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Luca Guardabassi - One of the best experts on this subject based on the ideXlab platform.
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carprofen induced depletion of Proton Motive Force reverses tetk mediated doxycycline resistance in methicillin resistant staphylococcus pseudintermedius
Scientific Reports, 2019Co-Authors: Zofia Magnowska, Bimal Jana, Rikke Prejh Brochmann, Andrew Hesketh, Rene Lametsch, Cristian De Gobba, Luca GuardabassiAbstract:We previously showed that doxycycline (DOX) and carprofen (CPF), a veterinary non-steroidal anti-inflammatory drug, have synergistic antimicrobial activity against methicillin-resistant Staphylococcus pseudintermedius (MRSP) carrying the tetracycline resistance determinant TetK. To elucidate the molecular mechanism of this synergy, we investigated the effects of the two drugs, individually and in combination, using a comprehensive approach including RNA sequencing, two-dimensional differential in-gel electrophoresis, macromolecule biosynthesis assays and fluorescence spectroscopy. Exposure of TetK-positive MRSP to CPF alone resulted in upregulation of pathways that generate ATP and NADH, and promote the Proton gradient. We showed that CPF is a Proton carrier that dissipates the electrochemical potential of the membrane. In the presence of both CPF and DOX, the energy compensation strategy was attenuated by downregulation of all the processes involved, such as citric acid cycle, oxidative phosphorylation and ATP-providing arginine deiminase pathway. Furthermore, protein biosynthesis inhibition increased from 20% under DOX exposure alone to 75% upon simultaneous exposure to CPF. We conclude that synergistic interaction of the drugs restores DOX susceptibility in MRSP by compromising Proton-Motive-Force-dependent TetK-mediated efflux of the antibiotic. MRSP is unable to counterbalance CPF-mediated PMF depletion by cellular metabolic adaptations, resulting in intracellular accumulation of DOX and inhibition of protein biosynthesis.
David Kramer - One of the best experts on this subject based on the ideXlab platform.
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chloroplast atp synthase modulation of the thylakoid Proton Motive Force implications for photosystem i and photosystem ii photoprotection
Frontiers in Plant Science, 2017Co-Authors: Atsuko Kanazawa, Kaori Kohzuma, John E Froehlich, Elisabeth Ostendorf, Deserah D Strand, Mio Satocruz, Linda J Savage, Jeffrey A Cruz, Nicholas Fisher, David KramerAbstract:In wild type plants, decreasing CO2 lowers the activity of the chloroplast ATP synthase, slowing Proton efflux from the thylakoid lumen resulting in buildup of thylakoid Proton Motive Force (pmf). The resulting acidification of the lumen regulates both light harvesting, via the qE mechanism, and photosynthetic electron transfer through the cytochrome b6f complex. Here we show that the cfq mutant of Arabidopsis, harboring single point mutation in its γ-subunit of the chloroplast ATP synthase, increases the specific activity of the ATP synthase and disables its down-regulation under low CO2. The increased thylakoid Proton conductivity (gH+) in cfq results in decreased pmf and lumen acidification, preventing full activation of qE and more rapid electron transfer through the b6f complex, particularly under low CO2 and fluctuating light. These conditions favor the accumulation of electrons on the acceptor side of PSI, and result in severe loss of PSI activity. Comparing the current results with previous work on the pgr5 mutant suggests a general mechanism where increased PSI photodamage in both mutants is caused by loss of pmf, rather than inhibition of CEF per se. Overall, our results support a critical role for ATP synthase regulation in maintaining photosynthetic control of electron transfer to prevent photodamage.
