Extracellular electron transfer (EET) describes microbial bioelectrochemical processes in which electrons are transferred from the cytosol to the exterior of the cell1. Mineral-respiring bacteria use elaborate haem-based electron transfer mechanisms2,3,4 but the existence and mechanistic basis of other EETs remain largely unknown. Here we show that the food-borne pathogen Listeria monocytogenes uses a distinctive flavin-based EET mechanism to deliver electrons to iron or an electrode. By performing a forward genetic screen to identify L. monocytogenes mutants with diminished extracellular ferric iron reductase activity, we identified an eight-gene locus that is responsible for EET. This locus encodes a specialized NADH dehydrogenase that segregates EET from aerobic respiration by channelling electrons to a discrete membrane-localized quinone pool. Other proteins facilitate the assembly of an abundant extracellular flavoprotein that, in conjunction with free-molecule flavin shuttles, mediates electron transfer to extracellular acceptors. This system thus establishes a simple electron conduit that is compatible with the single-membrane structure of the Gram-positive cell. Activation of EET supports growth on non-fermentable carbon sources, and an EET mutant exhibited a competitive defect within the mouse gastrointestinal tract. Orthologues of the genes responsible for EET are present in hundreds of species across the Firmicutes phylum, including multiple pathogens and commensal members of the intestinal microbiota, and correlate with EET activity in assayed strains. These findings suggest a greater prevalence of EET-based growth capabilities and establish a previously underappreciated relevance for electrogenic bacteria across diverse environments, including host-associated microbial communities and infectious disease.
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The datasets generated during the current study are available from the corresponding author on reasonable request.
Shi, L. et al. Extracellular electron transfer mechanisms between microorganisms and minerals. Nat. Rev. Microbiol. 14, 651–662 (2016).
Myers, C. R. & Nealson, K. H. Bacterial manganese reduction and growth with manganese oxide as the sole electron acceptor. Science 240, 1319–1321 (1988).
Lovley, D. R. & Phillips, E. J. Novel mode of microbial energy metabolism: organic carbon oxidation coupled to dissimilatory reduction of iron or manganese. Appl. Environ. Microbiol. 54, 1472–1480 (1988).
Carlson, H. K. et al. Surface multiheme c-type cytochromes from Thermincola potens and implications for respiratory metal reduction by Gram-positive bacteria. Proc. Natl Acad. Sci. USA 109, 1702–1707 (2012).
Freitag, N. E., Port, G. C. & Miner, M. D. Listeria monocytogenes – from saprophyte to intracellular pathogen. Nat. Rev. Microbiol. 7, 623–628 (2009).
Deneer, H. G. & Boychuk, I. Reduction of ferric iron by Listeria monocytogenes and other species of Listeria. Can. J. Microbiol. 39, 480–485 (1993).
Kim, B. H., Kim, H. J., Hyun, M. S. & Park, D. H. Direct electrode reaction of Fe(III)-reducing bacterium, Shewanella putrefaciens. J. Microbiol. Biotechnol. 9, 127–131 (1999).
Marsili, E., Rollefson, J. B., Baron, D. B., Hozalski, R. M. & Bond, D. R. Microbial biofilm voltammetry: direct electrochemical characterization of catalytic electrode-attached biofilms. Appl. Environ. Microbiol. 74, 7329–7337 (2008).
Xu, S. J. Y. & El-Naggar, M. Y. Disentangling the roles of free and cytochrome-bound flavins in extracellular electron transport from Shewanella oneidensis MR-1. Electrochim. Acta 198, 49–55 (2016).
Karpowich, N. K., Song, J. M., Cocco, N. & Wang, D. N. ATP binding drives substrate capture in an ECF transporter by a release-and-catch mechanism. Nat. Struct. Mol. Biol. 22, 565–571 (2015).
Kerscher, S., Dröse, S., Zickermann, V. & Brandt, U. The three families of respiratory NADH dehydrogenases. Results Probl. Cell Differ. 45, 185–222 (2008).
