The EmhABC efflux pump decreases the efficiency of phenanthrene biodegradation by Pseudomonas fluorescens strain LP6a

Pseudomonas fluorescens strain LP6a, designated here as strain WEN (wild-type PAH catabolism, efflux positive), utilizes the polycyclic aromatic hydrocarbon phenanthrene as a carbon source but also extrudes it into the extracellular medium using the efflux pump EmhABC. Because phenanthrene is considered a nontoxic carbon source for P. fluorescens WEP, its energy-dependent efflux seems counter-productive. We hypothesized that the efflux of phenanthrene would decrease the efficiency of its biodegradation. Indeed, an emhB disruptant strain, wild-type PAH catabolism, efflux negative (WEN), biodegraded 44% more phenanthrene than its parent strain WEP during a 6-day incubation. To determine whether efflux affected the degree of oxidation of phenanthrene, we quantified the conversion of ¹⁴C-phenanthrene to radiolabeled polar metabolites and ¹⁴CO₂. The emhB ^? WEN strain produced approximately twice as much ¹⁴CO₂ and radiolabeled water-soluble metabolites as the WEP strain. In contrast, the mineralization of ¹⁴C-glucose, which is not a known EmhB efflux substrate, was equivalent in both strains. An early open-ring metabolite of phenanthrene, trans-4-(1-hydroxynaphth-2-yl)-2-oxo-3-butenoic acid, also was found to be a substrate of the EmhABC pump and accumulated in the supernatant of WEP but not WEN cultures. The analogous open-ring metabolite of dibenzothiophene, a heterocyclic analog of phenanthrene, was extruded by EmhABC plus a putative alternative efflux pump, whereas the end product 3-hydroxy-2-formylbenzothiophene was not actively extruded from either WEP or WEN cells. These results indicate that the active efflux of phenanthrene and its early metabolite(s) decreases the efficiency of phenanthrene degradation by the WEP strain. This activity has implications for the bioremediation and biocatalytic transformation of polycyclic aromatic hydrocarbons and heterocycles.     

Na+/solute symport in membrane vesicles from Bacillus alcalophilus

The characteristics of α-aminoisobutyric acid translocation were examined in membrane vesicles from obligately alkalophilic Bacillus alcalophilus and its non-alkalophilic mutant derivative, KM23. Vesicles from both strains exhibited α-aminoisobutyric acid uptake upon energization with ascorbate and N,N,N′,N′-tetramethyl-p-phenylenediamine. The presence of Na⁺ caused a pronounced reduction in the Km for α-aminoisobutyric acid in wild-type but not KM23 vesicles; the maximum velocity (V) was unaffected in vesicles from both strains. Passive efflux and exchange of α-aminoisobutyric acid from wild-type vesicles were Na⁺-dependent and occurred at comparable rates (with efflux slightly faster than exchange). This latter observation suggests that the return of the unloaded carrier to the inner surface is not rate-limiting for efflux. The rates of α-aminoisobutyric acid efflux and exchange were also comparable in KM23 vesicles, but were Na⁺-independent. Furthermore, in vesicles from the two strains, both efflux and exchange were inhibited by generation of a transmembrane electrochemical gradient of protons, outside positive. This suggests that the ternary complex between solute, carrier, and coupling ion bears a positive charge in both strains even though the coupling ion is changed. Evidence from experiments with an alkalophilic strain that was deficient in l-methionine transport indicated that the porters, i.e., the solute-translocating elements, used by non-alkalophilic mutants are not genetically distinct from those used by the alkalophilic parent; that is, the change in coupling ion cannot be explained by the expression of a completely new set of Na⁺-independent, H⁺-coupled porters upon mutation of B. alcalophilus to non-alkalophily.     

Permeability changes in membranes of Neurospora crassa after freezing and thawing.

Conidia of Neurospora crassa which are in different physiological states show different rates of survival after freezing and thawing. [¹⁴C]adenine uptake by frozen and thawed conidia in different physiological states show a correlation with their survival. The uptake method was extended to study the survival of mycelium in log phase and stationary phase. From the uptake data it appears that log phase mycelium is extremely sensitive to all rates of freezing and thawing studied, while the stationary phase mycelium showed slight tolerance to freezing, if freezing was done at a slow rate. A study of the efflux of labeled compounds from the conidia in various physiological states or from the mycelia after freezing and thawing showed that, although efflux followed the same general trend as survival in conidia, it did not relate to the survival in mycelium, suggesting that the death of conidia or mycelium in the freeze-thaw treatments is not due to efflux of compounds.     

