Analysis of the Brucella suis Twin Arginine Translocation System and Its Substrates Shows That It Is Essential for Viability.

Bacteria use the twin arginine translocator (Tat) system to export folded proteins from the cytosol to the bacterial envelope or to the extracellular environment. As with most Gram-negative bacteria, the Tat system of the zoonotic pathogen Brucella spp. is encoded by a three-gene operon, Our attempts, using several different strategies, to create a Brucella suis strain 1330 mutant were all unsuccessful.

Author Correction: Cryo-EM structure of the bacterial Ton motor subcomplex ExbB-ExbD provides information on structure and stoichiometry.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cryo-EM structure of the bacterial Ton motor subcomplex ExbB-ExbD provides information on structure and stoichiometry.

The TonB-ExbB-ExbD molecular motor harnesses the proton motive force across the bacterial inner membrane to couple energy to transporters at the outer membrane, facilitating uptake of essential nutrients such as iron and cobalamine. TonB physically interacts with the nutrient-loaded transporter to exert a force that opens an import pathway across the outer membrane. Until recently, no high-resolution structural information was available for this unique molecular motor.

Structural insight into the role of the Ton complex in energy transduction.

In Gram-negative bacteria, outer membrane transporters import nutrients by coupling to an inner membrane protein complex called the Ton complex. The Ton complex consists of TonB, ExbB, and ExbD, and uses the proton motive force at the inner membrane to transduce energy to the outer membrane via TonB. Here, we structurally characterize the Ton complex from Escherichia coli using X-ray crystallography, electron microscopy, double electron-electron resonance (DEER) spectroscopy, and crosslinking.

Contribution of the Twin Arginine Translocation system to the exoproteome of Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa uses secretion systems to deliver exoproteins into the environment. These exoproteins contribute to bacterial survival, adaptation, and virulence. The Twin arginine translocation (Tat) export system enables the export of folded proteins into the periplasm, some of which can then be further secreted outside the cell.

Characterization of a novel Zn²⁺-dependent intrinsic imipenemase from Pseudomonas aeruginosa.

Objectives: Previous work showed that PA5542 inactivation increases Pseudomonas aeruginosa 59.20 susceptibility to carbapenems. The objective of the current study was to purify PA5542, to determine its role in carbapenem resistance and to analyse the kinetic constants of this putative new β-lactamase.

Construction of a Pseudomonas aeruginosa genomic DNA library.

Although the completion and annotation of the entire genomic DNA sequence of Pseudomonas aeruginosa PAO1 strain has been carried out, an important number of genes are still of unknown function and many genetic elements involved in various regulatory pathways like small RNA are still unrevealed. One of the strategies to identify gene function or genetic elements is the construction and utilization of DNA genomic library. Here, we describe the construction a P.

A method to capture large DNA fragments from genomic DNA.

The gene capture technique is a powerful tool that allows the cloning of large DNA regions (up to 80 kb), such as entire genomic islands, without using restriction enzymes or DNA amplification. This technique takes advantage of the high recombinant capacity of the yeast. A "capture" vector containing both ends of the target DNA region must first be constructed.

Monitoring lectin interactions with carbohydrates.

Protein-carbohydrate interactions are often involved in the first step of infection and Pseudomonas aeruginosa produces several proteins that are able to bind specifically to glycan epitopes present on host epithelia. The experimental approaches for studying protein-carbohydrate interaction have been inspired, with some adaptations, from those commonly used for protein-protein or protein-ligand interactions. A range of methods are described herein for detecting lectin activity, screening for monosaccharide or oligosaccharide specificity, determining the affinity of binding together with thermodynamics and kinetics parameters, and producing crystal of lectin-carbohydrate complexes for further structural studies.

Construction of Pseudomonas aeruginosa two-hybrid libraries for high-throughput assays.

In Pseudomonas aeruginosa, identification of new partners of a protein of interest could give precious clues to decipher a biological process in which this protein is involved. However, genes encoding for partners of a protein of interest are unknown and frequently scattered throughout the genome. We describe herein the construction and the use of pan-genomic bacterial two-hybrid libraries to identify new partners of a protein of interest encoded by P.

Cell fractionation.

Proteins within a cell are localized into specific cellular compartments, allowing compartmentalization of distinct tasks. If we consider lipid bilayers as compartments, then gram-negative bacteria such as Pseudomonas aeruginosa can target proteins to five distinct locations: the cytoplasm, the inner membrane, the periplasm, the outer membrane, and the extracellular environment. In this chapter, we describe how the different compartments can be selectively isolated by a combination of centrifugation and disruption techniques.

Gene transfer: transformation/electroporation.

Since Pseudomonas aeruginosa is a non-naturally competent bacterium, various methods have been developed to transfer exogenous DNA. Alternatively to transduction and conjugation, electroporation can also be used to transfer exogenous DNA molecules into Pseudomonas. Electroporation uses an electric field which generates pores in bacterial membranes allowing the entry of the exogenous DNA molecule.

Energetics of colicin import revealed by genetic cross-complementation between the Tol and Ton systems.

Colicins are bacterial toxins that parasitize OM (outer membrane) receptors to bind to the target cells, use an import system to translocate through the cell envelope and then kill sensitive cells. Colicins classified as group A (colicins A, E1-E9, K and N) use the Tol system (TolA, TolB, TolQ and TolR), whereas group B colicins (colicins B, D, Ia, M and 5) use the ExbB-ExbD-TonB system. Genetic evidence has suggested that TolQ and ExbB, as well as TolR and ExbD, are interchangeable, whereas this is not possible with TolA and TonB.

Type II-dependent secretion of a Pseudomonas aeruginosa DING protein.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that uses a wide range of protein secretion systems to interact with its host. Genes encoding the PAO1 Hxc type II secretion system are linked to genes encoding phosphatases (LapA/LapB). Microarray genotyping suggested that Pseudomonas aeruginosa clinical isolates, including urinary tract (JJ692) and blood (X13273) isolates, lacked the lapA/lapB genes.

Draft genome sequence of the purple photosynthetic bacterium Phaeospirillum molischianum DSM120, a particularly versatile bacterium.

Here we present the draft genome sequence of the versatile and adaptable purple photosynthetic bacterium Phaeospirillum molischianum DSM120. This study advances the understanding of the adaptability of this bacterium, as well as the differences between the Phaeospirillum and Rhodospirillum genera.

A bacterial two-hybrid genome fragment library for deciphering regulatory networks of the opportunistic pathogen Pseudomonas aeruginosa.

Bacterial gene regulation is controlled by complex regulatory cascades which integrate input environmental signals and adapt specific and adequate output cellular responses. These complex networks are far from being elucidated, in particular in Pseudomonas aeruginosa. In the present study, we developed bacterial two-hybrid genome fragment libraries of the P.

Shotgun genome sequence of the large purple photosynthetic bacterium Rhodospirillum photometricum DSM122.

Here, we present the shotgun genome sequence of the purple photosynthetic bacterium Rhodospirillum photometricum DSM122. The photosynthetic apparatus of this bacterium has been particularly well studied by microscopy. The knowledge of the genome of this oversize bacterium will allow us to compare it with the other purple bacterial organisms to follow the evolution of the photosynthetic apparatus.