Bacteria on the foundational kelp in kelp forest ecosystems: Insights from culturing, whole genome sequencing and metabolic assays.

In coastal marine ecosystems, kelp forests serve as a vital habitat for numerous species and significantly influence local nutrient cycles. Bull kelp, or Nereocystis luetkeana, is a foundational species in the iconic kelp forests of the northeast Pacific Ocean and harbours a complex microbial community with potential implications for kelp health. Here, we report the isolation and functional characterisation of 16 Nereocystis-associated bacterial species, comprising 13 Gammaproteobacteria, 2 Flavobacteriia and 1 Actinomycetia. Genome analyses of these isolates highlight metabolisms potentially beneficial to the host, such as B vitamin synthesis and nitrogen retention. Assays revealed that kelp-associated bacteria thrive on amino acids found in high concentrations in the ocean and in the kelp (glutamine and asparagine), generating ammonium that may facilitate host nitrogen acquisition. Multiple isolates have genes indicative of interactions with key elemental cycles in the ocean, including carbon, nitrogen and sulphur. We thus report a collection of kelp-associated microbial isolates that provide functional insight for the future study of kelp-microbe interactions.

Versatile roles of protein flavinylation in bacterial extracyotosolic electron transfer.

Bacteria perform diverse redox chemistries in the periplasm, cell wall, and extracellular space. Electron transfer for these extracytosolic activities is frequently mediated by proteins with covalently bound flavins, which are attached through post-translational flavinylation by the enzyme ApbE. Despite the significance of protein flavinylation to bacterial physiology, the basis and function of this modification remains unresolved. Here we apply genomic context analyses, computational structural biology, and biochemical studies to address the role of ApbE flavinylation throughout bacterial life. We find that ApbE flavinylation sites exhibit substantial structural heterogeneity. We identify two novel classes of flavinylation substrates that are related to characterized proteins with non-covalently bound flavins, providing evidence that protein flavinylation can evolve from a non-covalent flavoprotein precursor. We further find a group of structurally related flavinylation-associated cytochromes, including those with the domain of unknown function DUF4405, that presumably mediate electron transfer in the cytoplasmic membrane. DUF4405 homologs are widespread in bacteria and related to ferrosome iron storage organelle proteins that may facilitate iron redox cycling within ferrosomes. These studies reveal a complex basis for flavinylated electron transfer and highlight the discovery power of coupling comparative genomic analyses with high-quality structural models.

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

Respiratory reductases enable microorganisms to use molecules present in anaerobic ecosystems as energy-generating respiratory electron acceptors. Here we identify three taxonomically distinct families of human gut bacteria (Burkholderiaceae, Eggerthellaceae and Erysipelotrichaceae) that encode large arsenals of tens to hundreds of respiratory-like reductases per genome. Screening species from each family (Sutterella wadsworthensis, Eggerthella lenta and Holdemania filiformis), we discover 22 metabolites used as respiratory electron acceptors in a species-specific manner. Identified reactions transform multiple classes of dietary- and host-derived metabolites, including bioactive molecules resveratrol and itaconate. Products of identified respiratory metabolisms highlight poorly characterized compounds, such as the itaconate-derived 2-methylsuccinate. Reductase substrate profiling defines enzyme-substrate pairs and reveals a complex picture of reductase evolution, providing evidence that reductases with specificities for related cinnamate substrates independently emerged at least four times. These studies thus establish an exceptionally versatile form of anaerobic respiration that directly links microbial energy metabolism to the gut metabolome.

Bifidobacteria metabolize lactulose to optimize gut metabolites and prevent systemic infection in patients with liver disease.