Inhibition of efflux transporter-mediated fungicide resistance in Pyrenophora tritici-repentis by a derivative of 4'-hydroxyflavone and enhancement of fungicide activity

Populations of the causal agent of wheat tan spot, Pyrenophora tritici-repentis, that are collected from fields frequently treated with reduced fungicide concentrations have reduced sensitivity to strobilurin fungicides and azole fungicides (C14-demethylase inhibitors). Energy-dependent efflux transporter activity can be induced under field conditions and after in vitro application of sublethal amounts of fungicides. Efflux transporters can mediate cross-resistance to a number of fungicides that belong to different chemical classes and have different modes of action. Resistant isolates can grow on substrata amended with fungicides and can infect plants treated with fungicides at levels above recommended field concentrations. We identified the hydroxyflavone derivative 2-(4-ethoxy-phenyl)-chromen-4-one as a potent inhibitor of energy-dependent fungicide efflux transporters in P. tritici-repentis. Application of this compound in combination with fungicides shifted fungicide-resistant P. tritici-repentis isolates back to normal sensitivity levels and prevented infection of wheat leaves. These results highlight the role of energy-dependent efflux transporters in fungicide resistance and could enable a novel disease management strategy based on the inhibition of fungicide efflux to be developed.     

The rising threat of fungicide resistance in plant pathogenic fungi: Botrytis as a case study

The introduction of site-specific fungicides almost 50 years ago has revolutionized chemical plant protection, providing highly efficient, low toxicity compounds for control of fungal diseases. However, it was soon discovered that plant pathogenic fungi can adapt to fungicide treatments by mutations leading to resistance and loss of fungicide efficacy. The grey mould fungus Botrytis cinerea, a major cause of pre- and post-harvest losses in fruit and vegetable production, is notorious as a ‘high risk’ organism for rapid resistance development. In this review, the mechanisms and the history of fungicide resistance in Botrytis are outlined. The introduction of new fungicide classes for grey mould control was always followed by the appearance of resistance in field populations. In addition to target site resistance, B. cinerea has also developed a resistance mechanism based on drug efflux transport. Excessive spraying programmes have resulted in the selection of multiresistant strains in several countries, in particular in strawberry fields. The rapid erosion of fungicide activity against these strains represents a major challenge for the future of fungicides against Botrytis. To maintain adequate protection of intensive cultures against grey mould, strict implementation of resistance management measures are required as well as alternative strategies with non-chemical products.     

Expression of a plasmid-encoded gene for cadmium resistance of Pseudomonas putida GAM-1 in Escherichia coli

A Cd²⁺-resistant Escherichia coli C600 transformant harboring pGU100, which was derived from Cd²⁺-resistant Pseudomonas putida GAM-1, was able to grow in concentrations of CdCl₂ as high as 3.5 mM, whereas E. coli C600 could not grow in the presence of 1.5 mM CdCl₂. E. coli C600 (pGU100) possesses a Cd²⁺ efflux system. This efflux system was inhibited by 100 μM dicyclohexylcarbodiimide, indicating that the system seems to be energy-dependent. Further studies revealed that the Cd²⁺ efflux system of E. coli C600 (pGU100) can operate under proliferous conditions, but not under nonproliferous conditions.     

Enhancement of α-methylglucoside efflux by respiration in respiratory mutants of Escherichia coli K-12

The rate of α-methylglucoside efflux from wild-type cells of Escherichia coli K-12 is enhanced by different substrates, as long as they are readily respired. A similar enhancement takes place in strains with impaired oxidative phosphorylation (unc mutants), regardless of their being able (strains AN120, N₁₄₄, and AN382) or unable (strain NR70) to energize the membrane through respiratory electron flow. The uncouplers carbonylcyanide-m-chlorophenylhydrazone and tetrachlorosalicylanilide do not diminish the efflux acceleration in wild-type strains or unc mutants. However, the stimulation of α-methylglucoside efflux does not occur in the mutant AN59 which cannot perform a normal respiratory electron transport due to a defective synthesis of ubiquinone. The failure to stimulate the efflux is observed with succinate, which is a typical substrate of respiration, as well as with substrates which can yield ATP both at respiratory and substrate levels such as gluconate or glycerol. Moreover, potassium cyanide nullifies the acceleration of α-methylglucoside efflux caused in any type of strain and by any substrate. These results show that neither ATP nor an energized state of the membrane appears to be needed for respiration to accelerate α-methylglucoside release from E. coli cells, and question the existence of any energy-requiring reaction for αMG exit, previously proposed by other authors.     

