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Plasma membrane H+-ATPases (PMAs) provide an H+ electrochemical gradient to establish a proton motive force and membrane potential for the transport of various nutrients across the plasma membrane. PMAs are regulated at multiple levels, and genetic modulation of PMA expression could enhance nutrient acquisition and use efficiency in plants. PMAs are involved in the coordination of the uptake and assimilation of both nitrate and ammonium. PMAs are involved in nutrient utilization by regulating root growth, facilitating organic anion exudation, and promoting the acquisition of nutrients through H+ extrusion-mediated apoplast and rhizosphere acidification. PMAs play essential roles in mycorrhizal symbioses in plant roots for nutrient acquisition. Plasma membrane H+-ATPases (PMAs) pump H+ out of the cytoplasm by consuming ATP to generate a membrane potential and proton motive force for the transmembrane transport of nutrients into and out of plant cells. PMAs are involved in nutrient acquisition by regulating root growth, nutrient uptake, and translocation, as well as the establishment of symbiosis with arbuscular mycorrhizas. Under nutrient stresses, PMAs are activated to pump more H+ and promote organic anion excretion, thus improving nutrient availability in the rhizosphere. Herein we review recent progress in the physiological functions and the underlying molecular mechanisms of PMAs in the efficient acquisition and utilization of various nutrients in plants. We also discuss perspectives for the application of PMAs in improving crop production and quality. [ABSTRACT FROM AUTHOR]

Escherichia coli is a common host for biotechnology and synthetic biology applications. During growth and fermentation, the microbes are often exposed to stress conditions, such as variations in pH or solvent concentrations. Bacterial membranes play a key role in response to abiotic stresses. Ornithine lipids (OLs) are a group of membrane lipids whose presence and synthesis have been related to stress resistance in bacteria. We wondered if this stress resistance could be transferred to bacteria not encoding the capacity to form OLs in their genome, such as E. coli. In this study, we engineered different E. coli strains to produce unmodified OLs and hydroxylated OLs by expressing the synthetic operon olsFC. Our results showed that OL formation improved pH resistance and increased biomass under phosphate limitation. Transcriptome analysis revealed that OL-forming strains differentially expressed stress- and membrane-related genes. OL-producing strains also showed better growth in the presence of the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), suggesting reduced proton leakiness in OL-producing strains. Furthermore, our engineered strains showed improved heterologous violacein production at phosphate limitation and also at low pH. Overall, this study demonstrates the potential of engineering the E. coli membrane composition for constructing robust hosts with an increased abiotic stress resistance for biotechnology and synthetic biology applications. Key points: • Ornithine lipid production in E. coli increases biomass yield under phosphate limitation. • Engineered strains show an enhanced production phenotype under low pH stress. • Transcriptome analysis and CCCP experiments revealed reduced proton leakage. [ABSTRACT FROM AUTHOR]

The proton motive force (pmf) generated across the thylakoid membrane rotates the Fo-ring of ATP synthase in chloroplasts. The pmf comprises two components: membrane potential (∆Ψ) and proton concentration gradient (∆pH). Acidification of the thylakoid lumen resulting from ∆pH downregulates electron transport in the cytochrome b 6 f complex. This process, known as photosynthetic control, is crucial for protecting photosystem I (PSI) from photodamage in response to fluctuating light. To optimize the balance between efficient photosynthesis and photoprotection, it is necessary to regulate pmf. Cyclic electron transport around PSI and pseudo-cyclic electron transport involving flavodiiron proteins contribute to the modulation of pmf magnitude. By manipulating the ratio between the two components of pmf, it is possible to modify the extent of photosynthetic control without affecting the pmf size. This adjustment can be achieved by regulating the movement of ions (such as K+ and Cl−) across the thylakoid membrane. Since ATP synthase is the primary consumer of pmf in chloroplasts, its activity must be precisely regulated to accommodate other mechanisms involved in pmf optimization. Although fragments of information about each regulatory process have been accumulated, a comprehensive understanding of their interactions is lacking. Here, I summarize current knowledge of the network for pmf regulation, mainly based on genetic studies. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria (LAB) are Gram positive bacteria frequently used in the food industry for fermentation, mainly transformation of carbohydrates into lactic acid. In addition, these bacteria also have the capacity to metabolize citrate, an organic acid commonly found in food products. Its fermentation leads to the production of 4-carbon compounds such as diacetyl, resulting in a buttery flavor desired in dairy products. Citrate metabolism is known to have several beneficial effects on LAB physiology. Nevertheless, a controversial effect of citrate has been described on the acid tolerance of the wine bacterium Oenococcus oeni. This observation raises questions about the effect of citrate on the capacity of O. oeni to conduct malolactic fermentation in highly acidic wines. This review aims to summarize the current understanding of citrate metabolism in LAB, with a focus on the wine bacterium O. oeni. Metabolism with the related enzymes is detailed, as are the involved genes organized in cit loci. The known systems of cit locus expression regulation are also described. Finally, the beneficial effects of citrate catabolism on LAB physiology are reported and the negative impact observed in O. oeni is discussed. [ABSTRACT FROM AUTHOR]

Bacteriophage endolysins are bacteriolytic enzymes that have been explored as potential weapons to fight antibiotic-resistant bacteria. Despite several studies support the application of endolysins as enzybiotics, detailed knowledge on cellular and enzymatic factors affecting their lytic activity is still missing. The bacterial membrane proton motive force (PMF) and certain cell wall glycopolymers of Gram-positive bacteria have been implicated in some tolerance to endolysins. Here, we studied how the anti-staphylococcal endolysin Lys11, a modular enzyme with two catalytic domains (peptidase and amidase) and a cell binding domain (CBD11), responded to changes in the chemical and/or electric gradients of the PMF (ΔpH and Δψ, respectively). We show that simultaneous dissipation of both gradients enhances endolysin binding to cells and lytic activity. The collapse of ΔpH is preponderant in the stimulation of Lys11 lytic action, while the dissipation of Δψ is mainly associated with higher endolysin binding. Interestingly, this binding depends on the amidase domain. The peptidase domain is responsible for most of the Lys11 bacteriolytic activity. Wall teichoic acids (WTAs) are confirmed as major determinants of endolysin tolerance, in part by severely hindering CBD11 binding activity. In conclusion, the PMF and WTA interfere differently with the endolysin functional domains, affecting both the binding and catalytic efficiencies. [ABSTRACT FROM AUTHOR]

