Identification of distinct physical activity profiles through adolescence: a longitudinal qualitative description study.

Introduction: We aimed to better understand longitudinal physical activity experiences among initially active adolescents and to identify and describe distinct physical activity profiles. Methods: A sample of 23 physically active participants [52% female; mean age = 12.2 (0.6) years at study inception] were selected from the MATCH study to take part in this nested qualitative descriptive study. Participants were interviewed once a year for six years. Following individual-level analyses, profiles were identified based on similarity of longitudinal experiences. Results: Four profiles captured participants' experiences: Independents (those who progressively seek activities that cater to their pursuit of autonomy); Multitaskers (those who participate in many different sports as an integral part of their lifestyle); Specialists (those who are dedicated to becoming the best they can be at one sport); Undecided (those who take part in physical activity to occupy time). Discussion: The exploration of longitudinal physical activity experiences led to the identification of distinct profiles that could be targets for tailored interventions, theory development, and participation models.

Substrate dependence of transport coupling and phenotype of a small multidrug resistance transporter in Pseudomonas aeruginosa.

Small multidrug resistance (SMR) transporters are key players in the defense of multidrug-resistant pathogens to toxins and other homeostasis-perturbing compounds. However, recent evidence demonstrates that EmrE, an SMR from Escherichia coli and a model for understanding transport, can also induce susceptibility to some compounds by drug-gated proton leak. This runs down the ∆pH component of the proton-motive force (PMF), reducing the viability of the affected bacteria. Proton leak may provide an unexplored drug target distinct from the targets of most known antibiotics. Activating proton leak requires an SMR to be merely present, rather than be the primary resistance mechanism, and dissipates the energy source for many other efflux pumps. PAsmr, an EmrE homolog from Pseudomonas aeruginosa, transports many EmrE substrates in cells and purified systems. We hypothesized that PAsmr, like EmrE, may confer susceptibility to some compounds via drug-gated proton leak. Growth assays of E. coli expressing PAsmr displayed substrate-dependent resistance and susceptibility phenotypes, and in vitro solid-supported membrane electrophysiology experiments revealed that PAsmr performs both antiport and substrate-gated proton uniport, demonstrating the same functional promiscuity observed in EmrE. Growth assays of P. aeruginosa strain PA14 demonstrated that PAsmr contributes resistance to some antimicrobial compounds, but no growth defect is observed with susceptibility substrates, suggesting P. aeruginosa can compensate for the proton leak occurring through PAsmr. These phenotypic differences between P. aeruginosa and E. coli advance our understanding of the underlying resistance mechanisms in P. aeruginosa and prompt further investigation into the role that SMRs play in antibiotic resistance in pathogens. IMPORTANCE: Small multidrug resistance (SMR) transporters are a class of efflux pumps found in many pathogens, although their contributions to antibiotic resistance are not fully understood. We hypothesize that these transporters may confer not only resistance but also susceptibility, by dissipating the proton-motive force. This means to use an SMR transporter as a target; it merely needs to be present (as opposed to being the primary resistance mechanism). Here, we test this hypothesis with an SMR transporter found in Pseudomonas aeruginosa and find that it can perform both antiport (conferring resistance) and substrate-gated proton leak. Proton leak is detrimental to growth in Escherichia coli but not P. aeruginosa, suggesting that P. aeruginosa responds differently to or can altogether prevent ∆pH dissipation.

Translation arrest cancellation of VemP, a secretion monitor in Vibrio, is regulated by multiple cis- and trans-factors, including SecY.

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.

Individual work-motive values: determinants and consequences for the appraisal of specific health-related work characteristics.