Unden, G. & Bongaerts, J. Alternative respiratory pathways of Escherichia coli: energetics and transcriptional regulation in response to electron acceptors. Biochim. Biophys. Acta 1320, 217–234 (1997).
Bertsova, Y. V. et al. Alternative pyrimidine biosynthesis protein ApbE is a flavin transferase catalyzing covalent attachment of FMN to a threonine residue in bacterial flavoproteins. J. Biol. Chem. 288, 14276–14286 (2013).
Deka, R. K., Brautigam, C. A., Liu, W. Z., Tomchick, D. R. & Norgard, M. V. Evidence for posttranslational protein flavinylation in the syphilis spirochete Treponema pallidum: structural and biochemical insights from the catalytic core of a periplasmic flavin-trafficking protein. MBio 6, e00519-15 (2015).
Zückert, W. R. Secretion of bacterial lipoproteins: through the cytoplasmic membrane, the periplasm and beyond. Biochim. Biophys. Acta 1843, 1509–1516 (2014).
Glasser, N. R., Saunders, S. H. & Newman, D. K. The colorful world of extracellular electron shuttles. Annu. Rev. Microbiol. 71, 731–751 (2017).
Brutinel, E. D. & Gralnick, J. A. Shuttling happens: soluble flavin mediators of extracellular electron transfer in Shewanella. Appl. Microbiol. Biotechnol. 93, 41–48 (2012).
Marsili, E. et al. Shewanella secretes flavins that mediate extracellular electron transfer. Proc. Natl Acad. Sci. USA 105, 3968–3973 (2008).
von Canstein, H., Ogawa, J., Shimizu, S. & Lloyd, J. R. Secretion of flavins by Shewanella species and their role in extracellular electron transfer. Appl. Environ. Microbiol. 74, 615–623 (2008).
Kotloski, N. J. & Gralnick, J. A. Flavin electron shuttles dominate extracellular electron transfer by Shewanella oneidensis. MBio 4, e00553-12 (2013).
Powers, H. J. Riboflavin (vitamin B-2) and health. Am. J. Clin. Nutr. 77, 1352–1360 (2003).
Hühner, J., Ingles-Prieto, Á., Neusüß, C., Lämmerhofer, M. & Janovjak, H. Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian model cells by CE with LED-induced fluorescence detection. Electrophoresis 36, 518–525 (2015).
Winter, S. E. et al. Gut inflammation provides a respiratory electron acceptor for Salmonella. Nature 467, 426–429 (2010).
Winter, S. E. et al. Host-derived nitrate boosts growth of E. coli in the inflamed gut. Science 339, 708–711 (2013).
Slobodkin, A. I. et al. Dissimilatory reduction of Fe(III) by thermophilic bacteria and archaea in deep subsurface petroleum reservoirs of western Siberia. Curr. Microbiol. 39, 99–102 (1999).
Roh, Y. et al. Isolation and characterization of metal-reducing thermoanaerobacter strains from deep subsurface environments of the Piceance Basin, Colorado. Appl. Environ. Microbiol. 68, 6013–6020 (2002).
Ogg, C. D. & Patel, B. K. Fervidicola ferrireducens gen. nov., sp. nov., a thermophilic anaerobic bacterium from geothermal waters of the Great Artesian Basin, Australia. Int. J. Syst. Evol. Microbiol. 59, 1100–1107 (2009).
Ogg, C. D. & Patel, B. K. Thermotalea metallivorans gen. nov., sp. nov., a thermophilic, anaerobic bacterium from the Great Artesian Basin of Australia aquifer. Int. J. Syst. Evol. Microbiol. 59, 964–971 (2009).
Ogg, C. D., Greene, A. C. & Patel, B. K. Thermovenabulum gondwanense sp. nov., a thermophilic anaerobic Fe(III)-reducing bacterium isolated from microbial mats thriving in a Great Artesian Basin bore runoff channel. Int. J. Syst. Evol. Microbiol. 60, 1079–1084 (2010).
Ogg, C. D. & Patel, B. K. Fervidicella metallireducens gen. nov., sp. nov., a thermophilic, anaerobic bacterium from geothermal waters. Int. J. Syst. Evol. Microbiol. 60, 1394–1400 (2010).
Masuda, M., Freguia, S., Wang, Y. F., Tsujimura, S. & Kano, K. Flavins contained in yeast extract are exploited for anodic electron transfer by Lactococcus lactis. Bioelectrochemistry 78, 173–175 (2010).
Zhang, E., Cai, Y., Luo, Y. & Piao, Z. Riboflavin-shuttled extracellular electron transfer from Enterococcus faecalis to electrodes in microbial fuel cells. Can. J. Microbiol. 60, 753–759 (2014).
Dong, Y. et al. Orenia metallireducens sp. nov. strain Z6, a novel metal-reducing member of the phylum Firmicutes from the deep subsurface. Appl. Environ. Microbiol. 82, 6440–6453 (2016).
Keogh, D. et al. Extracellular electron transfer powers Enterococcus faecalis biofilm metabolism. MBio 9, e00626-17 (2018).
Pankratova, G., Leech, D., Gorton, L. & Hederstedt, L. Extracellular electron transfer by the Gram-positive bacterium Enterococcus faecalis. Biochemistry 57, 4597–4603 (2018).
Pedersen, M. B., Gaudu, P., Lechardeur, D., Petit, M. A. & Gruss, A. Aerobic respiration metabolism in lactic acid bacteria and uses in biotechnology. Annu. Rev. Food Sci. Technol. 3, 37–58 (2012).
Hodgson, D. A. Generalized transduction of serotype 1/2 and serotype 4b strains of Listeria monocytogenes. Mol. Microbiol. 35, 312–323 (2000).
Zemansky, J. et al. Development of a mariner-based transposon and identification of Listeria monocytogenes determinants, including the peptidyl-prolyl isomerase PrsA2, that contribute to its hemolytic phenotype. J. Bacteriol. 191, 3950–3964 (2009).
Whiteley, A. T., Pollock, A. J. & Portnoy, D. A. The PAMP c-di-AMP is essential for Listeria monocytogenes growth in rich but not minimal media due to a toxic increase in (p)ppGpp. Cell Host Microbe 17, 788–798 (2015).
Xayarath, B., Alonzo, F. III & Freitag, N. E. Identification of a peptide-pheromone that enhances Listeria monocytogenes escape from host cell vacuoles. PLoS Pathog. 11, e1004707 (2015).
Burke, T. P. et al. Listeria monocytogenes is resistant to lysozyme through the regulation, not the acquisition, of cell wall-modifying enzymes. J. Bacteriol. 196, 3756–3767 (2014).
Lovley, D. R. & Phillips, E. J. Organic matter mineralization with reduction of ferric iron in anaerobic sediments. Appl. Environ. Microbiol. 51, 683–689 (1986).
Light, S. H., Cahoon, L. A., Halavaty, A. S., Freitag, N. E. & Anderson, W. F. Structure to function of an α-glucan metabolic pathway that promotes Listeria monocytogenes pathogenesis. Nat. Microbiol. 2, 16202 (2016).
Portnoy, D. A., Jacks, P. S. & Hinrichs, D. J. Role of hemolysin for the intracellular growth of Listeria monocytogenes. J. Exp. Med. 167, 1459–1471 (1988).
Bou Ghanem, E. N. et al. InlA promotes dissemination of Listeria monocytogenes to the mesenteric lymph nodes during food borne infection of mice. PLoS Pathog. 8, e1003015 (2012).
Becattini, S. et al. Commensal microbes provide first line defense against Listeria monocytogenes infection. J. Exp. Med. 214, 1973–1989 (2017).
Auerbuch, V., Lenz, L. L. & Portnoy, D. A. Development of a competitive index assay to evaluate the virulence of Listeria monocytogenes actA mutants during primary and secondary infection of mice. Infect. Immun. 69, 5953–5957 (2001).