Auxin is a crucial plant hormone that controls a multitude of developmental processes. The directional movement of auxin between cells is largely facilitated by canonical PIN-FORMED proteins in the plasma membrane. In contrast, non-canonical PIN-FORMED proteins and PIN-LIKES proteins appear to reside mainly in the endoplasmic reticulum. Despite recent progress in identifying the roles of the endoplasmic reticulum in cellular auxin responses, the transport dynamics of auxin at the endoplasmic reticulum are not well understood. PIN-LIKES are structurally related to PIN-FORMED proteins, and recently published structures of these transporters have provided new insights into PIN-FORMED proteins and PIN-LIKES function. In this review, we summarize current knowledge on PIN-FORMED proteins and PIN-LIKES in intracellular auxin transport. We discuss the physiological properties of the endoplasmic reticulum and the consequences for transport processes across the ER membrane. Finally, we highlight the emerging role of the endoplasmic reticulum in the dynamics of cellular auxin signalling and its impact on plant development. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria (LAB) are Gram positive bacteria frequently used in the food industry for fermentation, mainly transformation of carbohydrates into lactic acid. In addition, these bacteria also have the capacity to metabolize citrate, an organic acid commonly found in food products. Its fermentation leads to the production of 4-carbon compounds such as diacetyl, resulting in a buttery flavor desired in dairy products. Citrate metabolism is known to have several beneficial effects on LAB physiology. Nevertheless, a controversial effect of citrate has been described on the acid tolerance of the wine bacterium Oenococcus oeni. This observation raises questions about the effect of citrate on the capacity of O. oeni to conduct malolactic fermentation in highly acidic wines. This review aims to summarize the current understanding of citrate metabolism in LAB, with a focus on the wine bacterium O. oeni. Metabolism with the related enzymes is detailed, as are the involved genes organized in cit loci. The known systems of cit locus expression regulation are also described. Finally, the beneficial effects of citrate catabolism on LAB physiology are reported and the negative impact observed in O. oeni is discussed.

This study aimed to evaluate the antibacterial activities of α-terpineol against common foodborne pathogenic bacteria by agar well diffusion, broth microdilution, and colony counting assay. Propulsive research was conducted to reveal the antibacterial mechanisms, including morphology, infrared spectroscopy, membrane fluidity, membrane permeability, proton motive force, and oxidative phosphorylation. Results indicated that the antibacterial activity of α-terpineol decreased in the following order: Escherichia coli O157:H7, Salmonella typhimurium, Listeria monocytogenes, and Staphylococcus aureus. With an initial cell count of 8 log CFU/mL, α-terpineol at 0.8% (v/v) reduced E. coli O157:H7 and S. aureus by approximately 5.6 and 3.9 log CFU/mL within 1 h, respectively. Remarkable destruction in cell envelopes and intracellular organizations was observed. The hydroxyl of α-terpineol might form glycosidic bonds with carbohydrates and hydrogen bonds with PO2− and COO− via infrared spectroscopy analysis. Generalized polarization of Laurdan revealed that the polar head groups of phospholipids transformed into close packed. The anisotropy variations of trimethyl amino-diphenylhexatriene (TMA-DPH) and DPH suggested membrane fluidity decreased. The N-phenyl-1-naphthylamine intake assay indicated that α-terpineol impaired the cell wall. Propidium iodide staining was indicative of damaged plasma membranes. Electron transport in the cytoplasmic membrane was impaired, inducing reactive oxygen species accumulation. Both membrane electrical potential and membrane pH gradient collapsed. The disruption of proton motive force and the leakage of ATP resulted in a deficit of intracellular ATP. Our research revealed the interaction between the hydroxyl group of α-terpineol and bacteria affects membrane function contributing to the bacteria's death. Key points: • α-Terpineol hydroxy formed glycosidic bonds and hydrogen bonds with bacteria • α-Terpineol increased the membrane gelation and reduced the membrane fluidity • Proton motive force and oxidative phosphorylation were impaired [ABSTRACT FROM AUTHOR]

Introduction: Antimicrobial resistance, especially the development of multidrug)resistant strains, is an urgent public health threat. Antibiotic adjuvants have been shown to improve the treatment of resistant bacterial infections. Methods: We verified that exogenous L-arginine promoted the killing effect of gentamicin against Salmonella in vitro and in vivo, and measured intracellular ATP, NADH, and PMF of bacteria. Gene expression was determined using real-time quantitative PCR. Results: This study found that alkaline arginine significantly increased gentamicin, tobramycin, kanamycin, and apramycin-mediated killing of drug-resistant Salmonella, including multidrug-resistant strains. Mechanistic studies showed that exogenous arginine was shown to increase the proton motive force, increasing the uptake of gentamicin and ultimately inducing bacterial cell death. Furthermore, in mouse infection model, arginine effectively improved gentamicin activity against Salmonella typhimurium. Discussion: These findings confirm that arginine is a highly effective and harmless aminoglycoside adjuvant and provide important evidence for its use in combination with antimicrobial agents to treat drug-resistant bacterial infections. [ABSTRACT FROM AUTHOR]

Mitochondrial complex V (ATP synthase) is a remarkable molecular motor crucial in generating ATP and sustaining mitochondrial function. Its importance in cellular metabolism cannot be overstated, as malfunction of ATP synthase has been linked to various pathological conditions. Both natural and synthetic ATP synthase inhibitors have been extensively studied, revealing their inhibitory sites and modes of action. These findings have opened exciting avenues for developing new therapeutics and discovering new pesticides and herbicides to safeguard global food supplies. However, it is essential to remember that these compounds can also adversely affect human and animal health, impacting vital organs such as the nervous system, heart, and kidneys. This review aims to provide a comprehensive overview of mitochondrial ATP synthase, its structural and functional features, and the most common inhibitors and their potential toxicities.