Progression of chronic liver disease is precipitated by hepatocyte loss, inflammation and fibrosis. This process results in the loss of critical hepatic functions, increasing morbidity and the risk of infection. Medical interventions that treat complications of hepatic failure, including antibiotic administration for systemic infections and lactulose treatment for hepatic encephalopathy, can impact gut microbiome composition and metabolite production. Here, using shotgun metagenomic sequencing and targeted metabolomic analyses on 847 faecal samples from 262 patients with acute or chronic liver disease, we demonstrate that patients hospitalized for liver disease have reduced microbiome diversity and a paucity of bioactive metabolites, including short-chain fatty acids and bile acid derivatives, that impact immune defences and epithelial barrier integrity. We find that patients treated with the orally administered but non-absorbable disaccharide lactulose have increased densities of intestinal bifidobacteria and reduced incidence of systemic infections and mortality. Bifidobacteria metabolize lactulose, produce high concentrations of acetate and acidify the gut lumen in humans and mice, which, in combination, can reduce the growth of antibiotic-resistant bacteria such as vancomycin-resistant Enterococcus faecium in vitro. Our studies suggest that lactulose and bifidobacteria serve as a synbiotic to reduce rates of infection in patients with severe liver disease.

Purine-ifying uric acid by gut microbes.

Elevated bloodstream levels of uric acid, a mammalian purine degradation product, are associated with several noncommunicable diseases. Recent studies by Kasahara et al. and Liu et al. define purine-degrading activities of the gut microbiota that lower bloodstream uric acid in atherosclerosis and gout disease models, establishing a novel microbial role in host health.

Distinct Energy-Coupling Factor Transporter Subunits Enable Flavin Acquisition and Extracytosolic Trafficking for Extracellular Electron Transfer in Listeria monocytogenes.

A variety of electron transfer mechanisms link bacterial cytosolic electron pools with functionally diverse redox activities in the cell envelope and extracellular space. In Listeria monocytogenes, the ApbE-like enzyme FmnB catalyzes extracytosolic protein flavinylation, covalently linking a flavin cofactor to proteins that transfer electrons to extracellular acceptors. L. monocytogenes uses 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 EetB and FmnA proteins 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 of L. monocytogenes strains lacking different ECF transporter subunits demonstrate 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 that show that related ECF exporter genes are distributed across members of the phylum Firmicutes and frequently colocalize with genes encoding flavinylated extracytosolic proteins. These findings clarify the basis of ECF transporter export and extracytosolic flavin cofactor trafficking in Firmicutes. IMPORTANCE Bacteria import vitamins and other essential compounds from their surroundings but also traffic related compounds from the cytosol to the cell envelope where they serve various functions. Studying the foodborne pathogen Listeria monocytogenes, we find that the modular use of subunits from a prominent class of bacterial transporters enables the import of environmental vitamin B2 cofactors and the extracytosolic trafficking of a vitamin B2-derived cofactor that facilitates redox reactions in the cell envelope. These studies clarify the basis of bidirectional small-molecule transport across the cytoplasmic membrane and the assembly of redox-active proteins within the cell envelope and extracellular space.

Listeria monocytogenes requires cellular respiration for NAD+ regeneration and pathogenesis.

Cellular respiration is essential for multiple bacterial pathogens and a validated antibiotic target. In addition to driving oxidative phosphorylation, bacterial respiration has a variety of ancillary functions that obscure its contribution to pathogenesis. We find here that the intracellular pathogen Listeria monocytogenes encodes two respiratory pathways which are partially functionally redundant and indispensable for pathogenesis. Loss of respiration decreased NAD+ regeneration, but this could be specifically reversed by heterologous expression of a water-forming NADH oxidase (NOX). NOX expression fully rescued intracellular growth defects and increased L. monocytogenes loads >1000-fold in a mouse infection model. Consistent with NAD+ regeneration maintaining L. monocytogenes viability and enabling immune evasion, a respiration-deficient strain exhibited elevated bacteriolysis within the host cytosol and NOX expression rescued this phenotype. These studies show that NAD+ regeneration represents a major role of L. monocytogenes respiration and highlight the nuanced relationship between bacterial metabolism, physiology, and pathogenesis.

