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Colistin is a “last resort” antibiotic for treatment of infections caused by some multidrug resistant Gram-negative bacterial pathogens. Resistance to colistin varies between bacterial species. Some Gram-negative bacteria such as Burkholderia spp. are intrinsically resistant to very high levels of colistin with minimal inhibitory concentrations (MIC) often above 0.5 mg/ml. We have previously shown DedA family proteins YqjA and YghB are conserved membrane transporters required for alkaline tolerance and resistance to several classes of dyes and antibiotics in Escherichia coli. Here, we show that a DedA family protein in Burkholderia thailandensis (DbcA; DedA of Burkholderia required for colistin resistance) is a membrane transporter required for resistance to colistin. Mutation of dbcA results in >100-fold greater sensitivity to colistin. Colistin resistance is often conferred via covalent modification of lipopolysaccharide (LPS) lipid A. Mass spectrometry of lipid A of ΔdbcA showed a sharp reduction of aminoarabinose in lipid A compared to wild type. Complementation of colistin sensitivity of B. thailandensis ΔdbcA was observed by expression of dbcA, E. coli yghB or E. coli yqjA. Many proton-dependent transporters possess charged amino acids in transmembrane domains that take part in the transport mechanism and are essential for function. Site directed mutagenesis of conserved and predicted membrane embedded charged amino acids suggest that DbcA functions as a proton-dependent transporter. Direct measurement of membrane potential shows that B. thailandensis ΔdbcA is partially depolarized suggesting that loss of protonmotive force can lead to alterations in LPS structure and severe colistin sensitivity in this species.

Protein transport across the cytoplasmic membrane is coupled to energy derived from ATP hydrolysis or the proton motive force. A sophisticated, multi-component type III secretion system (T3SS) exports substrate proteins of both the bacterial flagellum and virulence-associated injectisome system of many Gram-negative pathogens. The T3SS is primarily a proton motive force-driven protein exporter. Here, we describe a method to investigate the export of substrate proteins of the flagellar T3SS into the culture supernatant under conditions that manipulate the proton motive force. Further, we describe methods to precisely quantify flagellar protein export into the culture supernatant using a split NanoLuc luciferase, and how fluorescence labeling of the extracellular flagellar filament can bring insights into the protein export rate of individual flagellar T3SS., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 (CBD 11 ), 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 CBD 11 binding activity. In conclusion, the PMF and WTA interfere differently with the endolysin functional domains, affecting both the binding and catalytic efficiencies.

Colistin is a "last resort" antibiotic for treatment of infections caused by some multidrug resistant Gram-negative bacterial pathogens. Resistance to colistin varies between bacterial species. Some Gram-negative bacteria such as Burkholderia spp. are intrinsically resistant to very high levels of colistin with minimal inhibitory concentrations (MIC) often above 0.5 mg/ml. We have previously shown DedA family proteins YqjA and YghB are conserved membrane transporters required for alkaline tolerance and resistance to several classes of dyes and antibiotics in Escherichia coli. Here, we show that a DedA family protein in Burkholderia thailandensis (DbcA; D edA of B urkholderia required for c olistin resistance) is a membrane transporter required for resistance to colistin. Mutation of dbcA results in >100-fold greater sensitivity to colistin. Colistin resistance is often conferred via covalent modification of lipopolysaccharide (LPS) lipid A. Mass spectrometry of lipid A of Δ dbcA showed a sharp reduction of aminoarabinose in lipid A compared to wild type. Complementation of colistin sensitivity of B. thailandensis Δ dbcA was observed by expression of dbcA , E. coli yghB or E. coli yqjA. Many proton-dependent transporters possess charged amino acids in transmembrane domains that take part in the transport mechanism and are essential for function. Site directed mutagenesis of conserved and predicted membrane embedded charged amino acids suggest that DbcA functions as a proton-dependent transporter. Direct measurement of membrane potential shows that B. thailandensis Δ dbcA is partially depolarized suggesting that loss of protonmotive force can lead to alterations in LPS structure and severe colistin sensitivity in this species. [ABSTRACT FROM AUTHOR]

