Distinct energy-coupling factor transporter subunits enable flavin acquisition and extracytosolic trafficking for extracellular electron transfer inListeria monocytogenes

Abstract

A variety of electron transfer mechanisms link bacterial cytosolic electron pools with functionally diverse redox activities in the cell envelope and extracellular space. InListeria monocytogenes, the ApbE-like enzyme FmnB catalyzes extracytosolic protein flavinylation, covalently linking a flavin cofactor to proteins that transfer electrons to extracellular acceptors.L. monocytogenesuses an energy-coupling factor (ECF) transporter complex that contains distinct substrate-binding, transmembrane, ATPase A, and ATPase A' subunits (RibU, EcfT, EcfA, and EcfA') to import environmental flavins, but the basis of extracytosolic flavin trafficking for FmnB flavinylation remains poorly defined. In this study, we show that the proteins EetB and FmnA are related to ECF transporter substrate-binding and transmembrane subunits, respectively, and are essential for exporting flavins from the cytosol for flavinylation. Comparisons of the flavin import versus export capabilities ofL. monocytogenesstrains lacking different ECF transporter subunits demonstrates a strict directionality of substrate-binding subunit transport but partial functional redundancy of transmembrane and ATPase subunits. Based on these results, we propose that ECF transporter complexes with different subunit compositions execute directional flavin import/export through a broadly conserved mechanism. Finally, we present genomic context analyses which show that related ECF exporter genes are distributed across the Firmicutes phylum and frequently co-localize with genes encoding flavinylated extracytosolic proteins. These findings clarify the basis of ECF transporter export and extracytosolic flavin cofactor trafficking in Firmicutes.

Enterococcus faecalis NADH Peroxidase-Defective Mutants Stain Falsely in Colony Zymogram Assay for Extracellular Electron Transfer to Ferric Ions

“…In a previous study, Ndh3, DMK, and EetA were shown to be important for the ferric reductase activity of washed E. faecalis cells [ 14 ]. The present work demonstrates that EetB is also required, which is consistent with other studies [ 26 , 27 , 37 ]. The direct electron donor(s) to ferric ion in the cell envelope of E. faecalis and other Gram-positive electrogenic bacteria, such as Listeria monocytogemes and Lactiplantibacillus plantarum , is not known.…”

“…However, PplA is not required for EET to Fe 3+ in E. faecalis [ 14 ] and recent experimental findings in Listeria monocytogenes show that EetB is a FAD-binding protein [ 37 ]. EetB is now suggested to be the substrate-binding component of an energy-coupled factor transporter that delivers FAD to FmnB, which is active on the outer side of the cytoplasmic membrane, catalyzing the covalent binding of FMN to a family of proteins [ 37 , 38 ]. The role of EetA is not known, but the protein seems tightly connected to the function of EetB [ 37 ].…”

“…EetB is now suggested to be the substrate-binding component of an energy-coupled factor transporter that delivers FAD to FmnB, which is active on the outer side of the cytoplasmic membrane, catalyzing the covalent binding of FMN to a family of proteins [ 37 , 38 ]. The role of EetA is not known, but the protein seems tightly connected to the function of EetB [ 37 ]. In a previous study, Ndh3, DMK, and EetA were shown to be important for the ferric reductase activity of washed E. faecalis cells [ 14 ].…”

“…The direct electron donor(s) to ferric ion in the cell envelope of E. faecalis and other Gram-positive electrogenic bacteria, such as Listeria monocytogemes and Lactiplantibacillus plantarum , is not known. It might be electron-conductive pili, one or more flavoproteins exposed on the outer side of the cytoplasmic membrane, and depend on small soluble redox compounds in the cell envelope [ 26 , 37 , 39 ]. The so-far-reported screens for ferric reductase-defective mutants of L. monocytogenes and E. faecalis based on colony Ferrozine zymogram staining have not identified any genes outside of the so called FLEET locus, comprising pplA , eetAB , and ndh2/3 , and dmkAB genes for quinone synthesis [ 14 , 27 ] (this work).…”

Dietary- and host-derived metabolites are used by diverse gut bacteria for anaerobic respiration

“…87 Recombinant reductase production and purification Hexa-histidine-tagged reductase constructs were expressed in E. coli Rosetta cells using either the pET28a or pMCSG53 expression vectors, as previously described. 88 Briefly, expression was performed using 500 mL cultures of Luria-Bertani broth (BD LB Broth, Miller #244610) + 100 µM riboflavin, grown to an OD of 0.7-1.0 at 37 °C, then induced with 1 mM β-d-1-thiogalactopyranoside overnight at 20 °C. Kanamycin (30 mg/mL, pET28a vector) or carbenicillin (100 mg/mL, pMCSG53 vector) were used for selection.…”