ABSTRACT Helicobacter pylori vacuolating cytotoxin A (VacA) is an intracellular-acting protein exotoxin that induces mitochondrial dysfunction and energy depletion within host cells. Although exposure to VacA results in mitochondrial dysfunction, one recent study revealed that, following limited exposure to VacA, mitochondrial function and cellular ATP levels were restored in a time-dependent manner. Studies performed to address the mechanism by which host cells detect and respond to intracellular VacA identified the adenosine monophosphate (AMP)-activated protein kinase (AMPK) as a sensor of toxin-dependent alterations in cellular energy status. Activation of AMPK in response to VacA was demonstrated to orchestrate alterations in mitochondrial dynamics which resulted in restoration of mitochondrial function. Specifically, upregulation of dynamin-related protein 1 (Drp-1)-dependent mitochondrial fission resulted in reversible fragmentation of filamentous mitochondria and time-dependent reduction in mitochondrial-associated VacA, suggesting that fragmentation is important for removal of VacA from mitochondria. Cells with reduced levels of Drp-1 were more susceptible to VacA-dependent cell death, suggesting that mitochondrial dynamics is important for maintaining cell viability through the reduction in mitochondrial-associated toxin. Collectively, these studies support a model that cellular recovery and survival in response to VacA-dependent mitochondrial dysfunction is linked to host cell modulation of mitochondrial dynamics. This study provides new insights into cellular recognition and responses to intracellular-acting toxin modulation of host cell function, which could be relevant for the growing list of pathogenic microbes and viruses identified that target mitochondria as part of their virulence strategies. IMPORTANCE Persistent human gastric infection with Helicobacter pylori is the single most important risk factor for development of gastric malignancy, which is one of the leading causes of cancer-related deaths worldwide. An important virulence factor for Hp colonization and severity of gastric disease is the protein exotoxin VacA, which is secreted by the bacterium and modulates functional properties of gastric cells. VacA acts by damaging mitochondria, which impairs host cell metabolism through impairment of energy production. Here, we demonstrate that intoxicated cells have the capacity to detect VacA-mediated damage, and orchestrate the repair of mitochondrial function, thereby restoring cellular health and vitality. This study provides new insights into cellular recognition and responses to intracellular-acting toxin modulation of host cell function, which could be relevant for the growing list of pathogenic microbes and viruses identified that target mitochondria as part of their virulence strategies.

ABSTRACT Bacterial antibiotic tolerance, a phenomenon first observed in 1944, is known to be responsible for both onset and exacerbation of recurrent and chronic bacterial infections. The development of antibiotic tolerance was previously thought to be due to a switch to physiological dormancy when bacteria encounter adverse growth conditions. Our recent laboratory findings, however, showed that a set of genes related to the maintenance of proton motive force (PMF) are up-regulated under starvation, indicating that the tolerant sub-population, which are commonly known as persisters, can actively maintain their tolerance phenotypes. In this study, we investigated the relative functional roles of proteins involved in the maintenance and active generation of PMF in mediating tolerance formation in bacteria and found that the PspA and RcsB proteins play a key role in PMF maintenance in persisters, as deletion of genes encoding these two proteins resulted in significantly lower tolerance levels. Consistently, expression of the OsmC and Bdm proteins, which is under regulation by RcsB, is required to maintain PMF and the antibiotic tolerance phenotypes. On the other hand, the NuoL, Ndh, AppC, CyoB, and NuoF proteins, which are electron transport chain (ETC) components, were also found to be actively expressed in persisters in order to generate PMF to support functioning of various tolerance mechanisms such as efflux activities. Our data show that active generation of PMF is even more important than the PMF maintenance functions of PspA and RcsB in the expression of antibiotic tolerance phenotypes in persisters. Assessment of double- and triple-gene knockout strains, in which the PMF maintenance genes and those encoding ETC components were simultaneously deleted, confirms that these two groups of genes are both required for the expression of antibiotic tolerance phenotypes and that a lack of these functions would result in complete PMF dissipation and accumulation of antibiotics in the intracellular compartment of persisters and eventually cell death. Products of these genes are, therefore, ideal targets for future development of anti-tolerance agents. IMPORTANCE In this work, bacteria were found to undergo active generation and maintenance of proton motive force (PMF) under adverse conditions, such as starvation so as to support a range of physiological functions in order to survive under such conditions for a prolonged period. The ability to maintain a substantial level of PMF was found to be directly linked to that exhibiting phenotypic antibiotic tolerance under nutrient starvation or other adverse conditions. These findings infer that bacteria do not simply become physiologically dormant when they become antibiotic tolerant, instead they need to produce a wide range of proteins including those which help prevent PMF dissipation, such as PspA and RcsB, and the electron transport chain components, such as NuoL and Ndh, that actively generate PMF even during long-term starvation. As antibiotic tolerant sub-population is known to play a role in eliciting recurrent and chronic infections, especially among patients with a weakened immune system, the PMF maintenance mechanisms identified in this work are potential targets for the development of new strategies to control recurrent and chronic infections.