Some growth peculiarities and membrane characteristics of probiotic strains of Escherichia coli

It is found that the probiotic strains of Escherichia coli, G35 N 59 isolated from “Okarin” and from “ASAP” drug formulas, and the commensal strain 5–1 isolated from healthy human intestine, manifest higher specific growth rates, lower acidifying capacity during glucose fermentation in the external medium, and lower rates of decrease in redox potential in comparison with the wild-type strain (MC4100). At the same time, despite similar values of their membrane potentials, these bacteria differ essentially in total and N,N′-dicyclohexylcarbodiimide-sensitive rates of energy-dependent transmembrane H⁺ and K⁺ transport and display a low level of H₂ production. It is suggested that the difference between their membrane characteristics reflects changes in the activity of proton-translocating F₀F₁-ATPase and is crucial for bacterial growth and probiotic activity of E. coli.     

Energy-dependent sodium efflux and sodium-dependent α-aminoisobutyrate transport in purple photosynthetic bacteria

Uptake of alanine and its nonmetabolizable analog α-aminoisobutyric acid (AIB) by the photosynthetic purple sulfur bacterium Chromatium vinosum is stimulated fivefold by Na⁺. Neither Li⁺ nor K⁺ have any stimulatory effect. AIB uptake can be supported by a Na⁺ gradient in the absence of other energy sources. AIB uptake is also accompanied by Na⁺ uptake. These results suggest that AIB is taken up by C. vinosum via a sodium symport. Cells of C. vinosum and the purple nonsulfur bacterium Rhodospirillum rubrum show energy-dependent Na⁺ efflux and Na⁺ uptake can be demonstrated with chromatophores prepared from these bacteria.     

Efflux of potassium ions from cells and spheroplasts of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> yeast treated with silver and copper ions

Silver ions induce the efflux of potassium from cells of the yeast Saccharomyces cerevisiae but have no such effect on spheroplasts. Copper ions and the natural fungicide 2-O-3-hydroxyhexanoyl-β-D-glucopyranosyl-(1→4)-(6-O-acetyl-β-D-glucopyranosyl-(1→16)-2,15,16-trihydroxyhexadecanoic acid) induce the efflux of potassium ions from both cells and spheroplasts of S. cerevisiae. Silver and copper ions inhibit the activity of the plasma membrane H⁺-ATPase during the treatment of both cells and spheroplasts. It is supposed that the inability of silver ions to stimulate potassium efflux from spheroplasts results from damage to some components of K⁺ transport systems during preparation of spheroplasts.     

Discovery of multidrug efflux pump inhibitors with a novel chemical scaffold

Multidrug efflux is a major contributor to antibiotic resistance in Gram-negative bacterial pathogens. Inhibition of multidrug efflux pumps is a promising approach for reviving the efficacy of existing antibiotics. Previously, inhibitors targeting both the efflux transporter AcrB and the membrane fusion protein AcrA in the Escherichia coli AcrAB-TolC efflux pump were identified. Here we use existing physicochemical property guidelines to generate a filtered library of compounds for computational docking. We then experimentally test the top candidate coumpounds using in vitro binding assays and in vivo potentiation assays in bacterial strains with controllable permeability barriers. We thus identify a new class of inhibitors of E. coli AcrAB-TolC. Six molecules with a shared scaffold were found to potentiate the antimicrobial activity of erythromycin and novobiocin in hyperporinated E. coli cells. Importantly, these six molecules were also active in wild-type strains of both Acinetobacter baumannii and Klebsiella pneumoniae, potentiating the activity of erythromycin and novobiocin up to 8-fold.     

Counterflow efflux of thiamin in Escherichia coli

A resting cell of Escherichia coli lacking thiamin kinase incorporated external thiamin with an energy-dependent counterflow efflux (C-efflux). This C-efflux could be separated from an energy-dependent exit by a selective inhibition of exit by $\text{2 · 10}{}^{\mathrm{?2}}\text{M}$ NaN₃. The extracellular thiamin could be replaced by thiamin diphosphate, resulting in the same rate of C-efflux, but the rate of C-efflux of intracellular thiamin diphosphate against the external thiamin was markedly low. This low rate of C-efflux of thiamin diphosphate could explain the higher accumulation of the compound than that of free thiamin in the thiamin-kinase-defective mutant as well as in its wild-type parent. Basic characteristics of free thiamin uptake and exit in E. coli W mutant were compared with those reported in K 12 mutant: a marked difference existed in the rate of exit. The low rate of exit in E. coli W 70-23-102 was inferred as the reason for the absence of an overshoot phenomenon of thiamin uptake in this strain.     

