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The Chichon volcano contains several thermal manifestations including an acidic crater lake. Here we report a metagenome-assembled genome of " Candidatus Aramenus sp. CH1," a Sulfolobales archaeon inhabiting the crater lake from the Chichon volcano. In this study, we generated a novel Aramenus genome sequence from a thermal area in Southern Mexico., Competing Interests: The authors declare no conflict of interest

Metabolic alterations are recognized as one of the hallmarks of cancer. Among these, alterations in mitochondrial function have been associated with an enhanced production of Reactive Oxygen Species (ROS), which activate ROS-regulated cancer cell signaling pathways. Breast cancer is the main cancer-related cause of death for women globally. It is a heterogeneous disease with subtypes characterized by specific molecular features and patient outcomes. With the purpose of identifying differences in energy metabolism and the oxidative stress management system in non-tumorigenic, estrogen receptor positive (ER+) and triple negative (TN) breast cancer cells, we evaluated ROS production, protein enzyme levels and activities and profiled energy metabolism. We found differences in energetic metabolism and ROS management systems between non-tumorigenic and cancer cells and between ER+ and TN breast cancer cells. Our results indicate a dependence on glycolysis despite different glycolytic ATP levels in all cancer cell lines tested. In addition, our data show that high levels of ROS in TN cells are a result of limited antioxidant capacity in the NADPH producing and GSH systems, mitochondrial dysfunction and non-mitochondrial ROS production, making them more sensitive to GSH synthesis inhibitors. Our data suggest that metabolic and antioxidant profiling of breast cancer will provide important targets for metabolic inhibitors or antioxidant treatments for breast cancer therapy.

The marine archaeon Methanosarcina acetivorans contains a putative NAD  +  -independent d-lactate dehydrogenase (D-iLDH/glycolate oxidase) encoded by the MA4631 gene, belonging to the FAD-oxidase C superfamily. Nucleotide sequences similar to MA4631 gene, were identified in other methanogens and Firmicutes with >90 and 35-40% identity, respectively. Therefore, the lactate metabolism in M. acetivorans is reported here. Cells subjected to intermittent pulses of oxygen (air-adapted; AA-Ma cells) consumed lactate only in combination with acetate, increasing methane production and biomass yield. In AA-Ma cells incubated with d-lactate plus [ 14 C]-l-lactate, the radioactive label was found in methane, CO 2 and glycogen, indicating that lactate metabolism fed both methanogenesis and gluconeogenesis. Moreover, d-lactate oxidation was coupled to O 2 -consumption which was sensitive to HQNO; also, AA-Ma cells showed high transcript levels of gene dld and those encoding subunits A (MA1006) and B (MA1007) of a putative cytochrome bd quinol oxidase, compared to anaerobic control cells. An E. coli mutant deficient in dld complemented with the MA4631 gene, grew with d-lactate as carbon source and showed membrane-bound d-lactate:quinone oxidoreductase activity. The product of the MA4631 gene is a FAD-containing monomer showing activity of iLDH with preference to d-lactate. The results suggested that air adapted M. acetivorans is able to co-metabolize lactate and acetate with associated oxygen consumption by triggering the transcription and synthesis of the D-iLDH and a putative cytochrome bd: methanophenazine (quinol) oxidoreductase. Biomass generation and O 2 consumption, suggest a potentially new oxygen detoxification mechanism coupled to energy conservation in this methanogen., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The crater lake at "El Chichón" volcano is an extreme acid-thermal environment with high concentrations of heavy metals. In this study, two bacterial strains with the ability to resist high concentrations of arsenic (As) were isolated from water samples from the crater lake. Staphylococcus ARSC1-P and Stenotrophomonas ARSC2-V isolates were identified by use of the 16S rDNA gene. Staphylococcus ARSC1-P was able to grow in 400 mM of arsenate [As(V)] under oxic and anoxic conditions. The IC 50 values were 36 and 382 mM for oxic and anoxic conditions, respectively. For its part, Stenotrophomonas ARSC2-V showed IC 50 values of 110 mM and 2.15 for As(V) and arsenite [As(III)], respectively. Arsenic accumulated intracellularly in both species [11-25 nmol As × mg cellular prot -1 in cells cultured in 50 mM As(V)]. The present study shows evidence of microbes that can potentially be a resource for the bio-treatment of arsenic in contaminated sites, which highlights the importance of the "El Chichón" volcano as a source of bacterial strains that are adaptable to extreme conditions., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Obesity has been linked to cognitive impairment through systemic low-grade inflammation. High fat and sugar diets (HFSDs) also induce systemic inflammation, either by induced Toll-like receptor 4 response, or by causing dysbiosis. This study aimed to evaluate the effect of symbiotics supplementation on spatial and working memory, butyrate concentration, neurogenesis, and electrophysiological recovery of HFSD-fed rats. In a first experiment, Sprague-Dawley male rats were given HFSD for 10 weeks, after which they were randomized into 2 groups ( n = 10 per group): water (control), or Enterococcus faecium + inulin (symbiotic) administration, for 5 weeks. In the fifth week, spatial and working memory was analyzed through the Morris Water Maze (MWM) and Eight-Arm Radial Maze (RAM) tests, respectively, with 1 week apart between tests. At the end of the study, butyrate levels from feces and neurogenesis at hippocampus were determined. In a second experiment with similar characteristics, the hippocampus was extracted to perform electrophysiological studies. Symbiotic-supplemented rats showed a significantly better memory, butyrate concentrations, and neurogenesis. This group also presented an increased firing frequency in hippocampal neurons [and a larger N -methyl-d-aspartate (NMDA)/α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) current ratio] suggesting an increase in NMDA receptors, which in turn is associated with an enhancement in long-term potentiation and synaptic plasticity. Therefore, our results suggest that symbiotics could restore obesity-related memory impairment and promote synaptic plasticity.