Pseudomonas aeruginosa is a ubiquitous nosocomial opportunistic pathogen that harbors many virulence determinants. Part of P. aeruginosa success colonizing a variety of habitats resides in its metabolic robustness and plasticity, which are the basis of its capability of adaptation to different nutrient sources and ecological conditions, including the infected host. Given this situation, it is conceivable that P. aeruginosa virulence might be, at least in part, under metabolic control, in such a way that virulence determinants are produced just when needed. Indeed, it has been shown that the catabolite repression control protein Crc, which together with the RNA chaperon Hfq regulates the P. aeruginosa utilization of carbon sources at the post-transcriptional level, also regulates, directly or indirectly, virulence-related processes in P. aeruginosa. Among them, Crc regulates P. aeruginosa cytotoxicity, likely by modulating the activity of the Type III Secretion System (T3SS), which directly injects toxins into eukaryotic host cells. The present work shows that the lack of Crc produces a Type III Secretion-defective phenotype in P. aeruginosa. The observed impairment is a consequence of a reduced expression of the genes encoding the T3SS, together with an impaired secretion of the proteins involved. Our results support that the impaired T3SS activity of the crc defective mutant is, at least partly, a consequence of a defective protein export, probably due to a reduced proton motive force. This work provides new information about the complex regulation of the expression and the activity of the T3SS in P. aeruginosa. Our results highlight the need of a robust bacterial metabolism, which is defective in the ∆crc mutant, to elicit complex and energetically costly virulence strategies, as that provided by the T3SS. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate collateral sensitivity and cross-resistance of antibiotic-induced resistant Salmonella Typhimurium to various antibiotics. S. Typhimurium ATCC 19585 (STWT) was exposed to ciprofloxacin, gentamicin, kanamycin, and tetracycline to induce antibiotic resistance, respectively, assigned as STCIP, STGEN, STKAN, and STTET. The susceptibilities of the antibiotic-induced resistant mutants to cefotaxime, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, polymyxin B, streptomycin, tetracycline, and tobramycin were determined in the absence and presence of CCCP and PAβN. STCIP showed the cross-resistance to tetracycline and collateral sensitivity to gentamicin (1/2 fold) and kanamycin (1/4 fold). STTET was also cross-resistant to ciprofloxacin (128-fold) and collateral sensitive to gentamicin (1/4-fold) and kanamycin (1/8-fold). The cross-resistance and collateral sensitivity of STCIP and STTET were associated with the AcrAB-TolC efflux pump and outer membrane porin proteins (OmpC). This study provides new insight into the collateral sensitivity phenomenon, which can be used for designing effective antibiotic treatment regimens to control antibiotic-resistant bacteria. [ABSTRACT FROM AUTHOR]

ATP synthase is a key protein in the oxidative phosphorylation process, as it aids in the effective production of ATP (Adenosine triphosphate) in all life's of kingdoms. ATP synthases have distinctive properties that contribute to efficient ATP synthesis. The ATP synthase of mycobacterium is of special relevance since it has been identified as a target for potential anti-TB molecules, especially Bedaquiline (BDQ). Better knowledge of how mycobacterial ATP synthase functions and its peculiar characteristics will aid in our understanding of bacterial energy metabolism adaptations. Furthermore, identifying and understanding the important distinctions between human ATP synthase and bacterial ATP synthase may provide insight into the design and development of inhibitors that target specific ATP synthase. In recent years, many potential candidates targeting the ATP synthase of mycobacterium have been developed. In this review, we discuss the druggable targets of the Electron transport chain (ETC) and recently identified potent inhibitors (including clinical molecules) from 2015 to 2022 of diverse classes that target ATP synthase of M. tuberculosis. [Display omitted] [ABSTRACT FROM AUTHOR]

Background and Aims The ATP yield of plant respiration (ATP/hexose unit respired) quantitatively links active heterotrophic processes with substrate consumption. Despite its importance, plant respiratory ATP yield is uncertain. The aim here was to integrate current knowledge of cellular mechanisms with inferences required to fill knowledge gaps to generate a contemporary estimate of respiratory ATP yield and identify important unknowns. Method A numerical balance sheet model combining respiratory carbon metabolism and electron transport pathways with uses of the resulting transmembrane electrochemical proton gradient was created and parameterized for healthy, non-photosynthesizing plant cells catabolizing sucrose or starch to produce cytosolic ATP. Key Results Mechanistically, the number of c subunits in the mitochondrial ATP synthase Fo sector c -ring, which is unquantified in plants, affects ATP yield. A value of 10 was (justifiably) used in the model, in which case respiration of sucrose potentially yields about 27.5 ATP/hexose (0.5 ATP/hexose more from starch). Actual ATP yield often will be smaller than its potential due to bypasses of energy-conserving reactions in the respiratory chain, even in unstressed plants. Notably, all else being optimal, if 25 % of respiratory O2 uptake is via the alternative oxidase – a typically observed fraction – ATP yield falls 15 % below its potential. Conclusions Plant respiratory ATP yield is smaller than often assumed (certainly less than older textbook values of 36–38 ATP/hexose) leading to underestimation of active-process substrate requirements. This hinders understanding of ecological/evolutionary trade-offs between competing active processes and assessments of crop growth gains possible through bioengineering of processes that consume ATP. Determining the plant mitochondrial ATP synthase c -ring size, the degree of any minimally required (useful) bypasses of energy-conserving reactions in the respiratory chain, and the magnitude of any 'leaks' in the inner mitochondrial membrane are key research needs. [ABSTRACT FROM AUTHOR]

Novel antibacterial therapies are urgently required to tackle the increasing number of multidrug‐resistant pathogens. Identification of new antimicrobial targets is critical to avoid possible cross‐resistance issues. Bacterial proton motive force (PMF), an energetic pathway located on the bacterial membrane, crucially regulates various biological possesses such as adenosine triphosphate synthesis, active transport of molecules, and rotation of bacterial flagella. Nevertheless, the potential of bacterial PMF as an antibacterial target remains largely unexplored. The PMF generally comprises electric potential (ΔΨ) and transmembrane proton gradient (ΔpH). In this review, we present an overview of bacterial PMF, including its functions and characterizations, highlighting the representative antimicrobial agents that specifically target either ΔΨ or ΔpH. At the same time, we also discuss the adjuvant potential of bacterial PMF‐targeting compounds. Lastly, we highlight the value of PMF disruptors in preventing the transmission of antibiotic resistance genes. These findings suggest that bacterial PMF represents an unprecedented target, providing a comprehensive approach to controlling antimicrobial resistance. [ABSTRACT FROM AUTHOR]