The EmhABC efflux pump in Pseudomonas fluorescens LP6a is involved in naphthalene tolerance but not efflux

The EmhABC efflux pump in Pseudomonas fluorescens LP6a effluxes polycyclic aromatic hydrocarbons (PAHs) such as phenanthrene and anthracene but not naphthalene. We previously showed that the presence of EmhABC decreased the efficiency of phenanthrene biodegradation. In this study, we determined whether P. fluorescens LP6a tolerance to naphthalene is a function of the EmhABC efflux pump and how its presence affects the efficiency of naphthalene biodegradation. Growth, membrane fatty acid (FA) composition, and cell morphology showed that 5-mmol?L^?1 naphthalene is inhibitory to P. fluorescens LP6a strains. The deleterious effect of naphthalene is suppressed in the presence of EmhABC, which suggests that, although naphthalene is not effluxed by EmhABC, this efflux pump is involved in tolerance of naphthalene toxicity. LP6a mutants lacking the EmhB efflux pump were unable to convert cis-unsaturated FAs to cyclopropane FAs, indicating that naphthalene interferes with the formation of cyclopropane FAs and supporting the proposal that EmhABC is involved in FA turnover in P. fluorescens LP6a strains. The EmhABC efflux pump increases the efficiency of naphthalene metabolism in strain LP6a, which may make naphthalene efflux unnecessary. Thus, the activity of hydrocarbon efflux pumps may be an important factor to consider when selecting bacterial strains for bioremediation or biocatalysis of PAHs.     

Characterization of amino Acid efflux from isolated soybean cells

Cells from reproductive soybean (Glycine max [L.] Merr.) plants were isolated using a mechanical-enzymic technique that produced a high yield of uniform, physiologically active cells. Cells were incubated in a pH 6.0 buffered solution and subjected to various treatments in order to determine the nature of net amino acid efflux. Total net amino acid (ninhydrinreactive substances) efflux was not affected by the following conditions: (a) darkness, (b) aeration, (c) K⁺ concentrations of 0.1, 1.0, 10, or 100 millimolar and (d) pH 4, 5, 6, 7, or 8. The Q₁₀ for net amino acid efflux between 10°C and 30°C was 1.6. Thus, it seems that net amino acid efflux requires neither current photosynthetic energy nor a pH/ion concentration gradient. Amino acid analyses of the intra-and extracellular fractions over time showed that each amino acid was exported linearly for at least 210 minutes, but that export rate was not necessarily related to internal amino acid pools. Amino acids that were exported fastest were alanine, lysine, leucine, and glycine. Addition of the inhibitor p-chloromercuriphenyl sulfonic acid, 3(3,4-dichlorophenyl)-1,1-dimethylurea, or carbonylcyanide p-trifluoromethoxyphenylhydrazone increased the rate of total amino acid efflux but had specific effects on the efflux of certain amino acids. For example, p-chloromercuriphenyl sulfonic acid greatly enhanced efflux of γ-aminobutyric acid, which is not normally exported rapidly even though a high concentration normally exists within cells. The data suggest that net amino acid efflux is a selective diffusional process. Because net efflux is the result of simultaneous efflux and influx, we propose that efflux is a facilitated diffusion process whereas influx involves energy-dependent carrier proteins.     

Novel sulfonamides against Botrytis cinerea with no positive cross-resistance to commercial fungicides: Design,synthesis and SAR study

Thirty-four novel compounds were synthesized using chesulfamide (N-(2-trifluoromethyl-4-chlorophenyl)-2-oxocyclohexyl sulfonamide), a high-profile fungicide, as the lead compound, and their structures were characterized by ¹H NMR, ¹³C NMR, MS and elemental analysis. Additionally, the structure of (1S,2R)-2-((3-bromophenethyl)amino)-N-(4-chloro-2-trifluoromethylphenyl)cyclohexane-1-sulfonamide (IV-9) was confirmed by X-ray single crystal diffraction. The mycelium inhibition tests, spore germination inhibition tests, tomato pot tests and field trials were performed against strains of B. cinerea. Bioassay results showed that most of target compounds had good fungicidal activity against B. cinerea, in particular, IV-9 exhibited similar or superior effects to procymidone, boscalid and pyrisoxazole in all in vitro and in vivo tests. Moreover, there was no positive cross-resistance found between the compound IV-9 and eight commercial fungicides (azoxystrobin, boscalid, chlorothalonil, diethofencarb, fludioxonil, procymidone, pyrimethanil and pyrisoxazole) in the cross-resistance validation test performed by an innovative method.     

