Antibacterial properties of phenothiazine derivatives against multidrug-resistant Acinetobacter baumannii strains.

AIM: As options to treat recalcitrant bacterial infections which are increasingly limited due to multidrug-resistant strains, searching for new, effective antibacterial compounds is necessary. One strategy is to generate treatment alternatives by drug repurposing. METHODS AND RESULTS: In this work, phenotypic microarrays were used for the screening of miscellaneous compounds against the growth and biofilm formation of Acinetobacter baumannii, an important emergent multidrug-resistant opportunistic pathogen. The results showed that the phenothiazine derivatives, such as promethazine, trifluoperazine, thioridazine, and chlorpromazine, inhibited the growth of antibiotic-sensitive and multidrug-resistant strains (showing minimal inhibitory concentrations ranging from 0·05 to 0·6 g l-1 and minimal bactericidal concentrations ranging from 0·1 to 2·5 g l-1 ). All phenothiazine derivatives were active against biofilm cells (with minimal biofilm eradication concentrations ranging from 0·5 to >3 g l-1 ). Chlorpromazine promoted reactive oxigen species (ROS) production, and cell membrane and DNA damage. Chlorpromazine showed synergy with antibiotics such as ceftazidime, meropenem, and colistin and was an effective treatment for experimentally infected Galleria mellonella when combined with ceftazidime. CONCLUSIONS: It was demonstrated that phenothiazine derivatives, especially chlorpromazine, are drugs with attractive antibacterial properties against nosocomial MDR strains of A. baumannii, by generating ROS and cell membrane and DNA damage. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study indicates that repurposing phenothiazine derivatives for treating recalcitrant infections by A. baumannii could be promising.

Increased synthesis of α-tocopherol, paramylon and tyrosine by Euglena gracilis under conditions of high biomass production.

AIMS: To analyse the production of different metabolites by dark-grown Euglena gracilis under conditions found to render high cell growth. METHODS AND RESULTS: The combination of glutamate (5 g l(-1) ), malate (2 g l(-1) ) and ethanol (10 ml l(-1) ) (GM + EtOH); glutamate (7·15 g l(-1) ) and ethanol (10 ml l(-1) ); or malate (8·16 g l(-1) ), glucose (10·6 g l(-1) ) and NH(4) Cl (1·8 g l(-1) ) as carbon and nitrogen sources, promoted an increase of 5·6, 3·7 and 2·6-fold, respectively, in biomass concentration in comparison with glutamate and malate (GM). In turn, the production of α-tocopherol after 120 h identified by LC-MS was 3·7 ± 0·2, 2·4 ± 0·1 and 2 ± 0·1 mg [g dry weight (DW)](-1) , respectively, while in the control medium (GM) it was 0·72 ± 0·1 mg (g DW)(-1) . For paramylon synthesis, the addition of EtOH or glucose induced a higher production. Amino acids were assayed by RP-HPLC; Tyr a tocopherol precursor and Ala an amino acid with antioxidant activity were the amino acids synthesized at higher concentration. CONCLUSIONS: Dark-grown E. gracilis Z is a suitable source for the generation of the biotechnologically relevant metabolites tyrosine, α-tocopherol and paramylon. SIGNIFICANCE AND IMPACT OF THE STUDY: By combining different carbon and nitrogen sources and inducing a tolerable stress to the cell by adding ethanol, it was possible to increase the production of biomass, paramylon, α-tocopherol and some amino acids. The concentrations of α-tocopherol achieved in this study are higher than others reported previously for Euglena, plant and algal systems. This work helps to understand the effect of different carbon sources on the synthesis of bio-molecules by E. gracilis and can be used as a basis for future works to improve the production of different metabolites of biotechnological importance by this organism.

Chromium uptake, retention and reduction in photosynthetic Euglena gracilis.

Photosynthetic Euglena gracilis grown with different K(2)CrO(4) concentrations was analyzed for its ability to take up, retain and reduce Cr(VI). For comparison, cells were also exposed to CrCl(3). Cellular Cr(VI) uptake at pH 7.2 showed a hyperbolic saturation pattern with K (m) of 1.1 mM, V (m) of 16 nmol (h x 10(7) cells)(-1), and K (i sulfate) of 0.4 mM. Kinetic parameters for sulfate uptake were similar, K (m) = 0.83 mM, V (m) = 15.9 nmol (h x 10(7)cells)(-1) and K (i chromate) = 0.3 mM. The capacity to accumulate chromium depended on the ionic species, external concentration and pH of the incubation medium. Cr(VI) or Cr(III) accumulation was negligible in the acidic (pH 3.5) culture medium, in which Cr(VI) was abiotically reduced to Cr(III). At pH 7.2 Cr(VI) was fully stable and high accumulation (>170 nmol/1 x 10(7) cells at 1 mM K(2)CrO(4)) was achieved; surprisingly, Cr(III) accumulation was also significant (>35 nmol/1 x 10(7) cells at 1 mM CrCl(3)). Cr(VI) was reduced by cells at pH 7.2, suggesting the presence of an external reductive activity. Cr(VI) induced an increased cysteine and glutathione content, but not in phytochelatins suggesting that chromium accumulation was mediated by monothiol compounds.