Cellular respiration is one of the main ways organisms make energy. It works by linking the oxidation of an electron donor (like sugar) to the reduction of an electron acceptor (like oxygen). Electrons pass between the two molecules along what is known as an 'electron transport chain'. This process generates a force that powers the production of adenosine triphosphate (ATP), a molecule that cells use to store energy. Respiration is a common way for cells to replenish their energy stores, but it is not the only way. A simpler process that does not require a separate electron acceptor or an electron transport chain is called fermentation. Many bacteria have the capacity to perform both respiration and fermentation and do so in a context-dependent manner. Research has shown that respiration can contribute to bacterial diseases, like tuberculosis and listeriosis (a disease caused by the foodborne pathogen Listeria monocytogenes). Indeed, some antibiotics even target bacterial respiration. Despite being often discussed in the context of generating ATP, respiration is also important for many other cellular processes, including maintaining the balance of reduced and oxidized nicotinamide adenine dinucleotide (NAD) cofactors. Because of these multiple functions, the exact role respiration plays in disease is unknown. To find out more, Rivera-Lugo, Deng et al. developed strains of the bacterial pathogen Listeria monocytogenes that lacked some of the genes used in respiration. The resulting bacteria were still able to produce energy, but they became much worse at infecting mammalian cells. The use of a genetic tool that restored the balance of reduced and oxidized NAD cofactors revived the ability of respiration-deficient L. monocytogenes to infect mammalian cells, indicating that this balance is what the bacterium requires to infect. Research into respiration tends to focus on its role in generating ATP. But these results show that for some bacteria, this might not be the most important part of the process. Understanding the other roles of respiration could change the way that researchers develop antibacterial drugs in the future. This in turn could help with the growing problem of antibiotic resistance.

RibU is an essential determinant of Listeria pathogenesis that mediates acquisition of FMN and FAD during intracellular growth.

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential riboflavin-derived cofactors involved in a myriad of redox reactions across all forms of life. Nevertheless, the basis of flavin acquisition strategies by riboflavin auxotrophic pathogens remains poorly defined. In this study, we examined how the facultative intracellular pathogen Listeria monocytogenes, a riboflavin auxotroph, acquires flavins during infection. A L. monocytogenes mutant lacking the putative riboflavin transporter (RibU) was completely avirulent in mice but had no detectable growth defect in nutrient-rich media. However, unlike wild type, the RibU mutant was unable to grow in defined media supplemented with FMN or FAD or to replicate in macrophages starved for riboflavin. Consistent with RibU functioning to scavenge FMN and FAD inside host cells, a mutant unable to convert riboflavin to FMN or FAD retained virulence and grew in cultured macrophages and in spleens and livers of infected mice. However, this FMN- and FAD-requiring strain was unable to grow in the gallbladder or intestines, where L. monocytogenes normally grows extracellularly, suggesting that these sites do not contain sufficient flavin cofactors to promote replication. Thus, by deleting genes required to synthesize FMN and FAD, we converted L. monocytogenes from a facultative to an obligate intracellular pathogen. Collectively, these data indicate that L. monocytogenes requires riboflavin to grow extracellularly in vivo but scavenges FMN and FAD to grow in host cells.

Extracellular electron transfer increases fermentation in lactic acid bacteria via a hybrid metabolism.

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.

Bacteria produce the energy they need to live through two processes, respiration and fermentation. While respiration is often more energetically efficient, many bacteria rely on fermentation as their sole means of energy production. Respiration normally depends on the presence of small soluble molecules, such as oxygen, that can diffuse inside the cell, but some bacteria can use metals or other insoluble compounds found outside the cell to perform 'extracellular electron transfer'. Lactic acid bacteria are a large group of bacteria that have several industrial uses and live in many natural environments. These bacteria survive using fermentation, but they also carry a group of genes needed for extracellular electron transfer. It is unclear whether they use these genes for respiration or if they have a different purpose. Tejedor-Sanz, Stevens et al. used a lactic acid bacterium called Lactiplantibacillus plantarum to study whether and how this group of bacteria use extracellular electron transfer. Analysis of L. plantarum and its effect on its surroundings showed that these bacteria use a hybrid process to produce energy: the cells use aspects of extracellular respiration to increase the yield and efficiency of fermentation. Combining these two approaches may allow L. plantarum to adapt to different environments and grow faster, allowing it to compete against other species. Tejedor-Sanz, Stevens et al. provide new information on a widespread group of bacteria that are often used in food production and industry. The next step will be to understand how the hybrid system is controlled and how it varies among species. Understanding this process could result in new biotechnologies and foods that are healthier, produce less waste, or have different tastes and textures.