A challenge in the study of gastrointestinal microbiota (GITm) is the validation of the genomic data with metabolic studies of the microbial communities to understand how the microbial networks work during health and sickness. To gain insights into the metabolism of the GITm, feces from healthy and sick rats with cancer were inoculated in a defined synthetic medium directed for anaerobic prokaryote growth (INC-07 medium). Significant differences between cultures of healthy and sick individuals were found: 1) the consumption of the carbon source and the enzyme activity involved in their catabolism (e.g., sucrase, lactase, lipases, aminotransferases, and dehydrogenases); 2) higher excretion of acetic, propionic, isobutyric, butyric, valeric, and isovaleric acids; 3) methane production; 4) ability to form biofilms; and 5) up to 500 amplicon sequencing variants (ASVs) identified showed different diversity and abundance. Moreover, the bowel inflammation induced by cancer triggered oxidative stress, which correlated with deficient antioxidant machinery (e.g., NADPH-producing enzymes) determined in the GITm cultures from sick individuals in comparison with those from control individuals. Altogether, the data suggested that to preserve the microbial network between bacteria and methanogenic archaea, a complete oxidation of the carbon source may be essential for healthy microbiota. The correlation of 16S rRNA gene metabarcoding between cultures and feces, as well as metabolomic data found in cultures, suggest that INC-07 medium may be a useful tool to understand the metabolism of microbiota under gut conditions., 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 © 2022 Peña-Ocaña, Hoshiko, Silva-Flores, Maeda, Pérez-Torres, García-Contreras, Gutiérrez-Sarmiento, Hernández-Esquivel, Marín-Hernández, Sánchez-Thomas, Saavedra, Rodríguez-Zavala and Jasso-Chávez.)

Tepache is a native beverage from Mexico, which is usually elaborated with pineapple shells, brown cane sugar and is fermented naturally. Beneficial health effects have been attributed to its consumption; however, the total ecosystem of this beverage including chemicals (substrates for microbial growth, prebiotics, etc) and microbiota (probiotics), and potential functionality had not been studied. In this work, the analysis of the tepache beverage for its physicochemical characteristics, as well as its structure of microbial communities and the predictive metabolic functionalities was carried out. Chemical characterization was performed via enzymatic and GC-MS methods. The bacterial and fungal communities were identified by using 16S rRNA and ITS metabarcoding through Illumina MiSeq 2 × 300. The metabolic potential was predicted by in silico tools. This research showed that after 72 h of fermentation, the tepache physicochemical characteristics shifted to 9.5 Brix degrees and acidic pH. The content of ethanol, acetic and L-lactic acid increased significantly from 0.83 ± 0.02 to 3.39 ± 0.18 g/L, from 0.38 ± 0.04 to 0.54 ± 0.04 g/L and from 1.42 ± 0.75 to 8.77 ± 0.34 g/L, respectively. While, the total sugars was decreased from 123.43 ± 2.01 to 84.70 ± 2.34 g/L. The microbial diversity analysis showed a higher richness of bacterial communities and increased fungal evenness at the end of fermentation. At 72 h of fermentation the microbial community was dominated by Lactobacillus, Leuconostoc, Acetobacter and Lactococcus bacterial genera. As for the fungal community, Saccharomyces, Gibberella, Zygosaccharomyces, Candida, Meyerozyma, Talaromyces, Epicoccum and Kabatiella were found to be in most abundance. The predicted functionality profile evidenced a close-fitting relationship between fungal communities at 0 h with the bacterial communities at 72 h of fermentation. The metabolic potential showed that glycolysis and citrate cycle metabolism were predominant for fungal community, while glycolysis, fructose and tricarboxylic acid metabolism were more representative for the bacterial core. Tepache fermentation mainly occurred at two temporal successions. First, a lactic acid and ethanol fermentation dominated by lactic acid bacteria and yeast, and then an increase in acetogenic bacteria. This study revealed for the first time the physicochemical, microbiological changes and predictive functionality that are involved during tepache fermentation. These findings contributed to the knowledge of important microbial sources and could be essential to future efforts in manufacturing process. In addition, this work could help to analyze the health benefits that are empirically attributed to it by consumers., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Acetylation of proteins seems a widespread process found in the three domains of life. Several studies have shown that besides histones, acetylation of lysine residues also occurs in non-nuclear proteins. Hence, it has been suggested that this covalent modification is a mechanism that might regulate diverse metabolic pathways by modulating enzyme activity, stability, and/or subcellular localization or interaction with other proteins. However, protein acetylation levels seem to have low correlation with modification of enzyme activity and pathway fluxes. In addition, the results obtained with mutant enzymes that presumably mimic acetylation have frequently been over-interpreted. Moreover, there is a generalized lack of rigorous enzyme kinetic analysis in parallel to acetylation level determinations. The purpose of this review is to analyze the current findings on the impact of acetylation on metabolic enzymes and its repercussion on metabolic pathways function/regulation., (© 2021 Wiley Periodicals LLC.)

