pilus and flagellum expression did not inhibit Fe(III) oxide reduction, suggests that D. ferrophilus has adopted strategies for extracellular electron exchange that are different from those of intensively studied electroactive microbes like Shewanella and Geobacter species. Thus, the ability to genetically manipulate D. ferrophilus is likely to lead to new mechanistic concepts in electromicrobiology.
摘要：
硫酸盐还原微生物Desulfovibrio嗜铁性弧菌由于其相对罕见的能力也与Fe（III）氧化物作为电子受体一起生长以及对金属铁的快速腐蚀而受到关注。以前的研究已经提出了多种药剂为D.ferrophilus胞外电子交换，包括可溶性电子穿梭，导电绒毛，和外表面多血红素c型细胞色素。然而,以前缺乏一种策略的遗传操作，以D.feriphilus有限的机制研究。我们开发了一种电穿孔介导的转化方法，该方法能够通过双交换同源重组用抗生素抗性基因替换感兴趣的嗜铁性链球菌基因。鉴定了对基于鞭毛的运动性和两种类型的嗜铁性D.feriphilus菌毛的表达至关重要的基因。破坏基于鞭毛的运动性或两个菌毛中任何一个的表达都不会抑制Fe（III）氧化物的还原，也没有删除预测与外膜相关的多血红素c型细胞色素基因。尽管细胞色素或菌毛功能的冗余可能解释了其中一些表型，总的来说,结果与主要通过电子穿梭还原Fe（III）氧化物的嗜铁性D.这一发现，即D.feriphilus是遗传可处理的不仅将有助于阐明其机制的进一步细节的Fe（III）氧化物还原，但也提供了一个新的实验方法，为开发一个更好的理解其其他一些独特的特征，例如能够以高速率腐蚀金属铁并接受来自负平衡电极的电子。重要性嗜铁性脱硫弧菌是厌氧海洋环境中Fe（III）氧化物还原和含铁金属腐蚀的重要纯培养模型。这项研究表明，嗜铁性疟原虫是遗传可处理的，阐明它与细胞外电子受体和供体相互作用的机制的重要进展。结果表明，没有一种特定的外表面多血红素D.铁性c型细胞色素是Fe（III）氧化物还原所必需的。这个发现,再加上缺乏明显的孔蛋白-细胞色素导管编码在D.feriphilus基因组和发现删除基因的菌毛和鞭毛表达并不抑制Fe（III）氧化物还原，表明D.ferrophilus采用的细胞外电子交换策略不同于深入研究的电活性微生物，例如Shewanella和Geobacter物种。因此，基因操纵嗜铁性链球菌的能力可能会导致电微生物学中的新机制概念。