The dissemination of antibiotic resistance genes (ARGs), especially via the plasmid-mediated conjugation, is becoming a pervasive global health threat. This study reported that this issue can be worse by CO 2 , as increased CO 2 was found to facilitate the conjugative transfer of ARGs carried on plasmid RP4 by 2.4–9.0 and 1.3–3.8 fold within and across genera, respectively. Mechanistic studies revealed that CO 2 benefitted the cell-to-cell contact by increasing cell surface hydrophobicity and decreasing cell surface charge, both of which resulted in the reduced intercellular repulsion. Besides, the transcriptional expression of genes responsible for global regulator (korA , korB and trbA), plasmid transfer and replication system (trfAp), and mating pair formation system (traF and traG) were all influenced by CO 2 , facilitating the mobilization and channel transfer of plasmid. Furthermore, the presence of CO 2 induced the release of intracellular Ca2+ and increased the transmembrane potential of recipients, which contributed to the increased proton motive force (PMF), providing more power for DNA uptake. This is the first study addressing the potential risks of increased CO 2 on the propagation of ARGs, which provides a new insight into the concerns of anthropogenic CO 2 emissions and CO 2 storage. • CO 2 promotes the conjugative transfer of ARGs. • Oxidative stress induced by CO 2 promotes the conjugative transfer of ARGs. • Intercellular contact and PMF contribute to the conjugative transfer of ARGs. • Carbon emission causes a new public health issue. [ABSTRACT FROM AUTHOR]

We investigated antibacterial properties of a recently described membrane-active lipopeptide, C 10 OOc 12 O (decanoyl-ornithyl-ornithyl-dodecanoyl-ornithyl-amide) against Gram-positive bacteria (GPB). Minimal inhibitory concentrations (MICs) and kinetics were compared in culture media and plasma. Chemo-sensitization to antibiotics was determined using the checkerboard assay. Membrane damages were estimated using diverse membrane potential sensitive dyes. ATP levels and relevant enzymes activities were measured using commercial bioassay kits. While relatively weakly active in simple culture media, sub-MIC levels (~ten-fold) of C 10 OOc 12 O have significantly improved the antibacterial function of Human plasma. Mechanistic studies indicated that C 10 OOc 12 O-treated bacteria have sustained mild membrane damage(s) in association with rapid (within 2 min) but low (<10%) dissipation of the trans-membrane potential; Intracellular ATP levels were transiently reduced (~20%) whereas extracellular ATP increased only at MIC values; Sub-inhibitory concentrations were sufficient for inhibiting major agr -regulated virulence factors (lipase and α-toxin) and for sensitizing MRSA USA300 to the antibiotic oxacillin to the point of reverting the bacteria status from oxacillin-resistant to oxacillin-sensitive (i.e., oxacillin MIC was reduced from 32 to 0.1 mg/l). These findings argue that by means of mild depolarization, C 10 OOc 12 O affects the quorum sensing regulator in a manner that transiently weakens bacterial defenses, thereby enforcing studies that support the potential usefulness of fighting S. aureus (and possibly other GPB) infections, by targeting its virulence. Unlabelled Image • At sub-MIC, a mildly hydrophobic lipopeptide causes mild membrane depolarization. • The membrane-active lipopeptide sensitizes MRSA USA300 to plasma and antibiotics. • Mild depolarization can inhibit major virulence factors. [ABSTRACT FROM AUTHOR]