The objectives of the present study were to determine whether (I) work-motive values influence the appraisal of specific work characteristics of significance for health and function and (II) subject variables impact work-motive values. Two aspects of work-motive values were studied: values that assign importance to pursuing one's personal goals and interests, internally based work-motive values (IntWMVs), and values that assign importance to external factors, externally based work-motive values (ExtWMVs). These aspects of motive values, age, gender, skill level, managerial role, and specific psychosocial work characteristics were analyzed in a cross-sectional sample of 12,994 employees in 101 private and public organizations. Two-year follow-up prospective data from 6,252 employees in 69 organizations elucidated whether associations were stable over time. The results showed that IntWMV influenced reports of levels of control of decisions, empowering leadership, innovative climate, quantitative demands, feedback from work, and self-leadership. ExtWMVs were most consistently associated with role clarity. Skill level and managerial role were associated with reporting higher levels of IntWMVs and lower ExtWMVs. In conclusion, the present data support the assumption that work-motive values influence the appraisal, reporting, and consequently measurements of work characteristics. Managers differ from subordinates in work-motive values and may face challenges in ascertaining and supporting subordinates' needs.

Discovery of antibacterial diketones against gram-positive bacteria.

The rapid rise of antibiotic resistance calls for the discovery of new antibiotics with distinct antibacterial mechanisms. New target mining is indispensable for developing antibiotics. Plant-microbial antibiotics are appealing to underexplored sources due to a dearth of comprehensive understanding of antibacterial activity and the excavation of new targets. Here, a series of phloroglucinol derivatives of plant-root-associated Pseudomonas fluorescens were synthesized for structure-activity relationship analysis. Notably, 2,4-diproylphloroglucinol (DPPG) displayed efficient bactericidal activity against a wide range of gram-positive bacteria. Importantly, mechanistic study exhibits that DPPG binds to type II NADH dehydrogenase (NDH-2), an essential enzyme catalyzing the transfer of electrons from NADH to quinones in the electron transport chain (ETC), blocking electron transfer in S. aureus. Last, we validated the efficacy of DPPG in vivo through animal infection models. Our findings not only provide a distinct antibiotic lead to treat multidrug resistant pathogens but also identify a promising antibacterial target.

The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR): Physiological role, structure and function of a redox-driven, molecular machine.

Many bacterial processes are powered by the sodium motive force (smf) and in case of pathogens, the smf contributes to virulence. Vibrio cholerae, the causative agent of Cholera disease, possesses a Na+-translocating NADH:quinone oxidoreductase (NQR), a six-subunit membrane protein assembly. The 3D structure of NQR revealed the arrangement of the six subunits NqrABCDEF, the position of all redox cofactors (four flavins, two [2Fe-2S] centers) and the binding sites for the substrates NADH (in NqrF) and ubiquinone (in NqrB). Upon oxidation of NADH, electrons are shuttled twice across the membrane, starting with cytoplasmic FADNqrF and electron transfer to the [2Fe2S] clusterNqrF and from there to an intra-membranous [2Fe-2S] clusterNqrDE, periplasmic FMNNqrC, FMNNqrB and from there to riboflavinNqrB. This riboflavin is located at the cytoplasmic entry site of the sodium channel in NqrB, and it donates electrons to ubiquinone-8 positioned at the cytoplasmic side of NqrB. Targeting the substrate binding sites of NQR is a promising strategy to identify new inhibitors against many bacterial pathogens. Detailed structural information on the binding mode of natural inhibitors and small molecules in the active sites of NQR is now available, paving the way for the development of new antibiotics. The NQR shows different conformations as revealed in recent cryo-EM and crystallographic studies combined with spectroscopic analyses. These conformations represent distinct steps in the catalytic cycle. Considering the structural and functional data available, we propose a mechanism of Na+-NQR based on conformational coupling of electron transfer and Na+ translocation reaction steps.

Realization of affiliation goals, interpersonal identity development, and well-being: effects of the implicit affiliation motive among German and Zambian adolescents.