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the thylakoid Proton Motive Force in vivo quantitative non invasive probes energetics and regulatory consequences of light induced pmf
Biochimica et Biophysica Acta, 2007Co-Authors: Kenji Takizawa, Atsuko Kanazawa, Jeffrey A Cruz, David KramerAbstract:Abstract Endogenous probes of light-induced transthylakoid Proton Motive Force (pmf), membrane potential (Δψ) and ΔpH were used in vivo to assess in Arabidopsis the lumen pH responses of regulatory components of photosynthesis. The accumulation of zeaxanthin and Protonation of PsbS were found to have similar pKa values, but quite distinct Hill coefficients, a feature allowing high antenna efficiency at low pmf and fine adjustment at higher pmf. The onset of “energy-dependent’ exciton quenching (qE) occurred at higher lumen pH than slowing of plastoquinol oxidation at the cytochrome b6f complex, presumably to prevent buildup of reduced electron carriers that can lead to photodamage. Quantitative comparison of intrinsic probes with the electrochromic shift signal in situ allowed quantitative estimates of pmf and lumen pH. Within a degree of uncertainly of ∼ 0.5 pH units, the lumen pH was estimated to range from ∼ 7.5 (under weak light at ambient CO2) to ∼ 5.7 (under 50 ppm CO2 and saturating light), consistent with a ‘moderate pH’ model, allowing antenna regulation but preventing acid-induced photodamage. The apparent pKa values for accumulation of zeaxanthin and PsbS Protonation were found to be ∼ 6.8, with Hill coefficients of about 4 and 1 respectively. The apparent shift between in vitro violaxanthin deepoxidase Protonation and zeaxanthin accumulation in vivo is explained by steady-state competition between zeaxanthin formation and its subsequent epoxidation by zeaxanthin epoxidase. In contrast to tobacco, Arabidopsis showed substantial variations in the fraction of pmf (0.1–0.7) stored as Δψ, allowing a more sensitive qE response, possible as an adaptation to life at lower light levels.
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contribution of electric field δψ to steady state transthylakoid Proton Motive Force pmf in vitro and in vivo control of pmf parsing into δψ and δph by ionic strength
Biochemistry, 2001Co-Authors: Jeffrey A Cruz, Colette A Sacksteder, And Atsuko Kanazawa, David KramerAbstract:The observed levels of Delta G(ATP) in chloroplasts, as well as the activation behavior of the CF(1)CF(0)-ATP synthase, suggest a minimum transthylakoid Proton Motive Force (pmf) equivalent to a Delta pH of approximately 2.5 units. If, as is commonly believed, all transthylakoid pmf is stored as Delta pH, this would indicate a lumen pH of less than approximately 5. In contrast, we have presented evidence that the pH of the thylakoid lumen does not drop below pH approximately 5.8 [Kramer, D. M., Sacksteder, C. A., and Cruz, J. A. (1999) Photosynth. Res. 60, 151-163], leading us to propose that Delta psi can contribute to steady-state pmf. In this work, it is demonstrated, through assays on isolated thylakoids and computer simulations, that thylakoids can store a substantial fraction of pmf as Delta psi, provided that the activities of ions permeable to the thylakoid membrane in the chloroplast stromal compartment are relatively low and the buffering capacity (beta) for Protons of the lumen is relatively high. Measurements of the light-induced electrochromic shift (ECS) confirm the ionic strength behavior of steady-state Delta psi in isolated, partially uncoupled thylakoids. Measurements of the ECS in intact plants illuminated for 65 s were consistent with low concentrations of permeable ions and approximately 50% storage of pmf as Delta psi. We propose that the plant cell, possibly at the level of the inner chloroplast envelope, can control the parsing of pmf into Delta psi and Delta pH by regulating the ionic strength and balance of the chloroplast. In addition, this work demonstrates that, under certain conditions, the kinetics of the light-induced ECS can be used to estimate the fractions of pmf stored as Delta psi and Delta pH both in vitro and in vivo.
Lotte Sogaardandersen - One of the best experts on this subject based on the ideXlab platform.