F o portion of ATP synthase is a proton-motive rotary motor. The Coulombic attraction between the conserved acidic residues in the c -ring and the arginine in the a -subunit (a R) was early proposed to drive the c -ring rotation relative to the a -subunit, and has been actually observed in our previous molecular dynamics simulation with full atomistic description of F o embedded in the membrane. In this study, to quantify the driving force, we conducted the umbrella sampling (US) and obtained the free-energy landscape for the c -ring rotation. We first show that the free-energy gradient toward the ATP-synthesis direction appears in the deprotonated state of c E. Using the sampled snapshots that cover a wide range of the rotational angle, we further analyzed the rotational-angle dependence of the hydration and the protonation states and obtained the Coulomb-energy landscapes with a focus on the c E- a R interaction. The results indicate that both the Coulombic solvation energy of c E and the interaction energy between c E and a R contribute to the torque generation for the c -ring rotation. • Free-energy landscape for rotation of F o motor was obtained by umbrella sampling. • Coulomb interaction between conserved Glu and Arg generates the torque. • Solvation and protonation states of conserved Glu's on rotor were characterized. • Brownian ratchet due to desolvation penalty contributes to unidirectional rotation. [ABSTRACT FROM AUTHOR]

Following the discovery of streptomycin from Streptomyces griseus in the 1940s by Selman Waksman and colleagues, aminoglycosides were first used to treat tuberculosis and then numerous derivatives have since been used to combat a wide variety of bacterial infections. These bactericidal antibiotics were used as first-line treatments for several decades but were largely replaced by ß-lactams and fluoroquinolones in the 1980s, although widespread emergence of antibiotic-resistance has led to renewed interest in aminoglycosides. The primary site of action for aminoglycosides is the 30 S ribosomal subunit where they disrupt protein translation, which contributes to widespread cellular damage through a number of secondary effects including rapid uptake of aminoglycosides via elevated proton-motive force (PMF), membrane damage and breakdown, oxidative stress, and hyperpolarisation of the membrane. Several factors associated with aminoglycoside entry have been shown to impact upon bacterial killing, and more recent work has revealed a complex relationship between metabolic states and the efficacy of different aminoglycosides. Hence, it is imperative to consider the environmental conditions and bacterial physiology and how this can impact upon aminoglycoside entry and potency. This mini-review seeks to discuss recent advances in this area and how this might affect the future use of aminoglycosides. [ABSTRACT FROM AUTHOR]

Piceatannol (PIC) is a natural stilbene extracted from grape skins that exhibits biological activities such as antibacterial, antitumor, and antioxidant activities. The present study was carried out to further investigate the effect of PIC on the antibacterial activity of different antibiotics and to reveal the antibacterial mechanism of PIC. We found that PIC had an inhibitory effect against Staphylococcus aureus (S. aureus); its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 128 μg/mL and 256 μg/ mL, respectively. Additionally, we measured the fractional inhibitory concentration (FIC) of PIC combined with antibiotics via the checkerboard method. The results showed that when PIC and ciprofloxacin (CIP) were combined, they displayed a synergistic effect against S. aureus. Moreover, this synergistic effect was verified by time–kill assays. Further, the results of the membrane permeability assay and proton motive force assay revealed that PIC could enhance the sensitivity of S. aureus to CIP by dissipating the bacterial proton motive force (PMF), particularly the ∆ψ component, rather than increasing membrane permeability. PIC also inhibited bacterial adenosine triphosphate (ATP) synthesis and was less likely to induce bacterial resistance but exhibited slight hemolytic activity on mammalian erythrocytes. In summary, the combination of PIC and CIP is expected to become a new drug combination to combat S. aureus. [ABSTRACT FROM AUTHOR]

ABSTRACT Aminoglycosides (AG) have been used against Gram-negative bacteria for decades. Yet, how bacterial metabolism and environmental conditions modify AG toxicity is poorly understood. Here, we show that the level of AG susceptibility varies depending on the nature of the respiratory chain that Escherichia coli uses for growth, i.e., oxygen, nitrate, or fumarate. We show that all components of the fumarate respiratory chain, namely, hydrogenases 2 and 3, the formate hydrogenlyase complex, menaquinone, and fumarate reductase are required for AG-mediated killing under fumarate respiratory conditions. In addition, we show that the AAA+ ATPase RavA and its Von Wildebrand domain-containing partner, ViaA, are essential for AG to act under fumarate respiratory conditions. This effect was true for all AG that were tested but not for antibiotics from other classes. In addition, we show that the sensitizing effect of RavA-ViaA is due to increased gentamicin uptake in a proton motive force-dependent manner. Interestingly, the sensitizing effect of RavA-ViaA was prominent in poor energy conservation conditions, i.e., with fumarate, but dispensable under high energy conservation conditions, i.e., in the presence of nitrate or oxygen. We propose that RavA-ViaA can facilitate uptake of AG across the membrane in low-energy cellular states. IMPORTANCE Antibiotic resistance is a major public health, social, and economic problem. Aminoglycosides (AG) are known to be highly effective against Gram-negative bacteria, but their use is limited to life-threatening infections because of their nephrotoxicity and ototoxicity at therapeutic dose. Elucidation of AG-sensitization mechanisms in bacteria would allow reduced effective doses of AG. Here, we have identified the molecular components involved in anaerobic fumarate respiration that are required for AG to kill. In addition to oxidoreductases and menaquinone, this includes new molecular players, RavA, an AAA+ ATPase, and ViaA, its partner that has the VWA motif. Remarkably, the influence of RavA-ViaA on AG susceptibility varies according to the type of bioenergetic metabolism used by E. coli. This is a significant advance because anaerobiosis is well known to reduce the antibacterial activity of AG. This study highlights the critical importance of the relationship between culture conditions, metabolism, and antibiotic susceptibility.

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.