Introduction: Across various cultural contexts, success in goal realization relates to individuals' well-being. Moreover, commitment to and successful pursuance of goals are crucial when searching for a meaningful identity in adolescence. However, individuals' goals differ in how much they match their implicit motive dispositions. We hypothesized that successful pursuance of affiliation goals positively relates to commitment-related dimensions of interpersonal identity development (domain: close friends) that, in turn, predict adolescents' level of well-being. However, we further assumed that the links between goal success and identity commitment are particularly pronounced among adolescents who are characterized by a high implicit affiliation motive. Methods: To scrutinize the generalizability of the assumed relationships, data were assessed among adolescents in individualistic (Germany) and collectivistic (Zambia) cultural contexts. Results: Regardless of adolescents' cultural background, we found that commitment-related dimensions of interpersonal identity development mediate the link between successful attainment of affiliation goals and well-being, particularly among adolescents with a pronounced implicit affiliation motive; that is, the strength of the implicit affiliation motive moderates the association between goal success and identity commitment. Conclusion: We discuss findings concerning universal effects of implicit motives on identity commitment and well-being.

Dark triad traits, study and power motives among medical students-A cross-sectional study at a German medical faculty.

Background: A good physician should be empathic and altruistic, among other qualities. Therefore, the levels of socially undesirable personality traits (Dark Triad) as well as implicit motives of achievement, affiliation and power (Multi-Motive Grid) among medical students as future physicians were analyzed at two different points in their medical training. Methods: This study includes 380 medical students in their first year and 217 in their third year in Germany. All participants completed the Dirty Dozen (DD) and Multi-Motive Grid (MMG) questionnaires at the end of two different classes as paper-and-pencil tests. Relevant differences of the Dark Triad traits between the medical students and reference sample and the two different cohorts, as well as their implicit motives, the associations of Dark Triad traits and MMG components and gender differences of the Dark Triad traits were calculated. Results: There were no significant group differences between year one and year three medical students in narcissism, psychopathy and Machiavellianism (Dark Triad). There were no significant differences between the medical students and reference sample except in psychopathy. Male students scored significantly higher in the Dark Triad traits than female students. In the MMG, first-year students scored significantly higher levels in Fear of Rejection, and lower levels in Hope of Success and Hope of Power than the third-year students. Some associations were found between narcissism and Machiavelliansim with Hope of Success, Hope of Power and Fear of power. Conclusions: Dark Triad traits already appear to exist before the commencement of medical studies. These traits do not differ significantly between the medical students and reference sample; only a few MMG components seem to differ at different stages of their studies. This lack of differences between the medical students and validation cohort indicates that tests based on (undesirable) personality traits are not suitable criteria for the admission selection of medical students.

Nursing students' approaches to learning in selected Malawian nursing schools: a cross-sectional study.

BACKGROUND: Students' approaches to learning are of essence in nursing education. This is because nursing is a profession where classroom learning leads to clinical performance. Although the literature recognizes student's approaches to learning as a significant aspect affecting the quality of students' learning, studies suggest that quality of learning has not been highly achieved in Malawian nursing colleges. Currently, there is a scarcity of empirical data on the learning approaches that Malawian nursing and midwifery students in nursing colleges employ. This study assessed the different approaches to learning among nursing and midwifery students in selected Malawian nursing colleges. METHODS: This was a cross- sectional study that employed quantitative methods. The target population was nursing and midwifery students pursuing nursing diplomas from Nkhoma College of Nursing, Ekwendeni College of Health Sciences and Malawi College of Health Sciences. A total of 251 students were sampled randomly from the three nursing colleges. Data was collected through a self-administered questionnaire (R-SPQ-2 F) by Biggs. The data was analyzed using chi-square and binary logistic regression. In this study Cronbach's alpha was 0.6. RESULTS: Most students had used a deep approach to learning (M = 3.201, SD = 0.623) than the surface approach (M = 2.757, SD = 0.732). Being in the age category of 16-20 had more likelihood of adopting a surface approach to learning compared to other age categories (X2 = 7.669, DF 2, P = .02). Students from Malawi College of Health Sciences were more likely to adopt a surface approach to learning compared to students from Nkhoma Nursing College and Ekwendeni College of Health Sciences (X2 = 12.388, df = 2, P = .002). CONCLUSION: A deep approach to learning emerged as the most preferred approach to learning which indirectly implies that most students attain meaningful learning. Age and environment are some of the key determinants associated with different learning approaches. More attention should be given to younger students during teaching and learning to promote deep learning.

Drinking contexts, coping motive, simultaneous cannabis use, and high-intensity drinking among adults in the United States.