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a tonb dependent transporter is required for secretion of protease popc across the bacterial outer membrane
Nature Communications, 2019Co-Authors: Nuria Gomezsantos, Timo Glatter, Ralf Koebnik, Magdalena A świątekpolatynska, Lotte SogaardandersenAbstract:TonB-dependent transporters (TBDTs) are ubiquitous outer membrane β-barrel proteins that import nutrients and bacteriocins across the outer membrane in a Proton Motive Force-dependent manner, by directly connecting to the ExbB/ExbD/TonB system in the inner membrane. Here, we show that the TBDT Oar in Myxococcus xanthus is required for secretion of a protein, protease PopC, to the extracellular milieu. PopC accumulates in the periplasm before secretion across the outer membrane, and the Proton Motive Force has a role in secretion to the extracellular milieu. Reconstitution experiments in Escherichia coli demonstrate that secretion of PopC across the outer membrane not only depends on Oar but also on the ExbB/ExbD/TonB system. Our results indicate that TBDTs and the ExbB/ExbD/TonB system may have roles not only in import processes but also in secretion of proteins.
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a tonb dependent transporter is required for secretion of protease popc across the bacterial outer membrane
Nature Communications, 2019Co-Authors: Nuria Gomezsantos, Timo Glatter, Ralf Koebnik, Magdalena A świątekpolatynska, Lotte SogaardandersenAbstract:TonB-dependent transporters (TBDTs) are ubiquitous outer membrane β-barrel proteins that import nutrients and bacteriocins across the outer membrane in a Proton Motive Force-dependent manner, by directly connecting to the ExbB/ExbD/TonB system in the inner membrane. Here, we show that the TBDT Oar in Myxococcus xanthus is required for secretion of a protein, protease PopC, to the extracellular milieu. PopC accumulates in the periplasm before secretion across the outer membrane, and the Proton Motive Force has a role in secretion to the extracellular milieu. Reconstitution experiments in Escherichia coli demonstrate that secretion of PopC across the outer membrane not only depends on Oar but also on the ExbB/ExbD/TonB system. Our results indicate that TBDTs and the ExbB/ExbD/TonB system may have roles not only in import processes but also in secretion of proteins. TonB-dependent transporters (TBDTs) are outer membrane proteins that import nutrients and bacteriocins in bacteria. Here, Gomez-Santos et al. show that a TBDT is required for secretion of a protease in Myxococcus xanthus, suggesting that some TBDTs may be involved in protein secretion.
Andrew Hesketh - One of the best experts on this subject based on the ideXlab platform.
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carprofen induced depletion of Proton Motive Force reverses tetk mediated doxycycline resistance in methicillin resistant staphylococcus pseudintermedius
Scientific Reports, 2019Co-Authors: Zofia Magnowska, Bimal Jana, Rikke Prejh Brochmann, Andrew Hesketh, Rene Lametsch, Cristian De Gobba, Luca GuardabassiAbstract:We previously showed that doxycycline (DOX) and carprofen (CPF), a veterinary non-steroidal anti-inflammatory drug, have synergistic antimicrobial activity against methicillin-resistant Staphylococcus pseudintermedius (MRSP) carrying the tetracycline resistance determinant TetK. To elucidate the molecular mechanism of this synergy, we investigated the effects of the two drugs, individually and in combination, using a comprehensive approach including RNA sequencing, two-dimensional differential in-gel electrophoresis, macromolecule biosynthesis assays and fluorescence spectroscopy. Exposure of TetK-positive MRSP to CPF alone resulted in upregulation of pathways that generate ATP and NADH, and promote the Proton gradient. We showed that CPF is a Proton carrier that dissipates the electrochemical potential of the membrane. In the presence of both CPF and DOX, the energy compensation strategy was attenuated by downregulation of all the processes involved, such as citric acid cycle, oxidative phosphorylation and ATP-providing arginine deiminase pathway. Furthermore, protein biosynthesis inhibition increased from 20% under DOX exposure alone to 75% upon simultaneous exposure to CPF. We conclude that synergistic interaction of the drugs restores DOX susceptibility in MRSP by compromising Proton-Motive-Force-dependent TetK-mediated efflux of the antibiotic. MRSP is unable to counterbalance CPF-mediated PMF depletion by cellular metabolic adaptations, resulting in intracellular accumulation of DOX and inhibition of protein biosynthesis.
