How thermophilic Gram-positive organisms perform extracellular electron transfer: characterization of the cell surface terminal reductase OcwA

Abstract

Extracellular electron transfer is the key process underpinning the development of bioelectrochemical systems for the production of energy or added-value compounds.Thermincola potens JR is a promising Gram-positive bacterium to be used in these systems because it is thermophilic. In this paper we describe the structural and functional properties of the nonaheme cytochrome OcwA, which is the terminal reductase of this organism. The structure of OcwA, determined at 2.2Å resolution shows that the overall-fold and organization of the hemes are not related to other metal reductases and instead are similar to that of multiheme cytochromes involved in the biogeochemical cycles of nitrogen and sulfur. We show that, in addition to solid electron acceptors, OcwA can also reduce soluble electron shuttles and oxyanions. These data reveal that OcwA can take the role of a respiratory 'swiss-army knife' allowing this organism to grow in environments with rapidly changing availability of terminal electron acceptors without the need for transcriptional regulation and protein synthesis. ImportanceThermophilic Gram-positive organisms were recently shown to be a promising class of organisms to be used in bioelectrochemical systems for the production of electrical energy.These organisms present a thick peptidoglycan layer that was thought to preclude them to perform extracellular electron transfer (i.e. exchange catabolic electrons with solid electron acceptors outside of the cell). In this manuscript we describe the structure and functional mechanisms of the multiheme cytochrome OcwA, the terminal reductase of the Grampositive bacterium Thermincola potens JR found at the cell surface of this organism. The results presented here show that this protein is unrelated with terminal reductases found at the cell surface of other electroactive organisms. Instead, OcwA is similar to terminal reductases of soluble electron acceptors. Our data reveals that terminal oxidoreductases of soluble and insoluble substrates are evolutionarily related, providing novel insights into the evolutionary pathway of multiheme cytochromes.

Role of multiheme cytochromes involved in extracellular anaerobic respiration in bacteria

“…The putative extracellular electron transfer pathway was proposed to be composed of four proteins: a decaheme cytochrome (TherJR_1117 in T. potens and Tfer_0070 in T. ferriacetica ) that is anchored to the inner membrane and is proposed to receive electrons from the menaquinone pool; a periplasmic decaheme cytochrome (TherJR_0333 in T. potens and Tfer_1887 in T. ferriacetica ) proposed to transfer electrons within the periplasmic space of Thermincola ; a hexaheme cytochrome, proposed to be embedded in the peptidoglycan (TherJR_1122 in T. potens and Tfer_0075 in T. ferriacetica ), and a nonaheme cytochrome proposed to be the terminal reductase localized at the cell surface of the bacteria (TherJR_2595 in T. potens and Tfer_3197 in T. ferriacetica ) (Figure ). From these, only the cell‐surface multiheme cytochrome, named outer cell‐wall cytochrome A (OcwA) was characterized in detail …”

“…It contains five hemes with the typical bis‐histidine axial coordination (hemes 1, 3, 4, 6, 7, and 8), one heme with histidine‐methionine coordination (heme 9), and two hemes with a histidine as the proximal ligand and an open coordination side as the distal position (hemes 2 and 5). It was shown that these two high‐spin hemes, located at opposite ends of the nine heme arrangement may work as the putative active sites for substrate binding, which is a novelty within the family of multiheme cytochromes …”

Electroactivity across the cell wall of Gram-positive bacteria

“…Like Gram-negative bacteria, some Gram-positive bacteria rely on MHC to transfer electrons to solid electron acceptors outside of the cell. Up to date the only cell-surface cytochrome from Gram-positive bacteria that have been characterized in detailed was the OcwA from T. potens , showing that it can reduce solid electron acceptors, soluble electron shuttles and oxyanions [27] . Nevertheless, the way this protein is anchored and arranged at the cell surface and how electrons are transferred across the cell wall are still to be determined.…”

“…is composed by an inner-membrane MHC that receive electrons from the menaquinone pool, that then transfers the electrons to a periplasmic cytochrome. The transfer of electrons outside of the cell depends on a hexaheme cytochrome that was proposed to be embedded in the peptidoglycan at the cell wall and on the nine-heme cytochrome OcwA present at the cell-surface of these bacteria, responsible to reduce solid electron acceptors, electron shuttles and oxyanions [27] , [29] ( Fig. 2 B).…”

“…Nonetheless, Gram-positive bacteria have recently attracted the scientific attention, given their capacity in producing high levels of current in MFC [25] , [26] , and by being associated with infectious diseases in humans [16] , [17] , [22] , [23] . Given the importance of these organisms in BES, research has been dedicated in exploring their use as catalysts in BES and in the understanding of their EET processes [27] , [28] , [29] . Only by understanding how electroactive organisms perform EET and exchange electrons to electrodes, it is possible to use them in biotechnological processes and start implementing BES in real-world applications.…”