Changes In Proton Motive Force Research Articles

Transcriptomic and Biochemical Analysis of the Antimicrobial Mechanism of Lipopeptide Iturin W against Staphylococcus aureus.

Staphylococcus aureus is one of the most serious pathogens threatening food safety and public health. We have previously showed that iturin W exhibited obvious antifungal activity on plant pathogens. In the present study, we found iturin W, especially C14 iturin W, showed strong antimicrobial activity against S. aureus, and the antimicrobial mechanism of C14 iturin W was further investigated by transcriptomic analysis and a related biochemical experiment. The results showed that C14 iturin W can reduce the expression levels of genes associated with the reactive oxygen species (ROS) scavenging enzyme and genes involved in arginine biosynthesis, thus leading to the increase in ROS levels of S. aureus. Furthermore, C14 iturin W can also interfere with proton dynamics, which is crucial for cells to regulate various biological possesses. Therefore, ROS accumulation and change in proton motive force are import ways for C14 iturin W to exert the antimicrobial activity. In addition, C14 iturin W can also reduce the expression levels of genes related to virulence factors and decrease the production of enterotoxins and hemolysins in S. aureus, indicating that C14 iturin W has a good potential in food and pharmaceutical fields to reduce the harm caused by S. aureus in the future.

An energetics perspective on geroscience: mitochondrial protonmotive force and aging.

Mitochondria are organelles that provide energy to cells through ATP production. Mitochondrial dysfunction has long been postulated to mediate cellular declines that drive biological aging. Many well-characterized hallmarks of aging may involve underlying energetic defects that stem from loss of mitochondrial function with age. Why and how mitochondrial function declines with age is an open question and one that has been difficult to answer. Mitochondria are powered by an electrochemical gradient across the inner mitochondrial membrane known as the protonmotive force (PMF). This gradient decreases with age in several experimental models. However, it is unclear if a diminished PMF is a cause or a consequence of aging. Herein, we briefly review and define mitochondrial function, we summarize how PMF changes with age in several models, and we highlight recent studies that implicate PMF in aging biology. We also identify barriers that must be addressed for the field to progress. Emerging technology permits more precise in vivo study of mitochondria that will allow better understanding of cause and effect in metabolic models of aging. Once cause and effect can be discerned more precisely, energetics approaches to combat aging may be developed to prevent or reverse functional decline.

Neuronal AMPK coordinates mitochondrial energy sensing and hypoxia resistance in C. elegans.

Organisms adapt to their environment through coordinated changes in mitochondrial function and metabolism. The mitochondrial protonmotive force (PMF) is an electrochemical gradient that powers ATP synthesis and adjusts metabolism to energetic demands via cellular signaling. It is unknown how or where transient PMF changes are sensed and signaled due to the lack of precise spatiotemporal control in vivo. We addressed this by expressing a light‐activated proton pump in mitochondria to spatiotemporally “turn off” mitochondrial function through PMF dissipation in tissues with light. We applied our construct—mitochondria‐OFF (mtOFF)—to understand how metabolic status impacts hypoxia resistance, a response that relies on mitochondrial function. Activation of mtOFF induced starvation‐like behavior mediated by AMP‐activated protein kinase (AMPK). We found prophylactic mtOFF activation increased survival following hypoxia, and that protection relied on neuronal AMPK. Our study links spatiotemporal control of mitochondrial PMF to cellular metabolic changes that mediate behavior and stress resistance.

Energy Taxis in Escherichia Coli on the Single-Cell Level

Energy taxis allows bacteria to respond to external stresses that affect its electron transport system, and to navigate to the environment optimal for energy generation. For example, in an oxygen gradient bacteria accumulate in a sharply defined band at the oxygen concentration that maximizes their proton motive force (PMF) which is coupled to electron transport (Zhulin, 1996). Examples of energy taxis include aerotaxis, taxis to alternative electron acceptors, and taxis to respiratory substrates. In E. coli, energy sensing is mediated by Tar and Aer receptors: Tar senses changes in PMF, while Aer is likely a redox sensor (Edwards, 2007). It is therefore expected that in the spatial gradient of oxygen or alternative electron acceptors changes in bacterial swimming behavior, e.g. tumble bias, will be correlated with PMF, but this has never been demonstrated directly.We study taxis to nitrate - an alternative electron acceptor for E. coli - in bacteria under anaerobic conditions at the single cell level, using an optical trap (Min, 2009). We move bacteria in a defined gradient of nitrate inside the flow chamber, and measure resulting changes in both tumble bias and flagella rotation frequency, which is proportional to PMF under low viscous load that corresponds to free swimming bacteria (Gabel, 2003).Our preliminary results show that under anaerobic conditions E. coli respond to changes in nitrate concentration, which is reflected in both tumble bias and flagella rotation. Thus running response or decrease in tumble bias corresponds to the increase in flagella rotation frequency and vice versa. While changes in tumble bias are transient and are followed by adaptation, nitrate concentration change have long-lasting effect on flagella rotation which is expected if PMF depends on the absolute concentration of nitrate.Using our method we can measure preferred concentration of nitrate for individual bacteria at which their PMF is maximized and how it depends on other factors such as concentration of electron donor, energetic state of the bacteria and mode of metabolism.References J. C. Edwards et al. Mol Microbiol (2007). I. B. Zhulin et al. J Bacteriol (1996). T. L. Min et al. Nature Methods (2009). C. V Gabel & H. C. Berg. PNAS (2003).