Solution structure and dynamics of the mitochondrial-targeted GTPase-activating protein (GAP) VopE by an integrated NMR/SAXS approach.

The bacterial pathogen Vibrio cholerae use a type III secretion system to inject effector proteins into a host cell. Recently, a putative Toxic GTPase Activating Protein (ToxGAP) called Vibrio outer protein E (VopE) was identified as a T3SS substrate and virulence factor that affected host mitochondrial dynamics and immune response. However, biophysical and structural characterization has been absent. Here, we describe solution NMR structure of the putative GTPase-activating protein (GAP) domain (73-204) of VopE. Using size exclusion chromatography coupled with small-angle x-ray scattering and residual dipolar coupling data, we restrained the MD process to efficiently determine the overall fold and improve the quality of the output calculated structures. Comparing the structure of VopE with other ToxGAP's revealed a similar overall fold with several features unique to VopE. Specifically, the "Bulge 1," α1 helix, and noteworthy "backside linker" elements on the N-terminus are dissimilar to the other ToxGAP's. By using NMR relaxation dispersion experiments, we demonstrate that these regions undergo motions on a > 6 s-1 timescale. Based on the disposition of these mobile regions relative to the putative catalytic arginine residue, we hypothesize that the protein may undergo structural changes to bind cognate GTPases.

Conceptual Exchanges for Understanding Free-Living and Host-Associated Microbiomes.

Whether a microbe is free-living or associated with a host from across the tree of life, its existence depends on a limited number of elements and electron donors and acceptors. Yet divergent approaches have been used by investigators from different fields. The "environment first" research tradition emphasizes thermodynamics and biogeochemical principles, including the quantification of redox environments and elemental stoichiometry to identify transformations and thus an underlying microbe. The increasingly common "microbe first" research approach benefits from culturing and/or DNA sequencing methods to first identify a microbe and encoded metabolic functions. Here, the microbe itself serves as an indicator for environmental conditions and transformations. We illustrate the application of both approaches to the study of microbiomes and emphasize how both can reveal the selection of microbial metabolisms across diverse environments, anticipate alterations to microbiomes in host health, and understand the implications of a changing climate for microbial function.

Post-translational flavinylation is associated with diverse extracytosolic redox functionalities throughout bacterial life.

Disparate redox activities that take place beyond the bounds of the prokaryotic cell cytosol must connect to membrane or cytosolic electron pools. Proteins post-translationally flavinylated by the enzyme ApbE mediate electron transfer in several characterized extracytosolic redox systems but the breadth of functions of this modification remains unknown. Here, we present a comprehensive bioinformatic analysis of 31,910 prokaryotic genomes that provides evidence of extracytosolic ApbEs within ~50% of bacteria and the involvement of flavinylation in numerous uncharacterized biochemical processes. By mining flavinylation-associated gene clusters, we identify five protein classes responsible for transmembrane electron transfer and two domains of unknown function (DUF2271 and DUF3570) that are flavinylated by ApbE. We observe flavinylation/iron transporter gene colocalization patterns that implicate functions in iron reduction and assimilation. We find associations with characterized and uncharacterized respiratory oxidoreductases that highlight roles of flavinylation in respiratory electron transport chains. Finally, we identify interspecies gene cluster variability consistent with flavinylation/cytochrome functional redundancies and discover a class of 'multi-flavinylated proteins' that may resemble multi-heme cytochromes in facilitating longer distance electron transfer. These findings provide mechanistic insight into an important facet of bacterial physiology and establish flavinylation as a functionally diverse mediator of extracytosolic electron transfer.