ABSTRACT The emergence of bacterial drug resistance poses a severe threat to global public health. In particular, antimicrobial-resistant pathogens lead to a high rate of treatment failure and significantly increase mortality. Repurposing FDA-approved compounds to sensitize superbugs to conventional antibiotics provides a promising strategy to alleviate such crises. Pixantrone (PIX) has been approved for treating aggressive B-cell non-Hodgkin’s lymphoma. By high-throughput drug screening, we profiled the synergistic activity between PIX and rifampin (RFP) against Gram-negative extensively drug-resistant isolates by checkerboard assay. Mechanistic studies demonstrated that PIX impacted the flagellum assembly, induced irreversible intracellular reactive oxygen species accumulation and disrupted proton motive force. In addition, the combination of PIX with RFP possesses effective antimicrobial activity against multidrug-resistant strains in vivo without detected toxicity. Collectively, these results reveal the potential of PIX in combination with RFP as a therapy option for refractory infections caused by Gram-negative pathogens. IMPORTANCE Bacterial resistance has become increasingly serious because of the widespread use and abuse of antibiotics. In particular, the emergence of multidrug-resistant bacteria has posed a serious threat to human public health. Drug repurposing, the process of finding new uses for existing drugs, provide a promising pathway to solve antimicrobial resistance. Compared to the development of novel antibiotics, this strategy leverages well-characterized pharmacology and toxicology of known drugs and is more cost-effective.

Bacteria employ the flagellar type III secretion system (fT3SS) to construct flagellum, which acts as a supramolecular motility machine. The fT3SS of Salmonella enterica serovar Typhimurium is composed of a transmembrane export gate complex and a cytoplasmic ATPase ring complex. The transmembrane export gate complex is fueled by proton motive force across the cytoplasmic membrane and is divided into four distinct functional parts: a dual-fuel export engine; a polypeptide channel; a membrane voltage sensor; and a docking platform. ATP hydrolysis by the cytoplasmic ATPase complex converts the export gate complex into a highly efficient proton (H+)/protein antiporter that couples inward-directed H+ flow with outward-directed protein export. When the ATPase ring complex does not work well in a given environment, the export gate complex will remain inactive. However, when the electric potential difference, which is defined as membrane voltage, rises above a certain threshold value, the export gate complex becomes an active H+/protein antiporter to a considerable degree, suggesting that the export gate complex has a voltage-gated activation mechanism. Furthermore, the export gate complex also has a sodium ion (Na+) channel to couple Na+ influx with flagellar protein export. In this article, we review our current understanding of the activation mechanism of the dual-fuel protein export engine of the fT3SS. This review article is an extended version of a Japanese article, Membrane voltage-dependent activation of the transmembrane export gate complex in the bacterial flagellar type III secretion system, published in SEIBUTSU BUTSURI Vol. 62, p165–169 (2022).

ATP, produced by the light reactions of photosynthesis, acts as the universal cellular energy cofactor fuelling all life processes. Chloroplast ATP synthase produces ATP using the proton motive force created by solar energy-driven thylakoid electron transport reactions. Here we investigate how increasing abundance of ATP synthase affects leaf photosynthesis and growth of rice, Oryza sativa variety Kitaake. We show that overexpression of AtpD, the nuclear-encoded subunit of the chloroplast ATP synthase, stimulates both abundance of the complex, confirmed by immunodetection of thylakoid complexes separated by Blue Native-PAGE, and ATP synthase activity, detected as higher proton conductivity of the thylakoid membrane. Plants with increased AtpD content had higher CO2 assimilation rates when a stepwise increase in CO2 partial pressure was imposed on leaves at high irradiance. Fitting of the CO2 response curves of assimilation revealed that plants overexpressing AtpD had a higher electron transport rate (J) at high CO2, despite having wild-type-like abundance of the cytochrome b 6 f complex. A higher maximum carboxylation rate (V cmax) and lower cyclic electron flow detected in transgenic plants both pointed to an increased ATP production compared with wild-type plants. Our results present evidence that the activity of ATP synthase modulates the rate of electron transport at high CO2 and high irradiance. [ABSTRACT FROM AUTHOR]

Plants growing in nature often experience fluctuating irradiance. However, in the laboratory, the dynamics of photosynthesis are usually explored by instantaneously exposing dark-adapted plants to constant light and examining the dark-to-light transition, which is a poor approximation of natural phenomena. With the aim creating a better approximation, we exposed leaves of pea (Pisum sativum) to oscillating light and measured changes in the functioning of PSI and PSII, and of the proton motive force at the thylakoid membrane. We found that the dynamics depended on the oscillation period, revealing information about the underlying regulatory networks. As demonstrated for a selected oscillation period of 60 s, the regulation tries to keep the reaction centers of PSI and PSII open. We present an evaluation of the data obtained, and discuss the involvement of particular processes in the regulation of photosynthesis. The forced oscillations provided an information-rich fingerprint of complex regulatory networks. We expect future progress in understanding these networks from experiments involving chemical interventions and plant mutants, and by using mathematical modeling and systems identification and control tools. [ABSTRACT FROM AUTHOR]

Although a mechanism accounting for hyperthermic death at critical temperatures remains elusive, the mitochondria of crucial active excitable tissues (i.e. heart and brain) may well be key to this process. Mitochondria produce ∼90% of the ATP required by cells to maintain cellular integrity and function. They also integrate into biosynthetic pathways that support metabolism as a whole, allow communication within the cell, and regulate cellular health and death pathways. We have previously shown that cardiac and brain mitochondria demonstrate decreases in the efficiency of, and absolute capacity for ATP synthesis as temperatures rise, until ultimately there is too little ATP to support cellular demands, and organ failure follows. Importantly, substantial decreases in ATP synthesis occur at temperatures immediately below the temperature of heart failure, and this suggests a causal role of mitochondria in hyperthermic death. However, what causes mitochondria to fail? Here, we consider the answers to this question. Mitochondrial dysfunction at high temperature has classically been attributed to elevated leak respiration suspected to result from increased movement of protons (H+) through the inner mitochondrial membrane (IMM), thereby bypassing the ATP synthases. In this Commentary, we introduce some alternative explanations for elevated leak respiration. We first consider respiratory complex I and then propose that a loss of IMM structure occurs as temperatures rise. The loss of the cristae folds of the IMM may affect the efficiency of H+ transport, increasing H+ conductance either through the IMM or into the bulk water phases of mitochondria. In either case, O2 consumption increases while ATP synthesis decreases., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