AIMS: High-intensity drinking (HID), extreme drinking considerably above the level of heavy episodic drinking (HED), is associated with long-term health and social consequences. There is limited understanding of HID beyond young adulthood. This study aims to identify concurrent risk factors for HID, comparing age differences among all adults. METHODS: Multinomial logistic and linear regression modeling was performed using a nationally-representative sample of adults (analytic n = 7956) from the 2015 and 2020 National Alcohol Surveys. The outcomes were any HID of 8-11 drinks and 12+ drinks for men, and 8+ drinks for women, and corresponding frequencies. Concurrent risk factors included coping motive, sensation seeking, simultaneous use of alcohol and cannabis (SAC), and drinking at a bar or party. Analyses were stratified by age (18-29 vs. older) and sex. RESULTS: For younger men, sensation-seeking was significantly associated with HID (vs. no HED) at both levels and frequency of HID 8-11 drinks, while drinking to cope was only significant for 12+ drinks. For older men, drinking to cope was a consistent predictor for both HID level and its frequency, but sensation-seeking was not significant. Both coping and sensation-seeking were significantly associated with any HID for all women, while coping was significant for HID frequency for younger women. Frequent drinking at bars and parties were associated with greater odds of HID for all adults. With HED as referent, similar patterns of (though fewer significant) associations were observed. CONCLUSIONS: Younger and older adults share similar risk factors for HID, with coping more consistent for older men.

Diet quality and associations with motivation and ability to consume a healthy diet among adolescents from urban low-income households in Bangladesh.

In low- and middle-income countries, particularly in urban areas, adolescent diets consist mainly of energy-dense and nutrient-poor foods, putting them at risk of malnutrition and non-communicable diseases (NCD). In Bangladesh, little is known about the diet quality of adolescents, their food choices and the drivers of such choices. This study assessed motivations and ability to consume a healthy diet among adolescent girls and boys from low-income urban families and how these drivers were associated with dietary diversity and diet quality. A cross-sectional survey was conducted among 299 adolescents (15-19 years) from low-income households in Dhaka city during September-October 2020. The Diet Quality Questionnaire was used to collect non-quantitative food intake in the previous day or night to calculate diet quality indicators of food group diversity score, % of adolescents achieving minimum dietary diversity, NCD-Protect and NCD-Risk and the Global Dietary Recommendations score. Motivation was measured by 11 food choice motives. Ability was measured by belief in own ability to engage in healthy eating behaviors (self-efficacy). Adolescent diets showed a mean food group diversity of 4.9 out of 10, with 60% of adolescents achieving minimum dietary diversity, but lacked health-promoting foods (average of 2.7 out of 9 food groups) yet included few foods to avoid and limit (1.6 out of 9). Adolescents valued food choice motive 'safety' the most, followed by 'health', 'taste', 'price', 'convenience' and 'local or seasonal'. A higher motivation to consume 'local or seasonal' and a lower motivation driven by 'price', and a higher perceived self-efficacy were associated with better diet quality. Future interventions should address self-efficacy, concerns about food price and increase local and seasonal foods availability in the urban poor food environment of Dhaka to improve overall diet quality.

ATP regeneration by ATPases for in vitro biotransformation.

Adenosine triphosphate (ATP) regeneration is a significant step in both living cells and in vitro biotransformation (ivBT). 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.

Different self-damaging behaviours, similar motives? Testing measurement invariance of motives for nonsuicidal self-injury, disordered eating and substance misuse.