Experimental evolution of Escherichia coli K-12 with benzoate, a partial uncoupler of the proton motive force (PMF), selects for mutations that decrease antibiotic resistance. We conducted experimental evolution in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a strong uncoupler. Cultures were serially diluted daily 1:100 in LBK medium containing 20 to 150 μM CCCP buffered at pH 6.5 or at pH 8.0. After 1,000 generations, the populations tolerated up to 150 μM CCCP. Sequenced isolates had mutations in mprA (emrR), which downregulates the EmrABTolC pump that exports CCCP. A mprA::kanR deletion conferred growth at 60 μM CCCP, though not at the higher levels resisted by evolved strains (150 μM). Some mprA mutant strains also had point mutations affecting emrA, but deletion of emrA abolished the CCCP resistance. Thus, CCCP-evolved isolates contained additional adaptations. One isolate lacked emrA or mprA mutations but had mutations in cecR (ybiH), whose product upregulates drug pumps YbhG and YbhFSR, and in gadE, which upregulates the multidrug pump MdtEF. A cecR::kanR deletion conferred partial resistance to CCCP. Other multidrug efflux genes that had mutations included ybhR and acrAB. The acrB isolate was sensitive to the AcrAB substrates chloramphenicol and tetracycline. Other mutant genes in CCCP-evolved strains include rng (RNase G) and cyaA (adenylate cyclase). Overall, experimental evolution revealed a CCCPdependent fitness advantage for mutations increasing CCCP efflux via EmrA and for mutations that may deactivate proton-driven pumps for drugs not present (cecR, gadE, acrAB, and ybhR). These results are consistent with our previous report of drug sensitivity associated with evolved benzoate tolerance. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • IMRG4, a chalcone, displays anti-bacterial activity against multi-drug resistant S. aureus. • IMRG4 is non-toxic and effective in mice models of infection. • At lower concentrations, IMRG4 inhibits NorA efflux pump. • It can rejuvenate the activity of fluoroquinolones against VISA. Abstract Staphylococcus aureus is the leading cause of bacteraemia and the dwindling supply of effective antibacterials has exacerbated the problem of managing infections caused by this bacterium. Isoliquiritigenin (ISL) is a plant flavonoid that displays therapeutic potential against S. aureus. The present study identified a novel mannich base derivatives of ISL, IMRG4, active against Vancomycin intermediate S. aureus (VISA). IMRG4 damages the bacterial membranes causing membrane depolarization and permeabilization, as determined by loss of salt tolerance, flow cytometric analysis, propidium idodie and fluorescent microscopy. It reduces the intracellular invasion of HEK-293 cells by S. aureus and decreases the staphylococcal load in different organs of infected mice models. In addition to anti-staphylococcal activity, IMRG4 inhibits the multidrug efflux pump, NorA, which was determined by molecular docking and EtBr efflux assays. In combination, IMRG4 significantly reduces the MIC of norfloxacin for clinical strains of S. aureus including VISA. Development of resistance against IMRG4 alone and in combination with norfloxacin was low and IMRG4 prolongs the post-antibiotic effect of norfloxacin. These virtues combined with the low toxicity of IMRG4, assessed by MTT assay and haemolysis, makes it an ideal candidate to enter drug development pipeline against S. aureus. [ABSTRACT FROM AUTHOR]

We propose a model whereby microscopic tunnels form in basalt glass in response to a natural proton flux from seawater into the glass. This flux is generated by the alteration of the glass as protons from water replace cations in the glass. In our proton gradient model, cells are gateways through which protons enter and alter the glass and through which cations leave the glass. In the process, tunnels are formed, and cells derive energy from the proton and ion fluxes. Proton flux from seawater into basalt glass would have occurred on Earth as soon as water accumulated on the surface and would have preceded biological redox catalysis. Tunnels in modern basalts are similar to tunnels in Archean basalts, which may be our earliest physical evidence of life. Proton gradients like those described in this paper certainly exist on other planetary bodies where silicate rocks are exposed to acidic to slightly alkaline water. [ABSTRACT FROM AUTHOR]

Hydrogenases are metal-containing biocatalysts that reversibly convert protons and electrons to hydrogen gas. This reaction can contribute in different ways to the generation of the proton motive force (PMF) of a cell. One means of PMF generation involves reduction of protons on the inside of the cytoplasmic membrane, releasing H2 gas, which being without charge is freely diffusible across the cytoplasmic membrane, where it can be re-oxidized to release protons. A second route of PMF generation couples transfer of electrons derived from H2 oxidation to quinone reduction and concomitant proton uptake at the membrane-bound heme cofactor. This redox-loop mechanism, as originally formulated by Mitchell, requires a second, catalytically distinct, enzyme complex to re-oxidize quinol and release the protons outside the cell. A third way of generating PMF is also by electron transfer to quinones but on the outside of the membrane while directly drawing protons through the entire membrane. The cofactor-less membrane subunits involved are proposed to operate by a conformational mechanism (redox-linked proton pump). Finally, PMF can be generated through an electron bifurcation mechanism, whereby an exergonic reaction is tightly coupled with an endergonic reaction. In all cases the protons can be channelled back inside through a F1F0-ATPase to convert the 'energy' stored in the PMF into the universal cellular energy currency, ATP. New and exciting discoveries employing these mechanisms have recently been made on the bioenergetics of hydrogenases, which will be discussed here and placed in the context of their contribution to energy conservation. [ABSTRACT FROM AUTHOR]

The bacterial flagellum and the related virulence-associated injectisome system of pathogenic bacteria utilize a type III secretion system (T3SS) to export substrate proteins across the inner membrane in a proton motive force-dependent manner. The T3SS is composed of an export gate (FliPQR/FlhA/FlhB) located in the flagellar basal body and an associated soluble ATPase complex in the cytoplasm (FliHIJ). Here, we summarise recent insights into the structure, assembly and protein secretion mechanisms of the T3SS with a focus on energy transduction and protein transport across the cytoplasmic membrane.

