The differing roles of flavins and quinones in extracellular electron transfer in Lactiplantibacillus plantarum

Abstract

Lactiplantibacillus plantarum is a lactic acid bacteria that is commonly found in the human gut and fermented food products. Despite its overwhelmingly fermentative metabolism, this microbe can perform extracellular electron transfer (EET) when provided with an exogenous quinone, 1,4-dihydroxy-2-naphthoic acid (DHNA) and riboflavin. However, the separate roles of DHNA and riboflavin in EET in L. plantarum has remained unclear. Here we seek to understand the role of quinones and flavins for EET by monitoring iron and anode reduction in the presence and absence of these small molecules. We found that either addition of DHNA or riboflavin can support robust iron reduction, indicating electron transfer to extracellular iron occurs through both flavin-dependent and DHNA-dependent routes. Using genetic mutants of L. plantarum, we found that flavin-dependent iron reduction requires Ndh2 and EetA, while DHNA-dependent iron reduction largely relies on Ndh2 and PplA. In contrast to iron reduction, DHNA-containing media supported more robust anode reduction than riboflavin-containing media, suggesting electron transfer to an anode proceeds most efficiently through the DHNA-dependent pathway. Furthermore, we found that flavin-dependent anode reduction requires EetA, Ndh2, and PplA, while DHNA-dependent anode reduction requires Ndh2 and PplA. Taken together, we identify multiple EET routes utilized by L. plantarum and show that the EET route depends on access to environmental biomolecules and on the extracellular electron acceptor. This work expands our molecular-level understanding of EET in Gram-positive microbes and provides additional opportunities to manipulate EET for biotechnology. ImportanceLactic acid bacteria are named because of their nearly exclusive fermentative metabolism. Thus, the recent observation of EET activity - typically associated with anaerobic respiration - in this class of organisms has forced researchers to rethink the rules governing microbial metabolic strategies. Our identification of multiple routes for EET in L. plantarum that depend on two separate redox active small molecules expands our understanding of how microbes metabolically adapt to different environments to gain an energetic edge and how these processes can be manipulated for biotechnological uses. Understanding the role of EET in lactic acid bacteria is of great importance due to the significance of lactic acid bacteria in agriculture, bioremediation, food production, and gut health. Furthermore, the maintenance of multiple EET routes speak to the importance of this process to function in a variety of environmental conditions.