VemP is a secretory protein in the Vibrio species that monitors cellular protein-transport activity through its translation arrest, allowing expression of the downstream secD2-secF2 genes in the same operon, which encode components of the protein translocation machinery. When cellular protein-transport function is fully active, secD2/F2 expression remains repressed as VemP translation arrest is canceled immediately. The VemP arrest cancellation occurs on the SecY/E/G translocon in a late stage in the translocation process and requires both trans factors, SecD/F and PpiD/YfgM, and a cis element, Arg-85 in VemP; however, the detailed molecular mechanism remains elusive. This study aimed to elucidate how VemP passing through SecY specifically monitors SecD/F function. Genetic and biochemical studies showed that SecY is involved in the VemP arrest cancellation and that the arrested VemP is stably associated with a specific site in the protein-conducting pore of SecY. VemP-Bla reporter analyses revealed that a short hydrophobic segment adjacent to Arg-85 plays a critical role in the regulated arrest cancellation with its hydrophobicity correlating with the stability of the VemP arrest. We identified Gln-65 and Pro-67 in VemP as novel elements important for the regulation. We propose a model for the regulation of the VemP arrest cancellation by multiple cis elements and trans factors with different roles., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

ABSTRACT Amidochelocardin is a broad-spectrum antibiotic with activity against many Gram-positive and Gram-negative bacteria. According to recent data, the antibiotic effect of this atypical tetracycline is directed against the cytoplasmic membrane, which is associated with the dissipation of the membrane potential. Here, we investigated the effect of amidochelocardin on the proteome of Clostridioides difficile to gain insight into the membrane stress physiology of this important anaerobic pathogen. For the first time, the membrane-directed action of amidochelocardin was confirmed in an anaerobic pathogen. More importantly, our results revealed that aromatic compounds potentially play an important role in C. difficile upon dissipation of its membrane potential. More precisely, a simultaneously increased production of enzymes required for the synthesis of chorismate and two putative phenazine biosynthesis proteins point to the production of a hitherto unknown compound in response to membrane depolarization. Finally, increased levels of the ClnAB efflux system and its transcriptional regulator ClnR were found, which were previously found in response to cationic antimicrobial peptides like LL-37. Therefore, our data provide a starting point for a more detailed understanding of C. difficile’s way to counteract membrane-active compounds. IMPORTANCE C. difficile is an important anaerobe pathogen causing mild to severe infections of the gastrointestinal tract. To avoid relapse of the infection following antibiotic therapy, antibiotics are needed that efficiently eradicate C. difficile from the intestinal tract. Since C. difficile was shown to be substantially sensitive to membrane-active antibiotics, it has been proposed that membrane-active antibiotics might be promising for the therapy of C. difficile infections. Therefore, we studied the response of C. difficile to amidochelocardin, a membrane-active antibiotic dissipating the membrane potential. Interestingly, C. difficile’s response to amidochelocardin indicates a role of aromatic metabolites in mediating stress caused by dissipation of the membrane potential.

The objective of this study was to evaluate collateral sensitivity and cross-resistance of antibiotic-induced resistant Salmonella Typhimurium to various antibiotics. S. Typhimurium ATCC 19585 (STWT) was exposed to ciprofloxacin, gentamicin, kanamycin, and tetracycline to induce antibiotic resistance, respectively, assigned as STCIP, STGEN, STKAN, and STTET. The susceptibilities of the antibiotic-induced resistant mutants to cefotaxime, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, polymyxin B, streptomycin, tetracycline, and tobramycin were determined in the absence and presence of CCCP and PAβN. STCIP showed the cross-resistance to tetracycline and collateral sensitivity to gentamicin (1/2 fold) and kanamycin (1/4 fold). STTET was also cross-resistant to ciprofloxacin (128-fold) and collateral sensitive to gentamicin (1/4-fold) and kanamycin (1/8-fold). The cross-resistance and collateral sensitivity of STCIP and STTET were associated with the AcrAB-TolC efflux pump and outer membrane porin proteins (OmpC). This study provides new insight into the collateral sensitivity phenomenon, which can be used for designing effective antibiotic treatment regimens to control antibiotic-resistant bacteria.

Pseudomonas aeruginosa is a ubiquitous nosocomial opportunistic pathogen that harbors many virulence determinants. Part of P. aeruginosa success colonizing a variety of habitats resides in its metabolic robustness and plasticity, which are the basis of its capability of adaptation to different nutrient sources and ecological conditions, including the infected host. Given this situation, it is conceivable that P. aeruginosa virulence might be, at least in part, under metabolic control, in such a way that virulence determinants are produced just when needed. Indeed, it has been shown that the catabolite repression control protein Crc, which together with the RNA chaperon Hfq regulates the P. aeruginosa utilization of carbon sources at the post-transcriptional level, also regulates, directly or indirectly, virulence-related processes in P. aeruginosa. Among them, Crc regulates P. aeruginosa cytotoxicity, likely by modulating the activity of the Type III Secretion System (T3SS), which directly injects toxins into eukaryotic host cells. The present work shows that the lack of Crc produces a Type III Secretion-defective phenotype in P. aeruginosa. The observed impairment is a consequence of a reduced expression of the genes encoding the T3SS, together with an impaired secretion of the proteins involved. Our results support that the impaired T3SS activity of the crc defective mutant is, at least partly, a consequence of a defective protein export, probably due to a reduced proton motive force. This work provides new information about the complex regulation of the expression and the activity of the T3SS in P. aeruginosa. Our results highlight the need of a robust bacterial metabolism, which is defective in the ∆crc mutant, to elicit complex and energetically costly virulence strategies, as that provided by the T3SS.