The bacterial flagellar motor converts the energy of proton flow through the MotA/MotB complex into mechanical works required for motor rotation. The rotational force is generated by electrostatic interactions between the stator protein MotA and the rotor protein FliG. The Arg-90 and Glu-98 from MotA interact with Asp-289 and Arg-281 of FliG, respectively. An increase in the expression level of the wild-type MotA/MotB complex inhibits motility of the gfp-motBfliG(R281V) mutant but not the fliG(R281V) mutant, suggesting that the MotA/GFP-MotB complex cannot work together with wild-type MotA/MotB in the presence of the fliG(R281V) mutation. However, it remains unknown why. Here, we investigated the effect of the GFP fusion to MotB at its N-terminus on the MotA/MotB function. Over-expression of wild-type MotA/MotB significantly reduced the growth rate of the gfp-motBfliG(R281V) mutant. The over-expression of the MotA/GFP-MotB complex caused an excessive proton leakage through its proton channel, thereby inhibiting cell growth. These results suggest that the GFP tag on the MotB N-terminus affects well-regulated proton translocation through the MotA/MotB proton channel. Therefore, we propose that the N-terminal cytoplasmic tail of MotB couples the gating of the proton channel with the MotA–FliG interaction responsible for torque generation.

Soil salinization and water shortage cause ion imbalance and hyperosmoticity in plant cells, adversely impairing photosynthesis efficiency. How soil salinity-induced photosynthetic acclimation influences the cross-tolerance to drought conditions represents a promising research topic. This study was to reveal the photosynthetic mechanism of soil salinity-induced resistance to the subsequent drought stress in tomato leaves through comprehensive photosynthesis-related spectroscopy analysis. We conducted soil salinity pretreatment and subsequent drought stress experiments, including irrigation with 100 mL water, 100 mL 100 mM NaCl solution (NaCl100), 50 mL water, and 50 mL 100 mM NaCl solution (NaCl50) for five days, followed by five-day drought stress. The results showed that soil salinity treatment by NaCl decreased the rate of photosynthetic gas exchange but enhanced CO2 assimilation, along with photosystem II [PS(II)] and photosystem I [PS(I)] activity and photochemical efficiency in tomato plants compared with water pretreatment after subsequent drought stress. NaCl100 and NaCl50 had the capacity to maintain non-photochemical quenching (NPQ) of chlorophyll fluorescence and the cyclic electron (CEF) flow around PSI in tomato leaves after being subjected to subsequent drought stress, thus avoiding the decrease of photosynthetic efficiency under drought conditions. NaCl100 and NaCl50 pretreatment induced a higher proton motive force (pmf) and also alleviated the damage to the thylakoid membrane and adenosine triphosphate (ATP) synthase of tomato leaves caused by subsequent drought stress. Overall, soil salinity treatment could enhance drought resistance in tomato plants by inducing NPQ, maintaining CEF and pmf that tradeoff between photoprotection and photochemistry reactions. This study also provides a photosynthetic perspective for salt and drought cross-tolerance.

Chloroplastic ATP synthase plays a significant role in the regulation of proton motive force (pmf) and proton gradient (ΔpH) across the thylakoid membranes. However, the regulation of chloroplastic ATP synthase at chilling temperature and its role in photoprotection are little known. In our present study, we examined the chlorophyll fluorescence, P700 signal, and electrochromic shift signal at 25°C, and 6°C in tobacco (Nicotiana tabacum L. cv. Samsun). Although photosynthetic electron flow through both PSI and PSII were severely inhibited at 6°C, non-photochemical quenching and P700 oxidation ratio were largely increased. During the photosynthetic induction under high light, the formation of pmf at 6°C was similar to that at 25°C. However, the ΔpH was significantly higher at 6°C, owing to the decreased activity of chloroplastic ATP synthase (gH+). During illumination at 6°C and high light, a high ΔpH made PSI to be highly oxidized, preventing PSI from photoinhibition. These results indicate that the down-regulation of gH+ is critical to the buildup of ΔpH at low temperature, adjusting the redox state of PSI, and thus preventing photodamage to PSI. Our findings highlight the importance of chloroplastic ATP synthase in photoprotection at chilling temperature.