In bacteria, certain chemical reactions required for life do not take place directly inside the cells. For instance, 'redox' reactions essential to gather minerals, repair proteins and obtain energy are localised in the membranes and space that surround a bacterium. These chemical reactions involve electrons being transferred from one molecule to another in a cascade that connects the exterior of a cell to its internal space. The enzyme ApbE allows proteins to perform electron transfer by equipping them with ring-like compounds called flavins, through a process known as flavinylation. Yet, the prevelance of flavinylation in bacteria and the scope of redox reactions it facilitates has remained unclear. To investigate this question, Méheust, Huang et al. analysed over 30,000 bacterial genomes, finding genes essential for ApbE flavinylation in about half of all bacterial species across the tree of life. The role of ApbE-flavinylated proteins was then deciphered using a 'guilt by association' approach. In bacteria, genes that perform similar roles are often close to each other in the genome, which helps to infer the function of a protein coded by a specific gene. This approach revealed that flavinylation is involved in processes that allow bacteria to acquire iron and to use various energy sources. A number of interesting proteins were also identified, including a group that carry multiple flavins, and could therefore, in theory, transfer electrons over long distances. This discovery could be relevant to bioelectronic applications, which are already considering another class of bacterial electron-carrying molecules as candidates to form minuscule electric wires.

Bacterial Microcompartments Coupled with Extracellular Electron Transfer Drive the Anaerobic Utilization of Ethanolamine in Listeria monocytogenes.

Ethanolamine (EA) is a valuable microbial carbon and nitrogen source derived from cell membranes. EA catabolism is suggested to occur in a cellular metabolic subsystem called a bacterial microcompartment (BMC), and the activation of EA utilization (eut) genes is linked to bacterial pathogenesis. Despite reports showing that the activation of eut is regulated by a vitamin B12-binding riboswitch and that upregulation of eut genes occurs in mice, it remains unknown whether EA catabolism is BMC dependent in Listeria monocytogenes Here, we provide evidence for BMC-dependent anaerobic EA utilization via metabolic analysis, proteomics, and electron microscopy. First, we show vitamin B12-induced activation of the eut operon in L. monocytogenes coupled to the utilization of EA, thereby enabling growth. Next, we demonstrate BMC formation connected with EA catabolism with the production of acetate and ethanol in a molar ratio of 2:1. Flux via the ATP-generating acetate branch causes an apparent redox imbalance due to the reduced regeneration of NAD+ in the ethanol branch resulting in a surplus of NADH. We hypothesize that the redox imbalance is compensated by linking eut BMCs to anaerobic flavin-based extracellular electron transfer (EET). Using L. monocytogenes wild-type, BMC mutant, and EET mutant strains, we demonstrate an interaction between BMCs and EET and provide evidence for a role of Fe3+ as an electron acceptor. Taken together, our results suggest an important role of BMC-dependent EA catabolism in L. monocytogenes growth in anaerobic environments like the human gastrointestinal tract, with a crucial role for the flavin-based EET system in redox balancing.IMPORTANCE Listeria monocytogenes is a foodborne pathogen causing severe illness, and as such, it is crucial to understand the molecular mechanisms contributing to pathogenicity. One carbon source that allows L. monocytogenes to grow in humans is ethanolamine (EA), which is derived from phospholipids present in eukaryotic cell membranes. It is hypothesized that EA utilization occurs in bacterial microcompartments (BMCs), self-assembling subcellular proteinaceous structures and analogs of eukaryotic organelles. Here, we demonstrate that BMC-driven utilization of EA in L. monocytogenes results in increased energy production essential for anaerobic growth. However, exploiting BMCs and the encapsulated metabolic pathways also requires the balancing of oxidative and reductive pathways. We now provide evidence that L. monocytogenes copes with this by linking BMC activity to flavin-based extracellular electron transfer (EET) using iron as an electron acceptor. Our results shed new light on an important molecular mechanism that enables L. monocytogenes to grow using host-derived phospholipid degradation products.