El Chichón volcano is one of the most active volcanoes in Mexico. Previous studies have described its poly-extreme conditions and its bacterial composition, although the functional features of the complete microbiome have not been characterized yet. By using metabarcoding analysis, metagenomics, metabolomics and enzymology techniques, the microbiome of the crater lake was characterized in this study. New information is provided on the taxonomic and functional diversity of the representative Archaea phyla, Crenarchaeota and Euryarchaeota, as well as those that are representative of Bacteria, Thermotogales and Aquificae. With culture of microbial consortia and with the genetic information collected from the natural environment sampling, metabolic interactions were identified between prokaryotes, which can withstand multiple extreme conditions. The existence of a close relationship between the biogeochemical cycles of carbon and sulfur in an active volcano has been proposed, while the relationship in the energy metabolism of thermoacidophilic bacteria and archaea in this multi-extreme environment was biochemically revealed for the first time. These findings contribute towards understanding microbial metabolism under extreme conditions, and provide potential knowledge pertaining to "microbial dark matter", which can be applied to biotechnological processes and evolutionary studies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Under dysbiosis, a gut metabolic disorder, short-chain carboxylic acids (SCCAs) are secreted to the lumen, affecting colorectal cancer (CRC) development. Butyrate and propionate act as CRC growth inhibitors, but they might also serve as carbon source. In turn, the roles of acetate as metabolic fuel and protein acetylation promoter have not been clearly elucidated. To assess whether acetate favors CRC growth through active mitochondrial catabolism, a systematic study evaluating acetate thiokinase (AcK), energy metabolism, cell proliferation, and invasiveness was performed in two CRC cell lines incubated with physiological SCCAs concentrations. In COLO 205, acetate (+glucose) increased the cell density (50%), mitochondrial protein content (3-10 times), 2-OGDH acetylation, and oxidative phosphorylation (OxPhos) flux (36%), whereas glycolysis remained unchanged vs. glucose-cultured cells; the acetate-induced OxPhos activation correlated with a high AcK activity, content, and acetylation (1.5-6-fold). In contrast, acetate showed no effect on HCT116 cell growth, OxPhos, AcK activity, protein content, and acetylation. However, a substantial increment in the HIF-1α content, HIF-1α-glycolytic protein targets (1-2.3 times), and glycolytic flux (64%) was observed. Butyrate and propionate decreased the growth of both CRC cells by impairing OxPhos flux through mitophagy and mitochondrial fragmentation activation. It is described, for the first time, the role of acetate as metabolic fuel for ATP supply in CRC COLO 205 cells to sustain proliferation, aside from its well-known role as protein epigenetic regulator. The level of AcK determined in COLO 205 cells was similar to that found in human CRC biopsies, showing its potential role as metabolic marker., 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 © 2021 Rodríguez-Enríquez, Robledo-Cadena, Gallardo-Pérez, Pacheco-Velázquez, Vázquez, Saavedra, Vargas-Navarro, Blanco-Carpintero, Marín-Hernández, Jasso-Chávez, Encalada, Ruiz-Godoy, Aguilar-Ponce and Moreno-Sánchez.)

