Extracellular electron transfer (EET) describes microbial bioelectrochemical processes in which electrons are transferred from the cytosol to the exterior of the cell. Mineral-respiring bacteria use elaborate haem-based electron transfer mechanisms 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.

Zika flavivirus infection during pregnancy appears to produce higher risk of microcephaly, and also causes multiple neurological problems such as Guillain–Barré syndrome. The Zika virus is now widespread in Central and South America, and is anticipated to become an increasing risk in the southern United States. With continuing global travel and the spread of the mosquito vector, the exposure is expected to accelerate, but there are no currently approved treatments against the Zika virus. The Zika NS2B/NS3 protease is an attractive drug target due to its essential role in viral replication. Our studies have identified several compounds with inhibitory activity (IC50) and binding affinity (KD) of ∼5-10 μM against the Zika NS2B-NS3 protease from testing 71 HCV NS3/NS4A inhibitors that were initially discovered by high-throughput screening of 40,967 compounds. Competition surface plasmon resonance studies and mechanism of inhibition analyses by enzyme kinetics subsequently determined the best compound to be a competitive inhibitor with a Ki value of 9.5 μM. We also determined the X-ray structure of the Zika NS2B-NS3 protease in a “pre-open conformation”, a conformation never observed before for any flavivirus proteases. This provides the foundation for new structure-based inhibitor design.

Mineral-respiring bacteria use a process called extracellular electron transfer to route their respiratory electron transport chain to insoluble electron acceptors on the exterior of the cell. We recently characterized a flavin-based extracellular electron transfer system that is present in the foodborne pathogenListeria monocytogenes, as well as many other Gram-positive bacteria, and which highlights a more generalized role for extracellular electron transfer in microbial metabolism. Here we identify a family of putative extracellular reductases that possess a conserved posttranslational flavinylation modification. Phylogenetic analyses suggest that divergent flavinylated extracellular reductase subfamilies possess distinct and often unidentified substrate specificities. We show that flavinylation of a member of the fumarate reductase subfamily allows this enzyme to receive electrons from the extracellular electron transfer system and supportL. monocytogenesgrowth. We demonstrate that this represents a generalizable mechanism by finding that aL. monocytogenesstrain engineered to express a flavinylated extracellular urocanate reductase uses urocanate by a related mechanism and to a similar effect. These studies thus identify an enzyme family that exploits a modular flavin-based electron transfer strategy to reduce distinct extracellular substrates and support a multifunctional view of the role of extracellular electron transfer activities in microbial physiology.

Energy conservation in microorganisms is classically categorized into respiration and fermentation; however, recent work shows some species can use mixed or alternative bioenergetic strategies. We explored the use of extracellular electron transfer for energy conservation in diverse lactic acid bacteria (LAB), microorganisms that mainly rely on fermentative metabolism and are important in food fermentations. The LAB Lactiplantibacillus plantarum uses extracellular electron transfer to increase its NAD+/NADH ratio, generate more ATP through substrate-level phosphorylation, and accumulate biomass more rapidly. This novel, hybrid metabolism is dependent on a type-II NADH dehydrogenase (Ndh2) and conditionally requires a flavin-binding extracellular lipoprotein (PplA) under laboratory conditions. It confers increased fermentation product yield, metabolic flux, and environmental acidification in laboratory media and during kale juice fermentation. The discovery of a single pathway that simultaneously blends features of fermentation and respiration in a primarily fermentative microorganism expands our knowledge of energy conservation and provides immediate biotechnology applications.

Present within bacteria, plants, and some lower eukaryotes 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS) catalyzes the first committed step in the synthesis of a number of metabolites, including the three aromatic amino acids phenylalanine, tyrosine, and tryptophan. Catalyzing the first reaction in an important biosynthetic pathway, DAHPS is situated at a critical regulatory checkpoint-at which pathway input can be efficiently modulated to respond to changes in the concentration of pathway outputs. Based on a phylogenetic classification scheme, DAHPSs have been divided into three major subtypes (Ia, Ib, and II). These subtypes are subjected to an unusually diverse pattern of allosteric regulation, which can be used to further subdivide the enzymes. Crystal structures of most of the regulatory subclasses have been determined. When viewed collectively, these structures illustrate how distinct mechanisms of allostery are applied to a common catalytic scaffold. Here, we review structural revelations regarding DAHPS regulation and make the case that the functional difference between the three major DAHPS subtypes relates to basic distinctions in quaternary structure and mechanism of allostery.