ATP plays an essential role in the substrate/product transmembrane transportation during whole-cell bioconversion. This study aimed to address the impact of ATP upon cadaverine synthesis by whole-cell biocatalysts. The results showed no significant change in the ATP content (P = 0.625), and the specific cadaverine yield (P = 0.374) was observed in enzyme-catalyzed cadaverine synthesis with exogenous addition of ATP, indicating that the enzyme-catalyzed process does not require the participation of ATP. Furthermore, a whole-cell biocatalyst co-overexpressed methionine adenosyltransferase (MetK), lysine decarboxylase (CadA), and lysine/cadaverine antiporter (CadB) was constructed and used to investigate the effect of ATP deficiency on the cadaverine production by conversion of L -methionine and L -lysine, simultaneously. The results showed no significant difference (P = 0.585) in the specific cadaverine content between high and low levels of intracellular ATP. In addition, the intra- and extracellular cadaverine concentration and the ratio of ATP/ADP of whole-cell biocatalyst were determined. Results showed that the extracellular cadaverine concentration was much higher than the intracellular concentration, and no significant changes in ATP/ADP ratio during cadaverine synthesis. In contrast, an inhibition effect of the proton motive force (PMF) inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) on cadaverine production was detected. These findings strongly suggest that cadaverine transport in whole-cell biocatalysts was energized by PMF rather than ATP. Finally, a model was proposed to describe cadaverine's PMF-driven transport under different external pHs during whole-cell biocatalysis. This study is the first to experimentally confirm that the cadaverine production by Escherichia coli whole-cell bioconversion is independent of intracellular ATP, which helps guide the subsequent construction of biocatalysts and optimize transformation conditions. • Whole-cell synthesis of cadaverine does not involve direct consumption of ATP. • ATP-deficient E.coli was generated by bioconversion of L -methionine. • The deficiency of intracellular ATP does not affect cadaverine production. • The cadaverine transport in whole-cell biocatalysts was energized by PMF rather than ATP. • A model was proposed to describe cadaverine's PMF-driven transport during whole-cell biocatalysis. [ABSTRACT FROM AUTHOR]

Protein integration into biomembranes is an essential biological phenomenon common to all organisms. While various factors involved in protein integration, such as SRP, SecYEG and YidC, are proteinaceous, we identified a glycolipid named MPIase (Membrane Protein Integrase), which is present in the cytoplasmic membrane of E. coli. In vitro experiments using inverted membrane vesicles prepared from MPIase-depleted strains, and liposomes containing MPIase showed that MPIase is required for insertion of a subset of membrane proteins, which has been thought to be SecYEG-independent and YidC-dependent. Also, SecYEG-dependent substrate membrane proteins require MPIase in addition. Furthermore, MPIase is also essential for insertion of proteins with multiple negative charges, which requires both YidC and the proton motive force (PMF). MPIase directly interacts with SecYEG and YidC on the membrane. MPIase not only cooperates with these factors but also has a molecular chaperone-like function specific to the substrate membrane proteins through direct interaction with the glycan chain. Thus, MPIase catalyzes membrane insertion by accepting nascent membrane proteins on the membrane through its chaperone-like function, i.e., direct interaction with the substrate proteins, and then MPIase functionally interacts with SecYEG and YidC for substrate delivery, and acts with PMF to facilitate and complete membrane insertion when necessary. In this review, we will outline the mechanisms underlying membrane insertion catalyzed by MPIase, which cooperates with proteinaceous factors and PMF.

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) strains are tolerant of conventional antibiotics, making them extremely dangerous. Previous studies have shown the effectiveness of proton motive force (PMF) inhibitors at killing bacterial cells; however, whether these agents can launch a new treatment strategy to eliminate antibiotic-tolerant cells mandates further investigation. Here, using known PMF inhibitors and two different MRSA isolates, we showed that the bactericidal potency of PMF inhibitors seemed to correlate with their ability to disrupt PMF and permeabilize cell membranes. By screening a small chemical library to verify this correlation, we identified a subset of chemicals (including nordihydroguaiaretic acid, gossypol, trifluoperazine, and amitriptyline) that strongly disrupted PMF in MRSA cells by dissipating either the transmembrane electric potential (ΔΨ) or the proton gradient (ΔpH). These drugs robustly permeabilized cell membranes and reduced MRSA cell levels below the limit of detection. Overall, our study further highlights the importance of cellular PMF as a target for designing new bactericidal therapeutics for pathogens. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) emerged as a major hypervirulent pathogen that causes severe health care-acquired infections. These pathogens can be multidrug-tolerant cells, which can facilitate the recurrence of chronic infections and the emergence of diverse antibiotic-resistant mutants. In this study, we aimed to investigate whether proton motive force (PMF) inhibitors can launch a new treatment strategy to eliminate MRSA cells. Our in-depth analysis showed that PMF inhibitors that strongly dissipate either the transmembrane electric potential or the proton gradient can robustly permeabilize cell membranes and reduce MRSA cell levels below the limit of detection.

Adenosine triphosphate (ATP) regeneration is a significant step in both living cells and in vitro biotransformation (iv BT). Rotary motor ATP synthases (ATPases), which regenerate ATP in living cells, have been widely assembled in biomimetic structures for in vitro ATP synthesis. In this review, we present a comprehensive overview of ATPases, including the working principle, orientation and distribution density properties of ATPases, as well as the assembly strategies and applications of ATPase-based ATP regeneration modules. The original sources of ATPases for in vitro ATP regeneration include chromatophores, chloroplasts, mitochondria, and inverted Escherichia coli (E. coli) vesicles, which are readily accessible but unstable. Although significant advances have been made in the assembly methods for ATPase-artificial membranes in recent decades, it remains challenging to replicate the high density and orientation of ATPases observed in vivo using in vitro assembly methods. The use of bioproton pumps or chemicals for constructing proton motive forces (PMF) enables the versatility and potential of ATPase-based ATP regeneration modules. Additionally, overall robustness can be achieved via membrane component selection, such as polymers offering great mechanical stability, or by constructing a solid supporting matrix through layer-by-layer assembly techniques. Finally, the prospects of ATPase-based ATP regeneration modules can be expected with the technological development of ATPases and artificial membranes. [ABSTRACT FROM AUTHOR]