Pseudomonas aeruginosa is a primary bacterial model to study cooperative behaviors because it yields exoproducts such as siderophores and exoproteases that act as public goods and can be exploited by selfish nonproducers behaving as social cheaters. Iron-limited growth medium, mainly casamino acids medium supplemented with transferrin, is typically used to isolate and study nonproducer mutants of the siderophore pyoverdine. However, using a protein as the iron chelator could inadvertently select mutants unable to produce exoproteases, since these enzymes can degrade the transferrin to facilitate iron release. Here we investigated the evolutionary dynamics of pyoverdine and exoprotease production in media in which iron was limited by using either transferrin or a cation chelating resin. We show that concomitant loss of pyoverdine and exoprotease production readily develops in media containing transferrin, whereas only pyoverdine loss emerges in medium treated with the resin. Characterization of exoprotease- and pyoverdine-less mutants revealed loss in motility, different mutations, and large genome deletions (13-33 kb) including Quorum Sensing (lasR, rsal, and lasI) and flagellar genes. Our work shows that using transferrin as an iron chelator imposes simultaneous selective pressure for the loss of pyoverdine and exoprotease production. The unintended effect of transferrin uncovered by our experiments can help to inform the design of similar studies., (© 2021. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

Under physiological conditions, cells produce low basal levels of reactive oxygen species (ROS); however, in pathologic conditions ROS production increases dramatically, generating high concentrations of toxic unsaturated aldehydes. Aldehyde dehydrogenases (ALDHs) are responsible for detoxification of these aldehydes protecting the cell. Due to the physiological relevance of these enzymes, it is important to design strategies to modulate their activity. It was previously reported that omeprazole activation of ALDH1A1 protected Escherichia coli cells overexpressing this enzyme, from oxidative stress generated by H 2 O 2 . In this work, omeprazole cell protection potential was evaluated in eukaryotic cells. AS-30D cell or hepatocyte suspensions were subjected to a treatment with omeprazole and exposure to light (that is required to activate omeprazole in the active site of ALDH) and then exposed to H 2 O 2 . Cells showed viability similar to control cells, total activity of ALDH was preserved, while cell levels of lipid aldehydes and oxidative stress markers were maintained low. Cell protection by omeprazole was avoided by inhibition of ALDHs with disulfiram, revealing the key role of these enzymes in the protection. Additionally, omeprazole also preserved ALDH2 (mitochondrial isoform) activity, diminishing lipid aldehyde levels and oxidative stress in this organelle, protecting mitochondrial respiration and transmembrane potential formation capacity, from the stress generated by H 2 O 2 . These results highlight the important role of ALDHs as part of the antioxidant system of the cell, since if the activity of these enzymes decreases under stress conditions, the viability of the cell is compromised., (© 2021 Federation of European Biochemical Societies.)

The repurposing of gallium nitrate as an antibacterial, a drug used previously for the treatment of hypercalcemia, is a plausible alternative to combat infections by Pseudomonas aeruginosa, since it has antipseudomonal properties in vitro and in vivo in animal models and in human lung infections. Furthermore, gallium nitrate tolerance in clinical isolates is very rare. Nevertheless, studies on the reference strains PA14 and PAO1 show that resistance against gallium nitrate is achieved by decreasing gallium intracellular levels by increasing the production of pyocyanin. In this work, we induced resistance in a cystic fibrosis P. aeruginosa isolate and explored its resistance mechanisms. This isolated strain, INP-58M, was not a pyocyanin producer, and its pyoverdine levels remained unchanged upon gallium addition. However, it showed higher activities of NADPH-producing enzymes and the antioxidant enzyme SOD when gallium was added, which suggests a better antioxidant response. Remarkably, gallium intracellular levels in the resistant isolate were higher than those of the parental strain at 20 h but lower after 24 h of culture, suggesting that this strain is capable of gallium efflux.

Phosphate metabolism was studied to determine whether polyphosphate (polyP) pools play a role in the enhanced resistance against Cd 2+ and metal-removal capacity of Cd 2+ -preadapted (CdPA) Methanosarcina acetivorans . Polyphosphate kinase (PPK), exopolyphosphatase (PPX) and phosphate transporter transcript levels and their activities increased in CdPA cells compared to control (Cnt) cells. K + inhibited recombinant Ma-PPK and activated Ma-PPX, whereas divalent cations activated both enzymes. Metal-binding polyP and thiol-containing molecule contents, Cd 2+ -removal, and biofilm synthesis were significantly higher in CdPA cells >Cnt cells plus a single addition of Cd 2+ >Cnt cells. Also, CdPA cells showed a higher number of cadmium, sulfur, and phosphorus enriched-acidocalcisomes than control cells. Biochemical and physiological phenotype exhibited by CdPA cells returned to that of Cnt cells when cultured without Cd 2+ . Furthermore, no differences in the sequenced genomes upstream and downstream of the genes involved in Cd 2+ resistance were found between CdPA and Cnt cells, suggesting phenotype loss rather than genome mutations induced by chronic Cd 2+ -exposure. Instead, a metabolic adaptation induced by Cd 2+ stress was apparent. The dynamic ability of M. acetivorans to change its metabolism, depending on the environmental conditions, may be advantageous to remove cadmium in nature and biodigesters., (Copyright © 2019 Jasso-Chávez, Lira-Silva, González-Sánchez, Larios-Serrato, Mendoza-Monzoy, Pérez-Villatoro, Morett, Vega-Segura, Torres-Márquez, Zepeda-Rodríguez and Moreno-Sánchez.)