Neilson, K. A. et al. Less label, more free: approaches in label-free quantitative mass spectrometry. Proteomics 11, 535–553 (2011).
Nahnsen, S., Bielow, C., Reinert, K. & Kohlbacher, O. Tools for label-free peptide quantification. Mol. Cell. Proteomics 12, 549–556 (2013).
Plumb, R. S. et al. UPLC/MSE; a new approach for generating molecular fragment information for biomarker structure elucidation. Rapid Commun. Mass Spectrom. 20, 1989–1994 (2006).
Shliaha, P. V., Bond, N. J., Gatto, L. & Lilley, K. S. Effects of traveling wave ion mobility separation on data independent acquisition in proteomics studies. J. Proteome Res. 12, 2323–2339 (2013).
Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).
Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).
Jones, D. T., Taylor, W. R. & Thornton, J. M. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8, 275–282 (1992).
Kumar, S., Stecher, G. & Tamura, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016).
We thank G. Chen, J.-D. Sauer, E. Stevens, M. Marco and N. Freitag for providing bacterial strains; H. Carlson, A. Williamson and J. Coates for helpful feedback; and N. Garelis for experimental assistance. Research reported in this publication was supported by funding from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (F32AI136389 to S.H.L., 1P01 AI063302 to D.A.P., and 1R01 AI27655 to D.A.P.), the Office of Naval Research (N0001417WX01603 to C.M.A.-F.), and the China Scholarship Council (no. 201606090098 to L.S.). A mass spectrometer used in this study was purchased with NIH support (grant 1S10OD020062-01). Work at the Molecular Foundry was supported by the Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under Contract No. DE-AC02-05CH11231.
Nature thanks N. Freitag, J. Gralnick, K. Nealson and G. Reguera for their contribution to the peer review of this work.
D.A.P. has a consulting relationship with and a financial interest in Aduro Biotech; both he and the company stand to benefit from the commercialization of this research.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, The double chamber cell used for electrochemical experiments. CE, counter electrode; CEM, cation exchange membrane; RE, reference electrode; WE, working electrode. Inlets and outlets for N2 gas are labelled. b, Cyclic voltammograms of wild-type and ndh2::tn strains of L. monocytogenes. ‘Abiotic’ refers to an uninoculated control. Arrows highlight the initiation of the catalytic wave. Results are representative of three independent experiments.
Ratio of NAD+ to NADH in wild-type and ndh2::tn strains supplemented with ferric ammonium citrate under aerobic or microaerophilic conditions. Results from three independent experiments are expressed as mean ± s.e.m. A statistically significant difference between microaerophilic cells incubated with or without iron is indicated; *P = 0.0015, unpaired two-sided t-test.
Extended Data Fig. 3 Evidence that a distinct menaquinone derivative functions in aerobic respiration.
a, Ferric iron reductase activity of mutants described in Fig. 2 demonstrates that genes essential for growth on aerobic respiration medium are dispensable for EET. Results from three independent experiments are expressed as mean ± s.e.m. b, The L. monocytogenes hep operon. Notably, menG—which encodes demethylmenaquinone transferase (the enzyme that converts demethylmenaquinone to menaquione) (Fig. 2b)—neighbours the hepT and hepS genes, which function in quinone biosynthesis and are essential for aerobic respiration (Fig. 2c).
a, b, Deconvoluted mass spectra from a single experiment of recombinant PplA (a) and recombinant PplA incubated with FAD + FmnB (b). The observed molecular weight change (877 Da) is consistent with two post-translational FMNylations (2 × 438.3 Da) on PplA.