During pregnancy, the placenta protects the fetus against the maternal immune response, as well as bacterial and viral pathogens. Bacterial pathogens that have evolved specific mechanisms of breaching this barrier, such as Listeria monocytogenes, present a unique opportunity for learning how the placenta carries out its protective function. We previously identified the L. monocytogenes protein Internalin P (InlP) as a secreted virulence factor critical for placental infection. Here, we show that InlP, but not the highly similar L. monocytogenes internalin Lmo2027, binds to human afadin (encoded by AF-6), a protein associated with cell-cell junctions. A crystal structure of InlP reveals several unique features, including an extended leucine-rich repeat (LRR) domain with a distinctive Ca2+-binding site. Despite afadin’s involvement in the formation of cell-cell junctions, MDCK epithelial cells expressing InlP displayed a decrease in the magnitude of the traction stresses they could exert on deformable substrates, similar to the decrease in traction exhibited by AF-6 knock-out MDCK cells. L. monocytogenes ΔinlP mutants were deficient in their ability to form actin-rich protrusions from the basal face of polarized epithelial monolayers, a necessary step in the crossing of such monolayers (transcytosis). A similar phenotype was observed for bacteria expressing an internal in-frame deletion in inlP (inlP ΔLRR5) that specifically disrupts its interaction with afadin. However, afadin deletion in the host cells did not rescue the transcytosis defect. We conclude that secreted InlP targets cytosolic afadin to specifically promote L. monocytogenes transcytosis across the basal face of epithelial monolayers, which may contribute to the crossing of the basement membrane during placental infection.

The biosynthetic shikimate pathway consists of seven enzymes that catalyze sequential reactions to generate chorismate, a critical branch point in the synthesis of the aromatic amino acids. The third enzyme in the pathway, dehydroquinate dehydratase (DHQD), catalyzes the dehydration of 3-dehydroquinate to 3-dehydroshikimate. We present three crystal structures of the type I DHQD from the intestinal pathogens Clostridium difficile and Salmonella enterica. Structures of the enzyme with substrate and covalent pre-and post-dehydration reaction intermediates provide snapshots of successive steps along the type I DHQD-catalyzed reaction coordinate. These structures reveal that the position of the substrate within the active site does not appreciably change upon Schiff base formation. The intermediate state structures reveal a reaction state-dependent behavior of His-143 in which the residue adopts a conformation proximal to the site of catalytic dehydration only when the leaving group is present. We speculate that His-143 is likely to assume differing catalytic roles in each of its observed conformations. One conformation of His-143 positions the residue for the formation/hydrolysis of the covalent Schiff base intermediates, whereas the other conformation positions the residue for a role in the catalytic dehydration event. The fact that the shikimate pathway is absent from humans makes the enzymes of the pathway potential targets for the development of non-toxic antimicrobials. The structures and mechanistic insight presented here may inform the design of type I DHQD enzyme inhibitors.Present in bacteria, fungi, and plants but absent in higher eukaryotes, the seven enzymes of the shikimate pathway catalyze sequential reactions to generate chorismate. Chorismate serves as a precursor of many biologically important aromatic compounds including the ubiquinones, folates, and aromatic amino acids (1). The essential nature of these proteins in a number of species, in combination with an absence of human homologs, makes the shikimate pathway an attractive target for the development of non-toxic antimicrobials, anti-fungals, and herbicides (2-4). Given the complexities inherent to inhibitor design, a comprehensive understanding of the structural and mechanistic framework underlying the function of the shikimate pathway enzymes should aid in the development of novel shikimate-targeting inhibitors.The third step in the shikimate pathway, consisting of the dehydration of dehydroquinate to dehydroshikimate (Fig. 1), can be catalyzed by two unrelated enzymes, termed type I and type II dehydroquinate dehydratases (DHQDs).2 These two enzyme families lack sequence or structural homology and employ distinct reaction mechanisms (5-10). The type I DHQDs utilize a covalent Schiff base (imine) intermediate that results in a cis-elimination, whereas the type II reaction lacks a covalent intermediate and undergoes a trans-elimination (5-10). The phylogenetic distribution of the two enzyme types is somewhat disorderly, with closely related s...

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) catalyzes the first step in the biosynthesis of a number of aromatic metabolites. Likely because this reaction is situated at a pivotal biosynthetic gateway, several DAHPS classes distinguished by distinct mechanisms of allosteric regulation have independently evolved. One class of DAHPSs contains a regulatory domain with sequence homology to chorismate mutase-an enzyme further downstream of DAHPS that catalyzes the first committed step in tyrosine/phenylalanine biosynthesis-and is inhibited by chorismate mutase substrate (chorismate) and product (prephenate). Described in this work, structures of the Listeria monocytogenes chorismate/prephenate regulated DAHPS in complex with Mn 21 and Mn 21 1 phosphoenolpyruvate reveal an unusual quaternary architecture: DAHPS domains assemble as a tetramer, from either side of which chorismate mutase-like (CML) regulatory domains asymmetrically emerge to form a pair of dimers. This domain organization suggests that chorismate/prephenate binding promotes a stable interaction between the discrete regulatory and catalytic domains and supports a mechanism of allosteric inhibition similar to tyrosine/phenylalanine control of a related DAHPS class. We argue that the structural similarity of chorismate mutase enzyme and CML regulatory domain provides a unique opportunity for the design of a multitarget antibacterial.