To enhance our understanding of the control of archaeal carbon central metabolism, a detailed analysis of the regulation mechanisms of both fructose1,6-bisphosphatase (FruBPase) and ADP-phosphofructokinase-1 (ADP-PFK1) was carried out in the methanogen Methanosarcina acetivorans. No correlations were found among the transcript levels of the MA_1152 and MA_3563 (frubpase type II and pfk1) genes, the FruBPase and ADP-PFK1 activities, and their protein contents. The kinetics of the recombinant FruBPase II and ADP-PFK1 were hyperbolic and showed simple mixed-type inhibition by AMP and ATP, respectively. Under physiological metabolite concentrations, the FruBPase II and ADP-PFK1 activities were strongly modulated by their inhibitors. To assess whether these enzymes were also regulated by a phosphorylation/dephosphorylation process, the recombinant enzymes and cytosolic-enriched fractions were incubated in the presence of commercial protein phosphatase or protein kinase. De-phosphorylation of ADP-PFK1 slightly decreased its activity (i.e. Vmax) and did not change its kinetic parameters and oligomeric state. Thus, the data indicated a predominant metabolic regulation of both FruBPase and ADP-PFK1 activities by adenine nucleotides and suggested high degrees of control on the respective pathway fluxes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aging is associated with morphological, physiological and metabolic changes, leading to multiorgan degenerative pathologies, such as cognitive function decline. It has been suggested that memory loss also involves a decrease in neurotrophic factors, including brain-derived neurotrophic factor (BDNF). In recent years, microbiota has been proposed as an essential player in brain development, as it is believed to activate BDNF secretion through butyrate production. Thus, microbiota modulation by supplementation with probiotics and prebiotics may impact cognitive decline. This study aimed to evaluate the effects of probiotics and prebiotics supplementation on the memory of middle-aged rats. Sprague-Dawley male rats were randomized in four groups ( n = 13 per group): control (water), probiotic ( E. faecium ), prebiotic (agave inulin), symbiotic ( E. faecium + inulin), which were administered for 5 weeks by oral gavage. Spatial and associative memory was analyzed using the Morris Water Maze (MWM) and Pavlovian autoshaping tests, respectively. Hippocampus was obtained to analyze cytokines [interleukin (IL-1β) and tumor necrosis factor (TNF-α)], BDNF and γ-aminobutyric acid (GABA) by enzyme-linked immunosorbent assay (ELISA). Butyrate concentrations were also evaluated in feces. The symbiotic group showed a significantly better performance in MWM ( p < 0.01), but not in Pavlovian autoshaping test. It also showed significantly lower concentrations of pro-inflammatory cytokines ( p < 0.01) and the reduction in IL-1β correlated with a better performance of the symbiotic group in MWM ( p < 0.05). Symbiotic group also showed the highest BDNF and butyrate levels ( p < 0.0001). Finally, we compared the electrophysiological responses of control ( n = 8) and symbiotic ( n = 8) groups. Passive properties of CA1 pyramidal cells (PCs) exhibited changes in response to the symbiotic treatment. Likewise, this group showed an increase in the N -methyl-D-aspartate receptor (NMDA)/AMPA ratio and exhibited robust long-term potentiation (LTP; p < 0.01). Integrated results suggest that symbiotics could improve age-related impaired memory.