The emergence of multi- and extensively drug resistant tuberculosis worldwide poses a great threat to human health and highlight the need to discover and develop new, effective and inexpensive antituberculosis agents. High-throughput screening assays against well-validated drug targets and structure based drug design have been employed to discover new lead compounds. However, the great majority fail to demonstrate any antimycobacterial activity when tested against Mycobacterium tuberculosis in whole-cell screening assays. This is mainly due to some of the intrinsic properties of the bacilli, such as the extremely low permeability of its cell wall, slow growth, drug resistance, drug tolerance, and persistence. In this sense, understanding the pathways involved in M. tuberculosis drug tolerance, persistence, and pathogenesis, may reveal new approaches for drug development. Moreover, the need for compounds presenting a novel mode of action is of utmost importance due to the emergence of resistance not only to the currently used antituberculosis agents, but also to those in the pipeline. Cheminformatics studies have shown that drugs endowed with antituberculosis activity have the peculiarity of being more lipophilic than many other antibacterials, likely because this leads to improved cell penetration through the extremely waxy mycobacterial cell wall. Moreover, the interaction of the lipophilic moiety with the membrane alters its stability and functional integrity due to the disruption of the proton motive force, resulting in cell death. When a ligand-based medicinal chemistry campaign is ongoing, it is always difficult to predict whether a chemical modification or a functional group would be suitable for improving the activity. Nevertheless, in the “instruction manual” of medicinal chemists, certain functional groups or certain physicochemical characteristics (i.e., high lipophilicity) are considered red flags to look out for in order to safeguard drug-likeness and avoid attritions in the drug discovery process. In this review, we describe how antituberculosis compounds challenge established rules such as the Lipinski's “rule of five” and how medicinal chemistry for antituberculosis compounds must be thought beyond such dogmatic schemes.

Although photoinhibition of photosystem II (PSII) frequently occurs under natural growing conditions, knowledge about the effect of moderate photoinhibition on linear electron flow (LEF) remains controversial. Furthermore, mechanisms underlying the decrease in LEF upon PSII photoinhibition are not well clarified. We examined how selective PSII photoinhibition influenced LEF in the attached leaves of shade-demanding plant Panax notoginseng. After leaves were exposed to a high level of light (2258 μmol photons m-2 s-1) for 30 and 60 min, the maximum quantum yield of PSII (Fv/Fm) decreased by 17 and 23%, respectively, whereas the maximum photo-oxidizable P700 (Pm) remained stable. Therefore, this species displayed selective PSII photodamage under strong illumination. After these treatments, LEF was significantly decreased under all light levels but acidification of the thylakoid lumen changed only slightly. Furthermore, the decrease in LEF under low light was positively correlated with the extent of PSII photoinhibition. Thus, the decline in LEF was not caused by the enhancement of lumenal acidification, but was induced by a decrease in PSII activity. These results indicate that residual PSII activity is an important determinant of LEF in this shade-adapted species, and they provide new insight into how strong illumination affects the growth of shade-demanding plants.

Gram-negative bacteria import essential nutrients such as iron and vitamin B12 through outer membrane receptors. This process utilizes proton motive force harvested by the Ton system made up of three inner membrane proteins, ExbB, ExbD and TonB. ExbB and ExbD form the proton channel that energizes uptake through TonB. Recently, crystal structures suggest that the ExbB pentamer is the scaffold. Here, we present structures of hexameric complexes of ExbB and ExbD revealed by X-ray crystallography and single particle cryo-EM. Image analysis shows that hexameric and pentameric complexes coexist, with the proportion of hexamer increasing with pH. Channel current measurement and 2D crystallography support the existence and transition of the two oligomeric states in membranes. The hexameric complex consists of six ExbB subunits and three ExbD transmembrane helices enclosed within the central channel. We propose models for activation/inactivation associated with hexamer and pentamer formation and utilization of proton motive force.