Extracellular electron transfer powers flavinylated extracellular reductases in Gram-positive bacteria.

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 pathogen Listeria 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 support L. monocytogenes growth. We demonstrate that this represents a generalizable mechanism by finding that a L. monocytogenes strain 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.

A flavin-based extracellular electron transfer mechanism in diverse Gram-positive bacteria.

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 mechanisms2-4 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.

Listeria monocytogenes InlP interacts with afadin and facilitates basement membrane crossing.

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.

Transferase Versus Hydrolase: The Role of Conformational Flexibility in Reaction Specificity.

Active in the aqueous cellular environment where a massive excess of water is perpetually present, enzymes that catalyze the transfer of an electrophile to a non-water nucleophile (transferases) require specific strategies to inhibit mechanistically related hydrolysis reactions. To identify principles that confer transferase versus hydrolase reaction specificity, we exploited two enzymes that use highly similar catalytic apparatuses to catalyze the transglycosylation (a transferase reaction) or hydrolysis of α-1,3-glucan linkages in the cyclic tetrasaccharide cycloalternan (CA). We show that substrate binding to non-catalytic domains and a conformationally stable active site promote CA transglycosylation, whereas a distinct pattern of active site conformational change is associated with CA hydrolysis. These findings defy the classic view of induced-fit conformational change and illustrate a mechanism by which a stable hydrophobic binding site can favor transferase activity and disfavor hydrolysis. Application of these principles could facilitate the rational reengineering of transferases with desired catalytic properties.

Structure to function of an α-glucan metabolic pathway that promotes Listeria monocytogenes pathogenesis.

Here we employ a 'systems structural biology' approach to functionally characterize an unconventional α-glucan metabolic pathway from the food-borne pathogen Listeria monocytogenes (Lm). Crystal structure determination coupled with basic biochemical and biophysical assays allowed for the identification of anabolic, transport, catabolic and regulatory portions of the cycloalternan pathway. These findings provide numerous insights into cycloalternan pathway function and reveal the mechanism of repressor, open reading frame, kinase (ROK) transcription regulators. Moreover, by developing a structural overview we were able to anticipate the cycloalternan pathway's role in the metabolism of partially hydrolysed starch derivatives and demonstrate its involvement in Lm pathogenesis. These findings suggest that the cycloalternan pathway plays a role in interspecies resource competition-potentially within the host gastrointestinal tract-and establish the methodological framework for characterizing bacterial systems of unknown function.

An Unusual Cation-Binding Site and Distinct Domain-Domain Interactions Distinguish Class II Enolpyruvylshikimate-3-phosphate Synthases.

Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes a critical step in the biosynthesis of a number of aromatic metabolites. An essential prokaryotic enzyme and the molecular target of the herbicide glyphosate, EPSPSs are the subject of both pharmaceutical and commercial interest. Two EPSPS classes that exhibit low sequence homology, differing substrate/glyphosate affinities, and distinct cation activation properties have previously been described. Here, we report structural studies of the monovalent cation-binding class II Coxiella burnetii EPSPS (cbEPSPS). Three cbEPSPS crystal structures reveal that the enzyme undergoes substantial conformational changes that alter the electrostatic potential of the active site. A complex with shikimate-3-phosphate, inorganic phosphate (Pi), and K(+) reveals that ligand induced domain closure produces an unusual cation-binding site bordered on three sides by the N-terminal domain, C-terminal domain, and the product Pi. A crystal structure of the class I Vibrio cholerae EPSPS (vcEPSPS) clarifies the basis of differential class I and class II cation responsiveness, showing that in class I EPSPSs a lysine side chain occupies the would-be cation-binding site. Finally, we identify distinct patterns of sequence conservation at the domain-domain interface and propose that the two EPSPS classes have evolved to differently optimize domain opening-closing dynamics.