Twelve novel benzimidazole derivatives were synthesized and their in vitro activities against epimastigotes of Trypanosoma cruzi were evaluated. Two derivatives (6 and 7), which have 4-hydroxy-3-methoxyphenyl moiety in their structures, proved to be the most active in inhibiting the parasite growth. Compound 6 showed a trypanocidal activity higher than benznidazole (IC 50 =5µM and 7.5µM, respectively) and less than nifurtimox (IC 50 =3.6µM). In addition, the ability of 6 and 7 to modify the redox homeostasis in T cruzi epimastigote was studied; cysteine and glutathione increased in parasites exposed to both compounds, whereas trypanothione only increased with 7 treatment. These results suggest that the decrease in viability of T. cruzi may be attributed to the change in cellular redox balance caused by compound 6 or 7. Furthermore, compounds 6 and 7 showed CC 50 values of 160.64 and 160.66µM when tested in mouse macrophage cell line J774 and selectivity indexes (macrophage/parasite) of 32 and 20.1, respectively., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Given our vast methane reserves and the difficulty in transporting methane without substantial leaks, the conversion of methane directly into electricity would be beneficial. Microbial fuel cells harness electrical power from a wide variety of substrates through biological means; however, the greenhouse gas methane has not been used with much success previously as a substrate in microbial fuel cells to generate electrical current. Here we construct a synthetic consortium consisting of: (i) an engineered archaeal strain to produce methyl-coenzyme M reductase from unculturable anaerobic methanotrophs for capturing methane and secreting acetate; (ii) micro-organisms from methane-acclimated sludge (including Paracoccus denitrificans) to facilitate electron transfer by providing electron shuttles (confirmed by replacing the sludge with humic acids), and (iii) Geobacter sulfurreducens to produce electrons from acetate, to create a microbial fuel cell that converts methane directly into significant electrical current. Notably, this methane microbial fuel cell operates at high Coulombic efficiency.

Free-living microorganisms may become suitable models for removal of heavy metals from polluted water bodies, sediments, and soils by using and enhancing their metal accumulating abilities. The available research data indicate that protists of the genus Euglena are a highly promising group of microorganisms to be used in bio-remediation of heavy metal-polluted aerobic and anaerobic acidic aquatic environments. This chapter analyzes the variety of biochemical mechanisms evolved in E. gracilis to resist, accumulate and remove heavy metals from the environment, being the most relevant those involving (1) adsorption to the external cell pellicle; (2) intracellular binding by glutathione and glutathione polymers, and their further compartmentalization as heavy metal-complexes into chloroplasts and mitochondria; (3) polyphosphate biosynthesis; and (4) secretion of organic acids. The available data at the transcriptional, kinetic and metabolic levels on these metabolic/cellular processes are herein reviewed and analyzed to provide mechanistic basis for developing genetically engineered Euglena cells that may have a greater removal and accumulating capacity for bioremediation and recycling of heavy metals.

The multisubunit cation/proton antiporter 3 family, also called Mrp, is widely distributed in all three phylogenetic domains (Eukarya, Bacteria, and Archaea). Investigations have focused on Mrp complexes from the domain Bacteria to the exclusion of Archaea, with a consensus emerging that all seven subunits are required for Na + /H + antiport activity. The MrpA subunit from the MrpABCDEFG Na + /H + antiporter complex of the archaeon Methanosarcina acetivorans was produced in antiporter-deficient Escherichia coli strains EP432 and KNabc and biochemically characterized to determine the role of MrpA in the complex. Both strains containing MrpA grew in the presence of up to 500 mM NaCl and pH values up to 11.0 with no added NaCl. Everted vesicles from the strains containing MrpA were able to generate a NADH-dependent pH gradient (ΔpH), which was abated by the addition of monovalent cations. The apparent K m values for Na + and Li + were similar and ranged from 31 to 63 mM, whereas activity was too low to determine the apparent K m for K + Optimum activity was obtained between pH 7.0 and 8.0. Homology molecular modeling identified two half-closed symmetry-related ion translocation channels that are linked, forming a continuous path from the cytoplasm to the periplasm, analogous to the NuoL subunit of complex I. Bioinformatics analyses revealed genes encoding homologs of MrpABCDEFG in metabolically diverse methane-producing species. Overall, the results advance the biochemical, evolutionary, and physiological understanding of Mrp complexes that extends to the domain Archaea IMPORTANCE: The work is the first reported characterization of an Mrp complex from the domain Archaea, specifically methanogens, for which Mrp is important for acetotrophic growth. The results show that the MrpA subunit is essential for antiport activity and, importantly, that not all seven subunits are required, which challenges current dogma for Mrp complexes from the domain Bacteria A mechanism is proposed in which an MrpAD subcomplex catalyzes Na + /H + antiport independent of an MrpBCEFG subcomplex, although the activity of the former is modulated by the latter. Properties of MrpA strengthen proposals that the Mrp complex is of ancient origin and that subunits were recruited to evolve the ancestral complex I. Finally, bioinformatics analyses indicate that Mrp complexes function in diverse methanogenic pathways., (Copyright © 2016 American Society for Microbiology.)