a, Simplified adaptation of a previously proposed model of L. monocytogenes riboflavin uptake through the RibU, EcfT, EcfA and EcfA’ transporter10. According to this model, EcfT, EcfA and EcfA’ couple ATP hydrolysis with conformational changes that result in substrate bound to RibU being released into the cytosol. b, On the basis of protein homology (FmnA shares 50% sequence identity with EcfT) and the expectation that extracellular FAD is required for FmnB to catalyse FMNylation of PplA, we propose that the FmnA interacts with RibU to promote FAD secretion. c, Ferric iron reductase activity of strains incubated with 0.5 mM FAD for 1 h. The ability of exogenous FAD to specifically rescue ferric iron reductase activity in the fmnA::tn and ribU::tn strains is consistent with FmnA and RibU functioning in FAD secretion. Results from three independent experiments are expressed as mean ± s.e.m. Statistically significant differences between untreated and FAD-treated cells are indicated; *P = 0.038, **P < 0.0001, unpaired two-sided t-test.
a, Chronoamperometry results from L. monocytogenes-inoculated electrochemical reactors with 1 μM FMN injections at the indicated time points. Results are representative of three independent experiments. b, The effect of flavins on L. monocytogenes (Lm) ferric iron reductase activity with insoluble ferric (hydr)oxide (top) and soluble ferric ammonium citrate (bottom). With insoluble substrate the local iron concentration for most cells is low, whereas with soluble substrate the concentration of iron in the direct vicinity of cells is high (insets). Results from three independent experiments are expressed as mean ± s.e.m.
a, Growth following incubation of L. monocytogenes strains on xylitol medium without (left) or with (right) ferric iron under aerobic (top) or anaerobic (bottom) conditions. Results are representative of three independent experiments. Strain labels are coloured based on attributed deficiencies (Fig. 2d) in aerobic respiration (blue) or EET (red). Ndh1 and Ndh2 are probably functionally redundant under aerobic conditions, as a growth phenotype is only observed in the double mutant. Note the visual evidence of ferrous iron production in the agar adjoining anaerobically growing cells. b, CFUs of L. monocytogenes strains anaerobically incubated in xylitol medium without (−) or with (+) ferric supplementation. Results for soluble ferric ammonium citrate (top) and insoluble ferric (hydr)oxide (bottom) are shown. Dashed lines denote the number of cells at the start of the experiment. Results from three independent experiments are expressed as mean ± s.e.m. Statistically significant differences in the ferric iron-supplemented condition are noted; ***P < 0.0001, unpaired two-sided t-test.
a, Mouse bone-marrow-derived macrophages were infected with L. monocytogenes, and CFUs were enumerated at the indicated times. Results from three independent experiments are expressed as mean ± s.e.m. b, L. monocytogenes burdens in mouse organs (n = 5) 48 h after intravenous infection. Representative results from two independent experiments are expressed as median and s.e.
a, Phylogenetic tree constructed from select Ndh2 homologue sequences. A more comprehensive list of organisms that possess an EET locus is provided in Supplementary Table 3. Labels on the branches refer to the percentage of replicate trees that gave the depicted branch topology in a bootstrap test of 1,000 replicates. b, Distinct EET loci from select genomes are shown. Although the arrangement of genes varies, a locus with genes associated with EET is present in many genomes. Some loci contain ECF transporter ATPase subunits (homologous to those depicted in Extended Data Fig. 5a) that probably function with RibU and FmnA subunits in flavin transport. The dmkA-like gene found in Caldanaerobius fijiensis (and other genomes) lacks homology to dmkA, but is annotated as catalysing the same reaction. The pplA variant in some genomes contains a single FMNylated domain (rather than two) and this property is indicated by a shorter arrow. A few bacteria (including Lactococcus spp.) lack a recognizable locus and distribute genes associated with EET throughout the genome.
This file contains Supplementary Figure 1: Uncropped gel from Fig. 3c.
This file contains Supplementary Tables 1-4. Supplementary Table 1 provides proteomics evidence of the FMNylation of PplA. Supplementary Table 2 identifies surface-associated proteins in Listeria monocytogenes. Supplementary Table 3 identifies Firmicutes species with homologous EET genes. Supplementary Table 4 contains information about strains used in this study.
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Light, S.H., Su, L., Rivera-Lugo, R. et al. A flavin-based extracellular electron transfer mechanism in diverse Gram-positive bacteria. Nature 562, 140–144 (2018). https://doi.org/10.1038/s41586-018-0498-z
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