Quantification of flagellar motor stator dynamics through in vivo proton‐motive force control

The bacterial flagellar motor, one of the few rotary motors in nature, produces torque to drive the flagellar filament by ion translocation through membrane-bound stator complexes. We used the light-driven proton pump proteorhodopsin (pR) to control the proton-motive force (PMF) in vivo by illumination. pR excitation was shown to be sufficient to replace native PMF generation, and when excited in cells with intact native PMF generation systems increased motor speed beyond the physiological norm. We characterized the effects of rapid in vivo PMF changes on the flagellar motor. Transient PMF disruption events from loss of illumination caused motors to stop, with rapid recovery of their previous rotation rate after return of illumination. However, extended periods of PMF loss led to stepwise increases in rotation rate upon PMF return as stators returned to the motor. The rate constant for stator binding to a putative single binding site on the motor was calculated to be 0.06 s(-1). Using GFP-tagged MotB stator proteins, we found that transient PMF disruption leads to reversible stator diffusion away from the flagellar motor, showing that PMF presence is necessary for continued motor integrity, and calculated a stator dissociation rate of 0.038 s(-1).

Differentiation between electron transport sensing and proton motive force sensing by the Aer and Tsr receptors for aerotaxis

Aerotaxis (oxygen-seeking) behaviour in Escherichia coli is a response to changes in the electron transport system and not oxygen per se. Because changes in proton motive force (PMF) are coupled to respiratory electron transport, it is difficult to differentiate between PMF, electron transport or redox, all primary candidates for the signal sensed by the aerotaxis receptors, Aer and Tsr. We constructed electron transport mutants that produced different respiratory H+/e- stoichiometries. These strains expressed binary combinations of one NADH dehydrogenase and one quinol oxidase. We then introduced either an aer or tsr mutation into each mutant to create two sets of electron transport mutants. In vivo H+/e- ratios for strains grown in glycerol medium ranged from 1.46+/-0.18-3.04+/-0.47, but rates of respiration and growth were similar. The PMF jump in response to oxygen was proportional to the H+/e- ratio in each set of mutants (r2=0.986-0.996). The length of Tsr-mediated aerotaxis responses increased with the PMF jump (r2=0.988), but Aer-mediated responses did not correlate with either PMF changes (r2=0.297) or the rate of electron transport (r2=0.066). Aer-mediated responses were linked to NADH dehydrogenase I, although there was no absolute requirement. The data indicate that Tsr responds to changes in PMF, but strong Aer responses to oxygen are associated with redox changes in NADH dehydrogenase I.

Effects of Decreasing Mitochondrial Volume on the Regulation of the Permeability Transition Pore

The permeability transition pore (PTP) is a Ca(2+)-sensitive mitochondrial inner membrane channel involved in several models of cell death. Because the matrix concentration of PTP regulatory factors depends on matrix volume, we have investigated the role of the mitochondrial volume in PTP regulation. By incubating rat liver mitochondria in media of different osmolarity, we found that the Ca(2+) threshold required for PTP opening dramatically increased when mitochondrial volume decreased relative to the standard condition. This shrinkage-induced PTP inhibition was not related to the observed changes in protonmotive force, or pyridine nucleotide redox state and persisted when mitochondria were depleted of adenine nucleotides. On the other hand, mitochondrial volume did not affect PTP regulation when mitochondria were depleted of Mg(2+). By studying the effects of Mg(2+), cyclosporin A (CsA) and ubiquinone 0 (Ub(0)) on PTP regulation, we found that mitochondrial shrinkage increased the efficacy of Mg(2+) and Ub(0) at PTP inhibition, whereas it decreased that of CsA. The ability of mitochondrial volume to alter the activity of several PTP regulators represents a hitherto unrecognized characteristic of the pore that might lead to a new approach for its pharmacological modulation.

Behaviour of Azospirillum brasilense in a spatial gradient of oxygen and in a 'redox' gradient of an artificial electron acceptor

Azospirillum brasilense responds to a spatial gradient of oxygen by formation of a band in the zone of optimal oxygen concentration. The same behaviour was observed in a ‘redox’ gradient of tetramethyl-p-phenylenediamine (TMPD) oxidized by ferricyanide, under conditions where oxygen uptake was inhibited by cyanide. Inhibitors of the bc 1-complex of the redox chain did not inhibit aerotaxis. Cells inhibited by a high cyanide concentration still showed slow motility and weak attraction to oxygen, possibly as a result of cyanide-resistant respiration (3–5% of the initial level). It is suggested that electron flow through the redox chain (and/or consequent changes in protonmotive force), rather than binding of oxygen to terminal oxidases, may be a signal in the aerotactic response of A. brasilense.