Substrate specificity of the 3-methylcrotonyl coenzyme A (CoA) and geranyl-CoA carboxylases from Pseudomonas aeruginosa.

Biotin-containing 3-methylcrotonyl coenzyme A (MC-CoA) carboxylase (MCCase) and geranyl-CoA (G-CoA) carboxylase (GCCase) from Pseudomonas aeruginosa were expressed as His-tagged recombinant proteins in Escherichia coli. Both native and recombinant MCCase and GCCase showed pH and temperature optima of 8.5 and 37 degrees C. The apparent K(0.5) (affinity constant for non-Michaelis-Menten kinetics behavior) values of MCCase for MC-CoA, ATP, and bicarbonate were 9.8 microM, 13 microM, and 0.8 microM, respectively. MCCase activity showed sigmoidal kinetics for all the substrates and did not carboxylate G-CoA. In contrast, GCCase catalyzed the carboxylation of both G-CoA and MC-CoA. GCCase also showed sigmoidal kinetic behavior for G-CoA and bicarbonate but showed Michaelis-Menten kinetics for MC-CoA and the cosubstrate ATP. The apparent K(0.5) values of GCCase were 8.8 microM and 1.2 microM for G-CoA and bicarbonate, respectively, and the apparent K(m) values of GCCase were 10 microM for ATP and 14 microM for MC-CoA. The catalytic efficiencies of GCCase for G-CoA and MC-CoA were 56 and 22, respectively, indicating that G-CoA is preferred over MC-CoA as a substrate. The enzymatic properties of GCCase suggest that it may substitute for MCCase in leucine catabolism and that both the MCCase and GCCase enzymes play important roles in the leucine and acyclic terpene catabolic pathways.

[Microbial interactions with heavy metals]. / Interacciones microbianas con metales pesados.

Living organisms are exposed in nature to heavy metals, commonly present in their ionized species. These ions exert diverse toxic effects on microorganisms. Metal exposure both selects and maintains microbial variants able to tolerate their harmful effects. Varied and efficient metal resistance mechanisms have been identified in diverse species of bacteria, fungi and protists. The study of the interactions between microorganisms and metals may be helpful to understand the relations of toxic metals with higher organisms such as mammals and plants. Some microbial systems of metal tolerance have the potential to be used in biotechnological processes, such as the bioremediation of environmental metal pollution or the recovery of valuable metals. In this work we analyze several examples of the interactions of different types of microbes with heavy metals; these cases are related either with basic research or with possible practical applications.

Multisite control of the Crabtree effect in ascites hepatoma cells.

AS-30D hepatoma cells, a highly oxidative and fast-growing tumor line, showed glucose-induced and fructose-induced inhibition of oxidative phosphorylation (the Crabtree effect) of 54% and 34%, respectively. To advance the understanding of the underlying mechanism of this process, the effect of 5 mM glucose or 10 mM fructose on the intracellular concentration of several metabolites was determined. The addition of glucose or fructose lowered intracellular Pi (40%), and ATP (53%) concentrations, and decreased cytosolic pH (from 7.2 to 6.8). Glucose and fructose increased the content of AMP (30%), glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate (15, 13 and 50 times, respectively). The cytosolic concentrations of Ca2+ and Mg2+ were not modified. The addition of galactose or glycerol did not modify the concentrations of the metabolites. Mitochondria isolated from AS-30D cells, incubated in media with low Pi (0.6 mM) at pH 6.8, exhibited a 40% inhibition of oxidative phosphorylation. The data suggest that the Crabtree effect is the result of several small metabolic changes promoted by addition of exogenous glucose or fructose.

Modulation of 2-oxoglutarate dehydrogenase complex by inorganic phosphate, Mg(2+), and other effectors.