OBJECTIVES: Theory and research suggest that distinct self-damaging behaviours (SDBs; e.g., nonsuicidal self-injury [NSSI], restrictive eating, binge eating, drug misuse, alcohol misuse) share similar motives. However, few studies have used a common self-report inventory to investigate the shared relevance and relative salience of motives for SDBs. Accordingly, the present study: (1) examined whether self-report scales assessing intrapersonal motives (i.e., relieving negative emotions, enhancing positive emotions, punishing oneself) and interpersonal motives (i.e., bonding with others, conforming with others, communicating distress, communicating strength, reducing demands) have invariant factor structures across SDBs; and (2) compared the salience of these motives across SDBs. METHODS: 1018 adults (54.6% men, Mage = 35.41 years) with a history of SDBs were allocated to the following groups: NSSI (n = 213), restrictive eating (n = 200), binge eating (n = 200), drug misuse (n = 200) or alcohol misuse (n = 205). Participants reported on their motives for engaging in their allocated SDB. Measurement invariance analyses compared the factor structures and latent means of the motive scales across SDBs. RESULTS: The motive scales had comparable factor structures across SDBs. Intrapersonal motives were most strongly endorsed for NSSI and drug misuse. Interpersonal motives were most strongly endorsed for drug and alcohol misuse. All motives were least salient to restrictive eating. CONCLUSIONS: Results suggest that common motives underlie distinct SDBs and that they can be adequately assessed using a single self-report inventory. However, certain motives are more relevant to some SDBs than others, with restrictive eating being the most motivationally distinct SDB. This knowledge can inform transdiagnostic models and interventions for SDBs.

Plasma membrane H+-ATPases in mineral nutrition and crop improvement.

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.

Enhancing Escherichia coli abiotic stress resistance through ornithine lipid formation.

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.

Proton conductance and regulation of proton/potassium fluxes in Escherichia coli FhlA-lacking cells during fermentation of mixed carbon sources.

Escherichia coli uptake potassium ions with the coupling of proton efflux and energy utilization via proton FOF1-ATPase. In this study contribution of formate hydrogen lyase (FHL) complexes in the proton/potassium fluxes and the formation of proton conductance (CMH+) were investigated using fhlA mutant strain. The proton flux rate (JH+) decreased in fhlA by âˆ¼ 25 % and ∼70 % during the utilization of glucose and glycerol, respectively, at 20 h suggesting H+ transport via or through FHL complexes. The decrease in JK+ in fhlA by ∼40 % proposed the interaction between FHL and Trk secondary transport system during mixed carbon fermentation. Moreover, the usage of N,N'-dicyclohexylcarbodiimide (DCCD) demonstrated the mediation of FOF1-ATPase in this interaction. CMH+ was 13.4 nmol min-1 mV-1 in WT at 20 h, which decreased by 20 % in fhlA. Taken together, FHL complexes have a significant contribution to the modulation of H+/K+ fluxes and the CMH + for efficient energy transduction and regulation of the proton motive force during mixed carbon sources fermentation.

Macromolecular condensation organizes nucleolar sub-phases to set up a pH gradient.

Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass. In vitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs in vitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates.

Regulation of metabolism and proton motive force generation during mixed carbon fermentation by an Escherichia coli strain lacking the FOF1-ATPase.

Proton FOF1-ATPase is the key enzyme in E. coli under fermentative conditions. In this study the role of E. coli proton ATPase in the µ and formation of metabolic pathways during the fermentation of mixture of glucose, glycerol and formate using the DK8 (lacking FOF1) mutant strain was investigated. It was shown that the contribution of FOF1-ATPase in the specific growth rate was ∼45 %. Formate was not taken up in the DK8 strain during the initial hours of the growth. The utilization rates of glucose and glycerol were unchanged in DK8, however, the production of succinate, lactate and ethanol was decreased causing a reduction of the redox state up to -450 mV. Moreover, the contribution of FOF1-ATPase in the interplay between H+ and H2 cycles was described depending on the bacterial growth phase and main utilizing substrate. Besides, the H2 production rate in the DK8 strain was decreased by ∼60 % at 20 h and was absent at 72 h. Δp was decreased from -157 ± 4.8 mV to -140 ± 4.2 mV at 20 h and from -195 ± 5.9 mV to -148 ± 4.4 mV at 72 h, compared to WT. Taken together it can be concluded that during fermentation of mixed carbon sources metabolic cross talk between FOF1-ATPase-TrkA-Hyd-Fdh-H is taking place for maintaining the cell energy balance via regulation proton motive force.

Discovery and structural characterization of the D-box, a conserved TonB motif that couples an inner-membrane motor to outer-membrane transport.