The Regulation of Proton/Amino Acid Symport inRiccia fluitansL. by Cytosolic pH and Proton Pump Activity

In the aquatic liverwort Riccia fluitans the regulation of the plasma membrane H+/amino acid symport has been investigated. Cytosolic pH (pHc), membrane potential (Em) and membrane conductance have been measured and related to transport data, (i) The release of [14C]amino acids is strongly stimulated by cytosolic acidification, induced by the external addition of acetic acid, a decrease in external K + , and in the change from light to dark. On average, a decrease in pHc of 0-5 to 0-6 units corresponded with a 4-fold stimulation in amino acid efflux, (ii) External pH changes have far less effect on substrate transport than the cytosolic pH shifts of the same order, (iii) The inwardly directed positive current, induced by amino acids, is severely inhibited by cytosolic acidification, (iv) Fusicoccin (FC) stimulates amino acid uptake without considerable change in proton motive force, (v) When the proton motive force is kept constant, the uptake of amino acids into Riccia thalli is much lower than when the pump is deactivated. It is suggested that both the proton pump activity and cytosolic pH are the dominant factors in the regulation of the H+/amino acid symport across the plasma membrane of Riccia fluitans, and it is concluded that the proton motive force is not a reliable quantity to predict and interpret transport kinetics.

Signal transduction in chemotaxis to oxygen in Escherichia coli and Salmonella typhimurium.

Pathways previously proposed for sensory transduction in chemotaxis to oxygen (aerotaxis) involved either (i) cytochrome o, the electron transport system, and proton motive force or (ii) enzyme IIGlucose and the phosphoenolpyruvate:carbohydrate phosphotransferase system for active transport. This investigation distinguished between these possibilities. Aerotaxis was absent in a cyo cyd strain of Escherichia coli that lacked both cytochrome o and cytochrome d, which are the terminal oxidases for the branched electron transport system in E. coli. Aerotaxis, measured by either a spatial or temporal assay, was normal in E. coli strains that had a cyo+ or cyd+ gene or both. The membrane potential of all oxidase-positive strains was approximately -170 mV in aerated medium at pH 7.5. Behavioral responses to changes in oxygen concentration correlated with changes in proton motive force. Aerotaxis was normal in ptsG and ptsI strains that lack enzyme IIGlucose and enzyme I, respectively, and are deficient in the phosphotransferase system. A cya strain that is deficient in adenylate cyclase also had normal aerotaxis. We concluded that aerotaxis was mediated by the electron transport system and that either the cytochrome d or the cytochrome o branch of the pathway could mediate aerotaxis.

Role of proton motive force in phototactic and aerotactic responses of Rhodopseudomonas sphaeroides.

Rhodopseudomonas sphaeroides grown under nonrigorous anaerobic conditions in the light developed components of a branched respiratory electron transfer chain, and a photosynthetic electron transfer chain. Both respiratory pathways were sensitive to rotenone and high concentrations of cyanide, but oxygen uptake was only partially inhibited by the addition of low concentrations of cyanide or antimycin A. When incubated anaerobically in the dark, R. sphaeroides responded positively to an oxygen gradient in the absence of rotenone. In the presence of rotenone, aerotaxis only occurred when the antimycin A-sensitive branch of the pathway was functioning, although both branches still reduced oxygen. Although there was electron movement along the respiratory chain, aerotaxis only occurred in response to a change in proton motive force. When incubated anaerobically in the light, the movement of R. sphaeroides up a light gradient depended on photosynthetic electron transport. When incubated aerobically, high-intensity actinic illumination inhibited oxygen uptake and aerotaxis. In a low-intensity light gradient the phototactic response was inhibited by oxygen. These results are discussed in relation to the interaction of the electron transfer chains and their roles in controlling tactic responses in R. sphaeroides.

Transport of H+ against electrochemical gradients in turtle urinary bladder.

Active H+ transport (JH) by the isolated turtle bladder was inhibited by either an applied chemical gradient (deltapH) or an electrical gradient (deltapsi). The relation of JH to either deltapH or deltapsi was linear, and the slopes and the force gradients required to bring JH to zero were similar with both methods. The transport system was analyzed in terms of an equivalent circuit model comprising a proton motive force (PMF), an active conductance (LH) in series with the pump, and a parallel or passive conductance which may be ignored in this preparation. Increasing ambient PCO2 markedly increased JH and the active conductance (as deltaJH/deltadeltapH) but had no effect on the apparent PMF (PMF'). Similarly, acetazolamide caused comparable decreases in JH and LH without change in PMF'. Inhibition of metabolism by deoxygenation, deoxy-D-glucose, or depletion of metabolic substrate caused large decreases in JH and LH with reduction in PMF' of less than 14%. Glucose addition increased JH and LH but caused a slight decrease in PMF'. Thus, the experimental maneuvers affected the transport rate primarily through changes in the active conductance. Since PMF' was little affected, the force of the pump must be determined by factors other than the metabolic driving reaction alone. Conductance factors relating to transport as well as to metabolism participate in controlling PMF.