Bimal Jana - One of the best experts on this subject based on the ideXlab platform.
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carprofen induced depletion of Proton Motive Force reverses tetk mediated doxycycline resistance in methicillin resistant staphylococcus pseudintermedius
Scientific Reports, 2019Co-Authors: Zofia Magnowska, Bimal Jana, Rikke Prejh Brochmann, Andrew Hesketh, Rene Lametsch, Cristian De Gobba, Luca GuardabassiAbstract:We previously showed that doxycycline (DOX) and carprofen (CPF), a veterinary non-steroidal anti-inflammatory drug, have synergistic antimicrobial activity against methicillin-resistant Staphylococcus pseudintermedius (MRSP) carrying the tetracycline resistance determinant TetK. To elucidate the molecular mechanism of this synergy, we investigated the effects of the two drugs, individually and in combination, using a comprehensive approach including RNA sequencing, two-dimensional differential in-gel electrophoresis, macromolecule biosynthesis assays and fluorescence spectroscopy. Exposure of TetK-positive MRSP to CPF alone resulted in upregulation of pathways that generate ATP and NADH, and promote the Proton gradient. We showed that CPF is a Proton carrier that dissipates the electrochemical potential of the membrane. In the presence of both CPF and DOX, the energy compensation strategy was attenuated by downregulation of all the processes involved, such as citric acid cycle, oxidative phosphorylation and ATP-providing arginine deiminase pathway. Furthermore, protein biosynthesis inhibition increased from 20% under DOX exposure alone to 75% upon simultaneous exposure to CPF. We conclude that synergistic interaction of the drugs restores DOX susceptibility in MRSP by compromising Proton-Motive-Force-dependent TetK-mediated efflux of the antibiotic. MRSP is unable to counterbalance CPF-mediated PMF depletion by cellular metabolic adaptations, resulting in intracellular accumulation of DOX and inhibition of protein biosynthesis.
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exbb cytoplasmic loop deletions cause immediate Proton Motive Force independent growth arrest
Journal of Bacteriology, 2013Co-Authors: Charles M Bulathsinghala, Bimal Jana, Kristin R Baker, Kathleen PostleAbstract:ABSTRACT The Escherichia coli TonB system consists of the cytoplasmic membrane proteins TonB, ExbB, and ExbD and multiple outer membrane active transporters for diverse iron siderophores and vitamin B12. The cytoplasmic membrane proteins harvest and transmit the Proton Motive Force (PMF) to outer membrane transporters. This system, which spans the cell envelope, has only one component with a significant cytoplasmic presence, ExbB. Characterization of sequential 10-residue deletions in the ExbB cytoplasmic loop (residues 40 to 129; referred to as Δ10 proteins) revealed that it was required for all TonB-dependent activities, including interaction between the periplasmic domains of TonB and ExbD. Expression of eight out of nine of the Δ10 proteins at chromosomal levels led to immediate, but reversible, growth arrest. Arrest was not due to collapse of the PMF and did not require the presence of ExbD or TonB. All Δ10 proteins that caused growth arrest were dominant for that phenotype. However, several were not dominant for iron transport, indicating that growth arrest was an intrinsic property of the Δ10 variants, whether or not they could associate with wild-type ExbB proteins. The lack of dominance in iron transport also ruled out trivial explanations for growth arrest, such as high-level induction. Taken together, the data suggest that growth arrest reflected a changed interaction between the ExbB cytoplasmic loop and one or more unknown growth-regulatory proteins. Consistent with that, a large proportion of the ExbB cytoplasmic loop between transmembrane domain 1 (TMD1) and TMD2 is predicted to be disordered, suggesting the need for interaction with one or more cytoplasmic proteins to induce a final structure.
