A ubiquitous mobile genetic element changes the antagonistic weaponry of a human gut symbiont.

DNA transfer is ubiquitous in the human gut microbiota, especially among species of the order Bacteroidales. In silico analyses have revealed hundreds of mobile genetic elements shared between these species, yet little is known about the phenotypes they encode, their effects on fitness, or pleiotropic consequences for the recipient's genome. In this work, we show that acquisition of a ubiquitous integrative conjugative element (ICE) encoding a type VI secretion system (T6SS) shuts down the native T6SS of .

Versatile roles of protein flavinylation in bacterial extracyotosolic electron transfer.

Unlabelled: 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 remain unresolved.

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.

A ubiquitous mobile genetic element disarms a bacterial antagonist of the gut microbiota.

DNA transfer is ubiquitous in the gut microbiota, especially among species of Bacteroidales. analyses have revealed hundreds of mobile genetic elements shared between these species, yet little is known about the phenotypes they encode, their effects on fitness, or pleiotropic consequences for the recipient's genome. Here, we show that acquisition of a ubiquitous integrative and conjugative element encoding an antagonistic system shuts down the native contact-dependent antagonistic system of .

Maintains Concurrent Capability for Anaerobic and Nanaerobic Respiration.

species can use fumarate and oxygen as terminal electron acceptors during cellular respiration. In the human gut, oxygen diffuses from intestinal epithelial cells supplying "nanaerobic" oxygen levels. Many components of the anaerobic respiratory pathway have been determined, but such analyses have not been performed for nanaerobic respiration.

Cryo-EM structures of Na-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae.

The Na-pumping NADH-ubiquinone oxidoreductase (Na-NQR) couples electron transfer from NADH to ubiquinone with Na-pumping, generating an electrochemical Na gradient that is essential for energy-consuming reactions in bacteria. Since Na-NQR is exclusively found in prokaryotes, it is a promising target for highly selective antibiotics. However, the molecular mechanism of inhibition is not well-understood for lack of the atomic structural information about an inhibitor-bound state.

The side chain of ubiquinone plays a critical role in Na translocation by the NADH-ubiquinone oxidoreductase (Na-NQR) from Vibrio cholerae.

The Na-pumping NADH-ubiquinone (UQ) oxidoreductase (Na-NQR) is an essential bacterial respiratory enzyme that generates a Na gradient across the cell membrane. However, the mechanism that couples the redox reactions to Na translocation remains unknown. To address this, we examined the relation between reduction of UQ and Na translocation using a series of synthetic UQs with Vibrio cholerae Na-NQR reconstituted into liposomes.

Genetic and Biochemical Characterization of the Na/H Antiporters of Pseudomonas aeruginosa.

Pseudomonas aeruginosa has four Na/H antiporters that interconvert and balance Na and H gradients across the membrane. These gradients are important for bioenergetics and ionic homeostasis. To understand these transporters, we constructed four strains, each of which has only one antiporter, i.

Specific chemical modification explores dynamic structure of the NqrB subunit in Na-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae.

The Na-pumping NADH-ubiquinone oxidoreductase (Na-NQR) is a main ion transporter in many pathogenic bacteria. We previously proposed that N-terminal stretch of the NqrB subunit plays an important role in regulating the ubiquinone reaction at the adjacent NqrA subunit in Vibrio cholerae Na-NQR. However, since approximately three quarters of the stretch (NqrB-Met-Pro) was not modeled in an earlier crystallographic study, its structure and function remain unknown.

The Molecular Basis for Life in Extreme Environments.

Sampling and genomic efforts over the past decade have revealed an enormous quantity and diversity of life in Earth's extreme environments. This new knowledge of life on Earth poses the challenge of understandingits molecular basis in such inhospitable conditions, given that such conditions lead to loss of structure and of function in biomolecules from mesophiles. In this review, we discuss the physicochemical properties of extreme environments.

The three NADH dehydrogenases of Pseudomonas aeruginosa: Their roles in energy metabolism and links to virulence.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen which relies on a highly adaptable metabolism to achieve broad pathogenesis. In one example of this flexibility, to catalyze the NADH:quinone oxidoreductase step of the respiratory chain, P. aeruginosa has three different enzymes: NUO, NQR and NDH2, all of which carry out the same redox function but have different energy conservation and ion transport properties.

Metabolic engineering of E. coli for pyocyanin production.

Pyocyanin is a secondary metabolite from Pseudomonas aeruginosa that belongs to the class of phenazines, which are aromatic nitrogenous compounds with numerous biological functions. Besides its antifungal and antimicrobial activities, pyocyanin is a remarkable redox-active molecule with potential applications ranging from the pharma industry to the development of microbial fuel cells. Nevertheless, pyocyanin production has been restricted to P.

Nanaerobic growth enables direct visualization of dynamic cellular processes in human gut symbionts.

Mechanistic studies of anaerobic gut bacteria have been hindered by the lack of a fluorescent protein system to track and visualize proteins and dynamic cellular processes in actively growing bacteria. Although underappreciated, many gut "anaerobes" are able to respire using oxygen as the terminal electron acceptor. The oxygen continually released from gut epithelial cells creates an oxygen gradient from the mucus layer to the anaerobic lumen [L.

Inhibitors of a Na-pumping NADH-ubiquinone oxidoreductase play multiple roles to block enzyme function.

The Na-pumping NADH-ubiquinone (UQ) oxidoreductase (Na-NQR) is present in the respiratory chain of many pathogenic bacteria and is thought to be a promising antibiotic target. Whereas many details of Na-NQR structure and function are known, the mechanisms of action of potent inhibitors is not well-understood; elucidating the mechanisms would not only advance drug design strategies but might also provide insights on a terminal electron transfer from riboflavin to UQ. To this end, we performed photoaffinity labeling experiments using photoreactive derivatives of two known inhibitors, aurachin and korormicin, on isolated Na-NQR.

Genetic and Biochemical Analysis of Anaerobic Respiration in Bacteroides fragilis and Its Importance .

In bacteria, the respiratory pathways that drive molecular transport and ATP synthesis include a variety of enzyme complexes that utilize different electron donors and acceptors. This property allows them to vary the efficiency of energy conservation and to generate different types of electrochemical gradients (H or Na). We know little about the respiratory pathways in species, which are abundant in the human gut, and whether they have a simple or a branched pathway.

Glutamate 95 in NqrE Is an Essential Residue for the Translocation of Cations in Na-NQR.

The sodium-pumping NADH:quinone oxidoreductase (Na-NQR) is a bacterial enzyme that oxidizes NADH, reduces ubiquinone, and translocates Na across the membrane. We previously identified three acidic residues in the membrane-spanning helices, near the cytosol, NqrB-D397, NqrD-D133, and NqrE-E95, as candidates likely to be involved in Na uptake, and replacement of any one of them by a non-acidic residue affects the Na-dependent kinetics of the enzyme. Here, we have inquired further into the role of the NqrE-E95 residue by constructing a series of mutants in which this residue is replaced by amino acids with charges and/or sizes different from those of the glutamate of the wild-type enzyme.

Antibiotic Korormicin A Kills Bacteria by Producing Reactive Oxygen Species.

Korormicin is an antibiotic produced by some pseudoalteromonads which selectively kills Gram-negative bacteria that express the Na-pumping NADH:quinone oxidoreductase (Na-NQR.) We show that although korormicin is an inhibitor of Na-NQR, the antibiotic action is not a direct result of inhibiting enzyme activity. Instead, perturbation of electron transfer inside the enzyme promotes a reaction between O and one or more redox cofactors in the enzyme (likely the flavin adenine dinucleotide [FAD] and 2Fe-2S center), leading to the production of reactive oxygen species (ROS).