In higher plants, moderate photoinhibition of photosystem II (PSII) leads to a stimulation of cyclic electron flow (CEF) at low light, which is accompanied by an increase in the P700 oxidation ratio. However, the specific role of CEF stimulation at low light is not well known. Furthermore, the mechanism underlying this increase in P700 oxidation ratio at low light is unclear. To address these questions, intact leaves of the shade-adapted plant Panax notoginseng were treated at 2258 μmol photons m-2 s-1 for 30 min to induce PSII photoinhibition. Before and after this high-light treatment, PSI and PSII activity, the energy quenching in PSII, the redox state of PSI and proton motive force (pmf) at a low light of 54 μmol photons m-2 s-1 were determined at the steady state. After high-light treatment, electron flow through PSII (ETRII) significantly decreased but CEF was remarkably stimulated. The P700 oxidation ratio significantly increased but non-photochemical quenching changed negligibly. Concomitantly, the total pmf decreased significantly and the proton gradient (ΔpH) across the thylakoid membrane remained stable. Furthermore, the P700 oxidation ratio was negatively correlated with the value of ETRII. These results suggest that upon PSII photoinhibition, CEF is stimulated to increase the ATP synthesis, facilitating the rapid repair of photodamaged PSII. The increase in P700 oxidation ratio at low light cannot be explained by the change in pmf, but is primarily controlled by electron transfer from PSII.

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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

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., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhu, Zhou, Kang, Yang, Lin and Fang.)

Chloroplastic ATP synthase plays a significant role in the regulation of proton motive force (pmf) and proton gradient (ΔpH) across the thylakoid membranes. However, the regulation of chloroplastic ATP synthase at chilling temperature and its role in photoprotection are little known. In our present study, we examined the chlorophyll fluorescence, P700 signal, and electrochromic shift signal at 25°C, and 6°C in tobacco (Nicotiana tabacum L. cv. Samsun). Although photosynthetic electron flow through both PSI and PSII were severely inhibited at 6°C, non-photochemical quenching and P700 oxidation ratio were largely increased. During the photosynthetic induction under high light, the formation of pmf at 6°C was similar to that at 25°C. However, the ΔpH was significantly higher at 6°C, owing to the decreased activity of chloroplastic ATP synthase (g H+). During illumination at 6°C and high light, a high ΔpH made PSI to be highly oxidized, preventing PSI from photoinhibition. These results indicate that the down-regulation of g H+ is critical to the buildup of ΔpH at low temperature, adjusting the redox state of PSI, and thus preventing photodamage to PSI. Our findings highlight the importance of chloroplastic ATP synthase in photoprotection at chilling temperature. [ABSTRACT FROM AUTHOR]

In the present study, we studied the role of chloroplastic ATP synthase in photosynthetic regulation during leaf maturation. We measured gas exchange, chlorophyll fluorescence, P700 redox state, and the electrochromic shift signal in mature and immature leaves. Under high light, the immature leaves displayed high levels of non-photochemical quenching (NPQ) and P700 oxidation ratio, and higher values for proton motive force ( pmf ) and proton gradient (ΔpH) across the thylakoid membranes but lower values for the activity of chloroplastic ATP synthase ( g H + ) than the mature leaves. Furthermore, g H + was significantly and positively correlated with CO 2 assimilation rate and linear electron flow (LEF), but negatively correlated with pmf and ΔpH. ΔpH was significantly correlated with LEF and the P700 oxidation ratio. These results indicated that g H + was regulated to match photosynthetic capacity during leaf maturation, and the formation of pmf and ΔpH was predominantly regulated by the alterations in g H + . In the immature leaves, the high steady-state ΔpH increased lumen acidification, which, in turn, stimulated photoprotection for the photosynthetic apparatus via NPQ induction and photosynthetic control. Our results highlighted the importance of chloroplastic ATP synthase in optimizing the trade-off between CO 2 assimilation and photoprotection during leaf maturation. [ABSTRACT FROM AUTHOR]

Succinate-driven reverse electron transport (RET) is one of the main sources of mitochondrial reactive oxygen species (mtROS) in ischemia-reperfusion injury. RET is dependent on mitochondrial membrane potential (Δψm) and transmembrane pH difference (ΔpH), components of the proton motive force (pmf); a decrease in Δψm and/or ΔpH inhibits RET. In this study we aimed to determine which component of the pmf displays the more dominant effect on RET-provoked ROS generation in isolated guinea pig brain and heart mitochondria respiring on succinate or α-glycerophosphate (α-GP). Δψm was detected via safranin fluorescence and a TPP+ electrode, the rate of H2O2 formation was measured by Amplex UltraRed, the intramitochondrial pH (pHin) was assessed via BCECF fluorescence. Ionophores were used to dissect the effects of the two components of pmf. The K+/H+ exchanger, nigericin lowered pHin and ΔpH, followed by a compensatory increase in Δψm that led to an augmented H2O2 production. Valinomycin, a K+ ionophore, at low [K+] increased ΔpH and pHin, decreased Δψm, which resulted in a decline in H2O2 formation. It was concluded that Δψm is dominant over ∆pH in modulating the succinate- and α-GP-evoked RET. The elevation of extramitochondrial pH was accompanied by an enhanced H2O2 release and a decreased ∆pH. This phenomenon reveals that from the pH component not ∆pH, but rather absolute value of pH has higher impact on the rate of mtROS formation. Minor decrease of Δψm might be applied as a therapeutic strategy to attenuate RET-driven ROS generation in ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