The catalytic domain of Escherichia coli K-12 adenylate cyclase as revealed by deletion analysis of the cya gene

In Escherichia coli, adenylate cyclase activity is regulated by phosphorylated EnzymeIIA^Glc, a component of the phosphotransferase system for glucose transport. In strains deficient in EnzymeIIA^Glc, CAMP levels are very low. Adenylate cyclase containing the D414N substitution produces a low level of cAMP and it has been proposed that D414 may be involved in the process leading to activation by EnzymeIIA^Glc. In this work, spontaneous secondary mutants producing large amounts of cAMP in strains deficient in EnzymeIIA^Glc were obtained. The secondary mutations were all deletions located in the cya gene around the D414N mutation, generating adenylate cyclases truncated at the carboxyl end. Among them, a 48 kDa protein (half the size of wild-type adenylate cyclase) was shown to produce ten times more cAMP than wild-type adenylate cyclase in strains deficient in EnzymeIIA^Glc. In addition, this protein was not regulated in strains grown on glucose and diauxic growth was abolished. This allowed the definition of a catalytic domain that is not regulated by the phosphotransferase system and produces levels of cAMP similar to that of regulated wild-type adenylate cyclase in wild-type strains grown in the absence of glucose. Further analysis allowed the characterization of the COOH-terminal regulatory domain, which is proposed to be inhibitory to the activity of the catalytic domain.     

Control of the general amino acid permease of Penicillium chrysogenum by transinhibition and turnover

When nitrogen-starved mycelium of Penicillium chrysogenum is incubated with relatively high concentrations of labeled hydrophobic amino acids, influx is followed by efflux of the corresponding labeled α-ketoacid. In spite of the efflux, further transport activity is suppressed. Cell-free extracts contain a transaminase that accepts all those amino acids exhibiting α-ketoacid efflux. Transaminase activity is constitutive but is induced to a 2- to 3-fold higher level during a 2-hr preincubation period with a hydrophobic amino acid. Cycloheximide prevents efflux and also the induction of the transaminase. Cycloheximide itself stimulates a partial decay in transport activity but mycelium preincubated with l-leucine and cycloheximide together retain a greater fraction of the original transport activity than mycelium preincubated with l-leucine alone. The results suggest that transport is regulated partially by transinhibition but a significant part of the substrate-induced decay of transport activity is caused by either (a) the degradation of a permease component (perhaps facilitated by transinhibition), or (b) the induction by the substrate of a regulator protein (perhaps the transaminase).The uptake of labeled substrates by nutrient sufficient mycelium correlates well with lipid solubility of the substrates. This suggests that the nonsaturable uptake observed in these mycelia results from free diffusion of the uncharged species.     

Arf-like proteins (Arl1 and Arl2) are involved in mitochondrial homeostasis in Mucor circinelloides

Mucor circinelloides is an opportunistic dimorphic pathogen, with the dimorphic process controlled in parts by fermentative and oxidative metabolisms, which lead to yeast or mycelial growth, respectively. Dimorphic transition is important for pathogenesis since the mycelium represents the virulent morphology. We previously reported that the deletion of arl1 or arl2 stimulate anaerobic germination in M. circinelloides, suggesting an augmented fermentative metabolism. In the present study, we demonstrate that the heterokaryon Δarl1⁽⁺⁾⁽⁻⁾ and homokaryon Δarl2 strains contain low number of mitochondria, which possibly results in a dysfunctional oxidative metabolism, marked by a low oxygen consumption in glucose and poor growth in glycerol as the unique carbon source. This dysfunction is compensated for by an increase in the glycolysis and fermentation in aerobic conditions, demonstrating growth kinetics similar to that in the wild-type strain. Moreover, as a consequence a high fermentative activity, the Δarl1⁽⁺⁾⁽⁻⁾ and Δarl2 strains possibly increased the yeast cell growth during low oxygen concentrations in presence of glucose.To the best of our knowledge, this is the first study to demonstrate the control of members of Arf family on the mitochondrial population in a Mucor species.