Single Molecule Force Spectroscopy and Molecular Dynamics Simulations as a Combined Platform for Probing Protein Face-Specific Binding.

Biomolecular interactions frequently occur in orientation-specific manner. For example, prior nuclear magnetic resonance spectroscopy experiments in our lab have suggested the presence of a group of strongly binding residues on a particular face of the protein ubiquitin for interactions with Capto MMC multimodal ligands ("Capto" ligands) (Srinivasan, K.; et al.

Functional Studies on Membrane Proteins by Means of H/D Exchange in Infrared: Structural Changes in Na NQR from V. cholerae in the Presence of Lipids.

H/D exchange kinetics at the level of the amide proton in the mid infrared (1700-1500 cm) make it possible to study the conformational flexibility of membrane proteins, independent of size or the presence of detergent or lipids. Slow, medium, and fast exchanging domains are distinguished, which reveal a different accessibility to the solvent. Whereas amide hydrogens undergo rapid exchange with solvent in an open structure, hydrogens experience much slower exchange when involved in H-bonded structures or when sterically inaccessible to the solvent.

Occurrence of ferredoxin:NAD(+) oxidoreductase activity and its ion specificity in several Gram-positive and Gram-negative bacteria.

A ferredoxin:NAD(+) oxidoreductase was recently discovered as a redox-driven ion pump in the anaerobic, acetogenic bacterium Acetobacterium woodii. The enzyme is assumed to be encoded by the rnf genes. Since these genes are present in the genomes of many bacteria, we tested for ferredoxin:NAD(+) oxidoreductase activity in cytoplasmic membranes from several different Gram-positive and Gram-negative bacteria that have annotated rnf genes.

Auto Poisoning of the Respiratory Chain by a Quorum-Sensing-Regulated Molecule Favors Biofilm Formation and Antibiotic Tolerance.

Bacterial programmed cell death and quorum sensing are direct examples of prokaryote group behaviors, wherein cells coordinate their actions to function cooperatively like one organism for the benefit of the whole culture. We demonstrate here that 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), a Pseudomonas aeruginosa quorum-sensing-regulated low-molecular-weight excreted molecule, triggers autolysis by self-perturbing the electron transfer reactions of the cytochrome bc1 complex. HQNO induces specific self-poisoning by disrupting the flow of electrons through the respiratory chain at the cytochrome bc1 complex, causing a leak of reducing equivalents to O2 whereby electrons that would normally be passed to cytochrome c are donated directly to O2.

The Kinetic Reaction Mechanism of the Vibrio cholerae Sodium-dependent NADH Dehydrogenase.

The sodium-dependent NADH dehydrogenase (Na(+)-NQR) is the main ion transporter in Vibrio cholerae. Its activity is linked to the operation of the respiratory chain and is essential for the development of the pathogenic phenotype. Previous studies have described different aspects of the enzyme, including the electron transfer pathways, sodium pumping structures, cofactor and subunit composition, among others.

Complete topology of the RNF complex from Vibrio cholerae.

RNF is a redox-driven ion (Na(+) and in one case possibly H(+)) transporter present in many prokaryotes. It has been proposed that RNF performs a variety of reactions in different organisms, delivering low-potential reducing equivalents for specific cellular processes. RNF shares strong homology with the Na(+)-pumping respiratory enzyme Na(+)-NQR, although there are significant differences in subunit and redox cofactor composition.

A mutation in Na(+)-NQR uncouples electron flow from Na(+) translocation in the presence of K(+).

The sodium-pumping NADH:ubiquinone oxidoreductase (Na(+)-NQR) is a bacterial respiratory enzyme that obtains energy from the redox reaction between NADH and ubiquinone and uses this energy to create an electrochemical Na(+) gradient across the cell membrane. A number of acidic residues in transmembrane helices have been shown to be important for Na(+) translocation. One of these, Asp-397 in the NqrB subunit, is a key residue for Na(+) uptake and binding.