Genetic Manipulation of Desulfovibrio ferrophilus and Evaluation of Fe(III) Oxide Reduction Mechanisms

Abstract

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.

Keywords

• electromicrobiology
• c-type cytochromes
• e-pili
• iron reduction
• extracellular electron transfer
• electron transfer