The interplay of inorganic phosphate (Pi) with other ligands such as Mg(2+), ADP, ATP, and Ca(2+) on the activation of 2-oxoglutarate dehydrogenase complex (2-OGDH) in both isolated enzyme complex and mitochondrial extracts was examined. Pi alone activated the enzyme, following biphasic kinetics with high (K(0.5) = 1.96+/-0.42 mM) and low (K(0.5) = 9.8+/-0.4 mM) affinity components for Pi. The activation by Pi was highly pH-dependent; it increased when the pH raised from 7.1 to 7.6, but it was negligible at pH values below 7.1. Mg-Pi and Mg-ADP, but not Mg-ATP, were more potent activators of 2-OGDH than free Pi and free ADP. ATP inhibited the 2-OGDH activity by chelating the free Mg(2+) and also as a Mg-ATP complex. With or without Mg(2+), ADP, and Pi activated the 2-OGDH by increasing the affinity for 2-OG and the V(m) of the reaction; ATP diminished the V(m), but it increased the affinity for 2-OG in the mitochondrial extract. Pi did not modify the 2-OGDH activation by Ca(2+). The results above mentioned were similar for both preparations, except for hyperbolic kinetics in the isolated enzyme and sigmoidal kinetics in the mitochondrial extracts when 2-oxoglutarate was varied. The data of this study indicated that physiological concentrations of Pi may exert a significant activation of 2-OGDH, which was potentiated by Mg(2+) and high pH, but surpassed by ADP.

Sulfite and membrane energization induce two different active states of the Paracoccus denitrificans F0F1-ATPase.

Activation of the latent ATPase activity of inside-out vesicles from plasma membranes of Paracoccus denitrificans was studied. Several factors were found to induce activation: heat, membrane energization by succinate oxidation, methanol, oxyanions (sulfite, phosphate, arsenate, bicarbonate) and limited proteolysis with trypsin. Among the oxyanions, sulfite induced the higher increase in ATPase activity. Sulfite functioned as a nonessential activator that slightly modified the affinity for ATP and increased notoriously the Vmax. There was a competitive effect between sulfite, bicarbonate and phosphate for ATPase activation; their similar chemical geometry suggests that these oxyanions have a common binding site on the enzyme. Dithiothreitol did not affect the ATPase activity. ATPase activation by sulfite was decreased by uncoupler, enhanced by trypsin and inhibited by ADP, oligomycin and venturicidin. In contrast, activation induced by succinate was less sensitive to ADP, oligomycin, venturicidin and trypsin. It is proposed that the active states induced by sulfite and succinate reflect two conformations of the enzyme, in which the inhibitory subunit epsilon is differently exposed to trypsin.

Modulation of oxidative phosphorylation by Mg2+ in rat heart mitochondria.

The effect of varying the Mg2+ concentration on the 2-oxoglutarate dehydrogenase (2-OGDH) activity and the rate of oxidative phosphorylation of rat heart mitochondria was studied. The ionophore A23187 was used to modify the mitochondrial free Mg2+ concentration. Half-maximal stimulation (K0.5) of ATP synthesis by Mg2+ was obtained with 0.13 +/- 0.02 mM (n = 7) with succinate (+rotenone) and 0.48 +/- 0.13 mM (n = 6) with 2-oxoglutarate (2-OG) as substrates. Similar K0.5 values were found for NAD(P)H formation, generation of membrane potential, and state 4 respiration with 2-OG. In the presence of ADP, an increase in Pi concentration promoted a decrease in the K0.5 values of ATP synthesis, membrane potential formation and state 4 respiration for Mg2+ with 2-OG, but not with succinate. These results indicate that 2-OGDH is the main step of oxidative phosphorylation modulated by Mg2+ when 2-OG is the oxidizable substrate; with succinate, the ATP synthase is the Mg2+-sensitive step. Replacement of Pi by acetate, which promotes changes on intramitochondrial pH abolished Mg2+ activation of 2-OGDH. Thus, the modulation of the 2-OGDH activity by Mg2+ has an essential requirement for Pi (and ADP) in intact mitochondria which is not associated to variations in matrix pH.

Effect of intramitochondrial Mg2+ on citrulline synthesis in rat liver mitochondria.

A correlation was found between mitochondrial matrix free magnesium ([Mg2+]m), measured by means of the fluorescent indicator Mag-Fura-2, and citrulline synthesis in rat liver mitochondria. The variation of [Mg2+]m from 0.05 to 1.7 mM by changes in external Mg2+, induced a 20 +/- 8.5% increase in the rate of citrulline synthesis, whereas a further increase of [Mg2+]m to 3.3 mM induced a return to basal values. The increase in [Mg2+]m, as well as the diminution of external pH, also promoted an elevation of matrix free Ca2+ ([Ca2+]m). An increase in [Ca2+]m, at constant [Mg2+]m and pH, resulted in a 3-fold stimulation of citrulline synthesis. The data suggest that [Mg2+]m may modulate the rate of citrulline synthesis through a direct interaction with carbamoyl-phosphate synthase I (ammonia) and, indirectly, by changing the levels of matrix Ca2+.