Gluconeogenesis is an essential pathway in methanogens because they are unable to use exogenous hexoses as carbon source for cell growth. With the aim of understanding the regulatory mechanisms of central carbon metabolism in Methanosarcina acetivorans, the present study investigated gene expression, the activities and metabolic regulation of key enzymes, metabolite contents and fluxes of gluconeogenesis, as well as glycolysis and glycogen synthesis/degradation pathways. Cells were grown with methanol as a carbon source. Key enzymes were kinetically characterized at physiological pH/temperature. Active consumption of methanol during exponential cell growth correlated with significant methanogenesis, gluconeogenic flux and steady glycogen synthesis. After methanol exhaustion, cells reached the stationary growth phase, which correlated with the rise in glycogen consumption and glycolytic flux, decreased methanogenesis, negligible acetate production and an absence of gluconeogenesis. Elevated activities of carbon monoxide dehydrogenase/acetyl-CoA synthetase complex and pyruvate: ferredoxin oxidoreductase suggested the generation of acetyl-CoA and pyruvate for glycogen synthesis. In the early stationary growth phase, the transcript contents and activities of pyruvate phosphate dikinase, fructose 1,6-bisphosphatase and glycogen synthase decreased, whereas those of glycogen phosphorylase, ADP-phosphofructokinase and pyruvate kinase increased. Therefore, glycogen and gluconeogenic metabolites were synthesized when an external carbon source was provided. Once such a carbon source became depleted, glycolysis and methanogenesis fed by glycogen degradation provided the ATP supply. Weak inhibition of key enzymes by metabolites suggested that the pathways evaluated were mainly transcriptionally regulated. Because glycogen metabolism and glycolysis/gluconeogenesis are not present in all methanogens, the overall data suggest that glycogen storage might represent an environmental advantage for methanosarcinales when carbon sources are scarce. Also, the understanding of the central carbohydrate metabolism in methanosarcinales may help to optimize methane production., (© 2016 Federation of European Biochemical Societies.)

Pseudomonas aeruginosa colonizes the lungs of cystic fibrosis patients causing severe damage. This bacterium is intrinsically resistant to antibiotics and shows resistance against new antimicrobials and its virulence is controlled by the quorum-sensing response. Thus, attenuating its virulence by quorum quenching instead of inhibiting its growth has been proposed to minimize resistance; however, resistance against the canonical quorum quencher furanone C-30 can be achieved by mutations leading to increased efflux. In the present work, the effect of C-30 in the attenuation of the QS-controlled virulence factors elastase and pyocyanin was investigated in 50 isolates from cystic fibrosis patients. The results demonstrate that there is a high variability in the expression of both elastase and pyocyanin and that there are many naturally resistant C-30 strains. We report that the main mechanism of C-30 resistance in these strains was not due to enhanced efflux but a lack of permeability. Moreover, C-30 strongly inhibited the growth of several of the isolates studied, thus imposing high selective pressure for the generation of resistance., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The facultative protist Euglena gracilis, a heavy metal hyper-accumulator, was grown under photo-heterotrophic and extreme conditions (acidic pH, anaerobiosis and with Cd(2+)) and biochemically characterized. High biomass (8.5×10(6)cellsmL(-1)) was reached after 10 days of culture. Under anaerobiosis, photosynthetic activity built up a microaerophilic environment of 0.7% O₂, which was sufficient to allow mitochondrial respiratory activity: glutamate and malate were fully consumed, whereas 25-33% of the added glucose was consumed. In anaerobic cells, photosynthesis but not respiration was activated by Cd(2+) which induced higher oxidative stress. Malondialdehyde (MDA) levels were 20 times lower in control cells under anaerobiosis than in aerobiosis, although Cd(2+) induced a higher MDA production. Cd(2+) stress induced increased contents of chelating thiols (cysteine, glutathione and phytochelatins) and polyphosphate. Biosorption (90%) and intracellular accumulation (30%) were the mechanisms by which anaerobic cells removed Cd(2+) from medium, which was 36% higher versus aerobic cells. The present study indicated that E. gracilis has the ability to remove Cd(2+) under anaerobic conditions, which might be advantageous for metal removal in sediments from polluted water bodies or bioreactors, where the O₂ concentration is particularly low., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Methanosarcina acetivorans, considered a strict anaerobic archaeon, was cultured in the presence of 0.4-1% O2 (atmospheric) for at least 6 months to generate air-adapted cells; further, the biochemical mechanisms developed to deal with O2 were characterized. Methane production and protein content, as indicators of cell growth, did not change in air-adapted cells respect to cells cultured under anoxia (control cells). In contrast, growth and methane production significantly decreased in control cells exposed for the first time to O2. Production of reactive oxygen species was 50 times lower in air-adapted cells versus control cells, suggesting enhanced anti-oxidant mechanisms that attenuated the O2 toxicity. In this regard, (i) the transcripts and activities of superoxide dismutase, catalase and peroxidase significantly increased; and (ii) the thiol-molecules (cysteine + coenzyme M-SH + sulfide) and polyphosphate contents were respectively 2 and 5 times higher in air-adapted cells versus anaerobic-control cells. Long-term cultures (18 days) of air-adapted cells exposed to 2% O2 exhibited the ability to form biofilms. These data indicate that M. acetivorans develops multiple mechanisms to contend with O2 and the associated oxidative stress, as also suggested by genome analyses for some methanogens.