In natural variable environments, plants rapidly adjust photosynthesis for optimum balance between photochemistry and photoprotection. These adjustments mainly occur via changes in their proton motive force (pmf). Recent studies based on time resolved analysis of the Electro Chromic Signal (ECS) bandshift of photosynthetic pigments in the model plant Arabidopsis thaliana have suggested an active role of ion fluxes across the thylakoid membranes in the regulation of the pmf. Among the different channels and transporters possibly involved in this phenomenon, we previously identified the TPK3 potassium channel. Plants silenced for TPK3 expression displayed light stress signatures, with reduced Non Photochemical Quenching (NPQ) capacity and sustained anthocyanin accumulation, even at moderate intensities. In this work we re-examined the role of this protein in pmf regulation, starting from the observation that both TPK3 knock-down (TPK3 KD) and WT plants display enhanced anthocyanin accumulation in the light under certain growth conditions, especially in old leaves. We thus compared the pmf features of young “green” (without anthocyanins) and old “red” (with anthocyanins) leaves in both genotypes using a global fit analysis of the ECS. We found that the differences in the ECS profile measured between the two genotypes reflect not only differences in TPK3 expression level, but also a modified photosynthetic activity of stressed red leaves, which are present in a larger amounts in the TPK3 KD plants. [ABSTRACT FROM AUTHOR]

The proton motive force ( pmf ) across the thylakoid membranes plays a key role for photosynthesis in fluctuating light. However, the mechanisms underlying the regulation of pmf in fluctuating light are not well known. In this study, we aimed to identify the roles of chloroplastic ATP synthase and cyclic electron flow (CEF) around photosystem I (PSI) in the regulation of the pmf in fluctuating light. To do this, we measured chlorophyll fluorescence, P700 parameters, and the electrochromic shift signal in the fluctuating light alternating between 918 (high light) and 89 (low light) μmol photons m −2  s −1 every 5 min. We found that the activity of chloroplastic ATP synthase ( g H + ), pmf , CEF activity, non-photochemical quenching (NPQ), and the P700 redox state changed rapidly in fluctuating light. During transition from low to high light, the decreased g H + and the stimulation of CEF both contributed to the rapid formation of pmf , activating NPQ and optimizing the redox state of P700 in PSI. During the low-light phases, g H + rapidly increased and the pmf declined sharply, leading to the relaxation of NPQ and down-regulation of photosynthetic control. These findings indicate that in fluctuating light the g H + and CEF are finely regulated to modulate the pmf formation, avoiding the over-accumulation of reactive intermediates and maximizing energy use efficiency. [ABSTRACT FROM AUTHOR]

Bacterial persisters are phenotypic variants that survive the treatment of lethal doses of growth-targeting antibiotics without mutations. Although the mechanism underlying persister formation has been studied for decades, how the persister phenotype is switched on and protects itself from antibiotics has been elusive. In this study, we focused on the lactate dehydrogenase gene ( ldhA ) that was upregulated in an Escherichia coli persister-enriched population. A survival rate assay using an ldhA -overexpressing strain showed that ldhA expression induced persister formation. To identify ldhA -mediated persister formation at the single-cell level, time-lapse microscopy with a microfluidic device was used. Stochastic ldhA expression was found to induce dormancy and tolerance against high-dose ampicillin treatment (500 μg/ml). To better understand the underlying mechanism, we investigated the relationship between ldhA -mediated persister formation and previously reported persister formation through aerobic metabolism repression. As a result, ldhA expression enhanced the proton motive force (PMF) and ATP synthesis. These findings suggest that ldhA -mediated persister formation pathway is different from previously reported persister formation via repression of aerobic metabolism. [ABSTRACT FROM AUTHOR]

