Decoupling filamentous phage uptake and energy of the TolQRA motor in Escherichia coli .

International audienceFilamentous phages are non-lytic viruses that specifically infect bacteria, establishing a persistent association with their host. The phage particle has no machinery for generating energy and parasitizes its host existing structures in order to cross the bacterial envelope and deliver its genetic material. The import of filamentous phages across the bacterial periplasmic space requires some of the components of a macro-complex of the envelope known as the Tol system. This complex uses the energy provided by the proton-motive force of the inner membrane to perform essential and highly energy-consuming functions of the cell, such as envelope integrity maintenance and cell division. It has been suggested that phages take advantage of pmf-driven conformational changes in the Tol system to transit across the periplasm. However, this hypothesis has not been formally tested. In order to decouple the role of the Tol system in cell physiology and during phage parasitism, we used mutations on conserved essential residues known for inactivating pmf-dependent functions of the Tol system. We identified impaired Tol complexes that remain fully efficient for filamentous phage uptake. We further demonstrate that the TolQ-TolR homologous motor, ExbB-ExbD, normally operating with the TonB protein, is able to promote phage infection along with full length TolA

Multiple osmotic stress responses in acidihalobacter prosperus result in tolerance to chloride ions

Extremely acidophilic microorganisms (pH optima for growth of =3) are utilized for the extraction of metals from sulfide minerals in the industrial biotechnology of "biomining." A long term goal for biomining has been development of microbial consortia able to withstand increased chloride concentrations for use in regions where freshwater is scarce. However, when challenged by elevated salt, acidophiles experience both osmotic stress and an acidification of the cytoplasm due to a collapse of the inside positive membrane potential, leading to an influx of protons. In this study, we tested the ability of the halotolerant acidophile Acidihalobacter prosperus to grow and catalyze sulfide mineral dissolution in elevated concentrations of salt and identified chloride tolerance mechanisms in Ac. prosperus as well as the chloride susceptible species, Acidithiobacillus ferrooxidans. Ac. prosperus had optimum iron oxidation at 20 g L-1 NaCl while At. ferrooxidans iron oxidation was inhibited in the presence of 6 g L-1 NaCl. The tolerance to chloride in Ac. prosperus was consistent with electron microscopy, determination of cell viability, and bioleaching capability. The Ac. prosperus proteomic response to elevated chloride concentrations included the production of osmotic stress regulators that potentially induced production of the compatible solute, ectoine uptake protein, and increased iron oxidation resulting in heightened electron flow to drive proton export by the F0F1 ATPase. In contrast, At. ferrooxidans responded to low levels of Cl- with a generalized stress response, decreased iron oxidation, and an increase in central carbon metabolism. One potential adaptation to high chloride in the Ac. prosperus Rus protein involved in ferrous iron oxidation was an increase in the negativity of the surface potential of Rus Form I (and Form II) that could help explain how it can be active under elevated chloride concentrations. These data have been used to create a model of chloride tolerance in the salt tolerant and susceptible species Ac. prosperus and At. ferrooxidans, respectively. © 2017 The Authors

Sequence nucleotidique et etude de la transcription des genes de la colicine A, de la proteine d'immunite et de la proteine de lyse du plasmide col A

SIGLECNRS T 55968 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Le système Tol/Pal d'Escherichia coli (Rôle de la protéine ToIR dans le maintien de l'intégrité de la membrane externe et dans l'importation des colicines)

AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Le système Tol-Pal (l'intégrité membranaire et les interactions entre TolA, colicine A et G3p, protéine de capside des phages filamenteux)

AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Le Complexe membranaire Tol d'Escherichia coli (un modèle pour l'étude de l'organisation fonctionnelle d'un moteur moléculaire bactérien)

Le système protéique Tol-Pal est conservé chez les bactéries à Gram négatif. Chaque mutant tol ou pal provoque une déstabilisation de la membrane externe (ME). Les protéines TolA-Q-R forment un complexe de membrane interne (MI) ; Pal ancrée dans la ME interagit avec le peptidoglycane et la protéine périplasmique TolB. L interaction entre TolA et Pal dépend de TolQ-R et de la force proton-motrice (fpm). Les protéines TolA-Q-R relient ainsi MI, ME et le peptidoglycane. Le but de ce projet est de comprendre l organisation des segments transmembranaires (sTM) de TolQ-R et le mécanisme énergétique. Les interactions entre sTM ont été analysées ainsi que les résidus impliqués dans le passage d ions. Nous avons montré que le domaine C-terminal de TolR est dynamique en réponse à la fpm. Nous avons entrepris une mutagénèse systématique de chacun des résidus des sTM. Les résultats obtenus ont permis de dresser un modèle dynamique de l organisation des sTM du complexe TolQ-R.The Tol-Pal system is well conserved in Gram negative bacteria. Each of the tol- pal mutant exhibits outer membrane (OM) defects. The TolQ-R-A proteins form an inner membrane (IM) complex; Pal anchored in the OM interacts with the peptidoglycan and the periplasmic protein TolB. The TolA-Pal interaction depends on TolQ-R and on the proton motive force (pmf). The TolQ-R-A proteins may form a molecular motor using the pmf to link IM, OM and the peptidoglycan. The aim of this work is to understand the organisation of the IM TolQ-R proteins and the utilisation of pmf. Using mutagenesis approaches, we defined helix organization and key residues of the putative ion channel. We demonstrated movements of the C-terminus of TolR dependent on the pmf. To define complex organisation and conformational changes of the transmembrane segments (TMs), we performed a cysteine scanning mutagenesis. Our results suggest a dynamic model of the TolQ-R TMs organisation.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Etude fonctionnelle d'un moteur moléculaire bactérien (l'exemple du complexe TolQRA d'Escherichia coli)

AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Anémie du prématuré (transfusions, érythropoïétine, supplémentation en fer)

MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF