Mechanism(s) of electron transfer and metabolic changes during iron reduction by Clostridium acetobutylicum

Microorganisms catalyze many of the redox transformations driving the iron cycle in the geosphere. For instance, iron cycling includes the reduction of solid-phase and soluble ferric iron (Fe(III)) to ferrous iron (Fe(II)). However, in order to reduce solid-phase Fe(III), the reduction requires the delivery of electrons extracellularly, requiring a specialized electron transfer system. The mechanism of iron reduction by Gram-negative bacteria has been de-scribed in some detail, but very little is known for Gram-positive bacteria. These microorgan-isms are likely to contribute to iron reduction significantly, particularly in environments where Gram-negative bacteria fare poorly, such as metal contaminated sites, or hydrother-mal environments. Some Gram-positive bacteria use metal reduction as a form of enhanced fermentation, where the metal is a sink for excess reducing equivalents. In these cases, metal reduction is facultative and is not a mechanism for direct energy conservation. Here, we use Clostridium acetobutylicum, a Gram-positive fermenter, as a model organism for its ability to reduce iron and contaminant heavy metals. A major advantage of this model organism is that genetic tools are available to study genes involved in the metal reduction process. We investigated the reduction of solid-phase and soluble Fe(III) by C. acetobutyli-cum and found that it reduces both forms of iron. The bacteria do not require direct contact with solid-phase Fe(III) (as hydrous ferric oxide, HFO) for the reduction to occur. In fact, flavins were identified as likely to be involved in the extracellular electron transfer (EET) in this organism. We found that HFO reduction is increased by utilizing exogenous (resazurin, resorufin, anthraquinone-2,6-disulfonate) or endogenous (riboflavin, RF) electron shuttles. Inactivation of a putative flavin transporter (CAC2841) decreased HFO and Fe(III)-citrate reduction. We proposed that a complex CAC2841/ CAC3100/ CAC3101/ CAC3102 imports RF and CAC2841/ CAC3100 exports flavin adenine dinucleotide (FAD). FAD might be hy-drolyzed by CAC2761 and CAC2766, which interact with membrane-anchored flavoproteins potentially involved in EET (CAC2762 and CAC2767). Additionally, we performed a transcriptomic analysis of Fe(III)-citrate- and HFO-reducing cultures and compared them to fermentation alone. We found two Fe-S oxidoreductases (CA1254 and CAC1021) upregulated with Fe(III)-citrate, which have an unknown function and could be putative Fe(III)-citrate reductases. Additional upregulated redox active pro-teins with potential involvement in solid-phase Fe(III) reduction were identified as CA1044 and CAC3371. These proteins should be considered for further studies. Furthermore, iron reduction impacts bacterial metabolism by buffering the pH and shifting the carbon and electron flow to less reduced metabolites as compared to fermentation. In addition to catabolic pathways, metabolic modeling showed that iron reduction influences anabolic pathways. Finally, metabolic modeling suggests that NADH is the physiological elec-tron donor for iron reduction in this system. This study contributes to a better understanding of the mechanism of iron reduction by Gram-positive bacteria and provides implications for further studies to understand the EET by fermenters in natural environments.