The emergence of multi- and extensively drug resistant tuberculosis worldwide poses a great threat to human health and highlight the need to discover and develop new, effective and inexpensive antituberculosis agents. High-throughput screening assays against well-validated drug targets and structure based drug design have been employed to discover new lead compounds. However, the great majority fail to demonstrate any antimycobacterial activity when tested against Mycobacterium tuberculosis in whole-cell screening assays. This is mainly due to some of the intrinsic properties of the bacilli, such as the extremely low permeability of its cell wall, slow growth, drug resistance, drug tolerance, and persistence. In this sense, understanding the pathways involved in M. tuberculosis drug tolerance, persistence, and pathogenesis, may reveal new approaches for drug development. Moreover, the need for compounds presenting a novel mode of action is of utmost importance due to the emergence of resistance not only to the currently used antituberculosis agents, but also to those in the pipeline. Cheminformatics studies have shown that drugs endowed with antituberculosis activity have the peculiarity of being more lipophilic than many other antibacterials, likely because this leads to improved cell penetration through the extremely waxy mycobacterial cell wall. Moreover, the interaction of the lipophilic moiety with the membrane alters its stability and functional integrity due to the disruption of the proton motive force, resulting in cell death. When a ligand-based medicinal chemistry campaign is ongoing, it is always difficult to predict whether a chemical modification or a functional group would be suitable for improving the activity. Nevertheless, in the “instruction manual” of medicinal chemists, certain functional groups or certain physicochemical characteristics (i.e., high lipophilicity) are considered red flags to look out for in order to safeguard drug-likeness and avoid attritions in the drug discovery process. In this review, we describe how antituberculosis compounds challenge established rules such as the Lipinski's “rule of five” and how medicinal chemistry for antituberculosis compounds must be thought beyond such dogmatic schemes. [ABSTRACT FROM AUTHOR]

The Tat machinery catalyzes the transport of folded proteins across the cytoplasmic membrane in bacteria and the thylakoid membrane in plants. Transport occurs only in the presence of an electric field (Δψ) and/or a pH (ΔpH) gradient, and thus, Tat transport is considered to be dependent on the proton motive force (pmf). This presents a fundamental and major challenge, namely, that the Tat system catalyzes the movement of large folded protein cargos across a membrane without collapse of ion gradients. Current models argue that the active translocon assembles de novo for each cargo transported, thus providing an effective gating mechanism to minimize ion leakage. A limited structural understanding of the intermediates occurring during transport and the role of the pmf in stabilizing and/or driving this process have hindered the development of more detailed models. A fundamental question that remains unanswered is whether the pmf is actually 'consumed', providing an energetic driving force for transport, or alternatively, whether its presence is instead necessary to provide the appropriate environment for the translocon components to become active. Including addressing this issue in greater detail, we explore a series of additional questions that challenge current models, and, hopefully, motivate future work. [ABSTRACT FROM AUTHOR]

Light-emitting diodes (LEDs) are widely used in horticultural facilities in recent years. However, the influence of light quality on photosynthesis still calls for further exploration. This study comprehensively analyzed the influence of different LED lighting (white: W, combination of red and blue 1:1: RB, blue: B, purple: P and red: R) on photosynthetic electron transport in tomato leaves. Plant height under B and P treatments was significantly higher than the other treatments and showed more compact development. Photosynthetic pigment content and net photosynthetic rate (Pn) of B and P treatments were significantly lower than the W treatment. The redox state of photosystem I was repressed by blue and purple light while the intrinsic PSII activity was not altered by the treatments. The ETR (II) and ETR (I) under B and P treatment were lower than the other treatments, while the cyclic electron flow (CEF), the quantum yield of regulated energy dissipation in PSII [Y(NPQ)] and the quantum yield of PSI non-photochemical energy dissipation due to the donor-side limitation [Y(ND)] were higher, indicating that B and P treatment induced non-photochemical quenching of PSII and excitation of PSI’s self-protection mechanisms in tomato plants. Blue and purple light exposure resulted in a higher proton gradient (ΔpH), leading to impaired thylakoid membrane integrity and inhibited the activity of ATP-synthase. In conclusion, the blue and purple light significantly reduced photosynthesis efficiency, enhancing CEF and inducing NPQ for photoprotection of PSII and PSI via lumen acidification. [ABSTRACT FROM AUTHOR]