Gram-negative bacteria use TonB-dependent transport to take up nutrients from the external environment, employing the Ton complex to import a variety of nutrients that are either scarce or too large to cross the outer membrane unaided. The Ton complex contains an inner-membrane motor (ExbBD) that generates force, as well as nutrient-specific transport proteins on the outer membrane. These two components are coupled by TonB, which transmits the force from the inner to the outer membrane. TonB contains an N-terminus anchored in the inner membrane, a C-terminal domain that binds the outer-membrane transporter, and a proline-rich linker connecting the two. While much is known about the interaction between TonB and outer-membrane transporters, the critical interface between TonB and ExbBD is less well understood. Here, we identify a conserved motif within TonB that we term the D-box, which serves as an attachment point for ExbD. We characterize the interaction between ExbD and the D-box both functionally and structurally, showing that a homodimer of ExbD captures one copy of the D-box peptide via beta-strand recruitment. We additionally show that both the D-box motif and ExbD are conserved in a range of Gram-negative bacteria, including members of the ESKAPE group of pathogens. The ExbD:D-box interaction is likely to represent an important aspect of force transduction between the inner and outer membranes. Given that TonB-dependent transport is an important contributor to virulence, this interaction is an intriguing potential target for novel antibacterial therapies.

Fitness trade-offs of multidrug efflux pumps in Escherichia coli K-12 in acid or base, and with aromatic phytochemicals.

Multidrug efflux pumps are the frontline defense mechanisms of Gram-negative bacteria, yet little is known of their relative fitness trade-offs under gut conditions such as low pH and the presence of antimicrobial food molecules. Low pH contributes to the proton-motive force (PMF) that drives most efflux pumps. We show how the PMF-dependent pumps AcrAB-TolC, MdtEF-TolC, and EmrAB-TolC undergo selection at low pH and in the presence of membrane-permeant phytochemicals. Competition assays were performed by flow cytometry of co-cultured Escherichia coli K-12 strains possessing or lacking a given pump complex. All three pumps showed negative selection under conditions that deplete PMF (pH 5.5 with carbonyl cyanide 3-chlorophenylhydrazone or at pH 8.0). At pH 5.5, selection against AcrAB-TolC was increased by aromatic acids, alcohols, and related phytochemicals such as methyl salicylate. The degree of fitness cost for AcrA was correlated with the phytochemical's lipophilicity (logP). Methyl salicylate and salicylamide selected strongly against AcrA, without genetic induction of drug resistance regulons. MdtEF-TolC and EmrAB-TolC each had a fitness cost at pH 5.5, but salicylate or benzoate made the fitness contribution positive. Pump fitness effects were not explained by gene expression (measured by digital PCR). Between pH 5.5 and 8.0, acrA and emrA were upregulated in the log phase, whereas mdtE expression was upregulated in the transition-to-stationary phase and at pH 5.5 in the log phase. Methyl salicylate did not affect pump gene expression. Our results suggest that lipophilic non-acidic molecules select against a major efflux pump without inducing antibiotic resistance regulons.IMPORTANCEFor drugs that are administered orally, we need to understand how ingested phytochemicals modulate drug resistance in our gut microbiome. Bacteria maintain low-level resistance by proton-motive force (PMF)-driven pumps that efflux many different antibiotics and cell waste products. These pumps play a key role in bacterial defense by conferring resistance to antimicrobial agents at first exposure while providing time for a pathogen to evolve resistance to higher levels of the antibiotic exposed. Nevertheless, efflux pumps confer energetic costs due to gene expression and pump energy expense. The bacterial PMF includes the transmembrane pH difference (ΔpH), which may be depleted by permeant acids and membrane disruptors. Understanding the fitness costs of efflux pumps may enable us to develop resistance breakers, that is, molecules that work together with antibiotics to potentiate their effect. Non-acidic aromatic molecules have the advantage that they avoid the Mar-dependent induction of regulons conferring other forms of drug resistance. We show that different pumps have distinct selection criteria, and we identified non-acidic aromatic molecules as promising candidates for drug resistance breakers.