Quorum sensing (QS) coordinates the expression of virulence factors and allows bacteria to counteract the immune response, partly by increasing their tolerance to the oxidative stress generated by immune cells. Despite the recognized role of QS in enhancing the oxidative stress response, the consequences of this relationship for the bacterial ecology remain unexplored. Here we demonstrate that QS increases resistance also to osmotic, thermal and heavy metal stress. Furthermore a QS-deficient lasR rhlR mutant is unable to exert a robust response against H2O2 as it has less induction of catalase and NADPH-producing dehydrogenases. Phenotypic microarrays revealed that the mutant is very sensitive to several toxic compounds. As the anti-oxidative enzymes are private goods not shared by the population, only the individuals that produce them benefit from their action. Based on this premise, we show that in mixed populations of wild-type and the mexR mutant (resistant to the QS inhibitor furanone C-30), treatment with C-30 and H2O2 increases the proportion of mexR mutants; hence, oxidative stress selects resistance to QS compounds. In addition, oxidative stress alone strongly selects for strains with active QS systems that are able to exert a robust anti oxidative response and thereby decreases the proportion of QS cheaters in cultures that are otherwise prone to invasion by cheats. As in natural environments stress is omnipresent, it is likely that this QS enhancement of stress tolerance allows cells to counteract QS inhibition and invasions by social cheaters, therefore having a broad impact in bacterial ecology.

The novel antimicrobial gallium is a nonredox iron III analogue with bacteriostatic and bactericidal properties, effective for the treatment of Pseudomonas aeruginosa in vitro and in vivo in mouse and rabbit infection models. It interferes with iron metabolism, transport, and presumably its homeostasis. As gallium exerts its antimicrobial effects by competing with iron, we hypothesized that it ultimately will lead cells to an iron deficiency status. As iron deficiency promotes the expression of virulence factors in vitro and promotes the pathogenicity of P. aeruginosa in animal models, it is anticipated that treatment with gallium will also promote the production of virulence factors. To test this hypothesis, the reference strain PA14 and two clinical isolates from patients with cystic fibrosis were exposed to gallium, and their production of pyocyanin, rhamnolipids, elastase, alkaline protease, alginate, pyoverdine, and biofilm was determined. Gallium treatment induced the production of all the virulence factors tested in the three strains except for pyoverdine. In addition, as the Ga-induced virulence factors are quorum sensing controlled, co-administration of Ga and the quorum quencher brominated furanone C-30 was assayed, and it was found that C-30 alleviated growth inhibition from gallium. Hence, adding both C-30 and gallium may be more effective in the treatment of P. aeruginosa infections., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

To assess what defence mechanisms are triggered by Cd(2+) stress in Methanosarcina acetivorans, cells were cultured at different cadmium concentrations. In the presence of 100 μM CdCl2, the intracellular contents of cysteine, sulfide and coenzyme M increased, respectively, 8, 27 and 7 times versus control. Cells incubated for 24 h in medium with less cysteine and sulfide removed up to 80% of Cd(2+) added, whereas their cysteine and coenzyme M contents increased 160 and 84 times respectively. Cadmium accumulation (5.2 μmol/10-15 mg protein) resulted in an increase in methane synthesis of 4.5 times in cells grown on acetate. Total phosphate also increased under high (0.5 mM) Cd(2+) stress. On the other hand, cells preadapted to 54 μM CdCl2 and further exposed to > 0.63 mM CdCl2 developed the formation of a biofilm with an extracellular matrix constituted by carbohydrates, DNA and proteins. Biofilm cells were able to synthesize methane. The data suggested that increased intracellular contents of thiol molecules and total phosphate, and biofilm formation, are all involved in the cadmium resistance mechanisms in this marine archaeon., (© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.)

Pseudomonas aeruginosa PA14 cells resistant to the novel antimicrobial gallium nitrate (Ga) were developed using transposon mutagenesis and by selecting spontaneous mutants. The mutants showing the highest growth in the presence of Ga were selected for further characterization. These mutants showed 4- to 12-fold higher Ga minimal inhibitory growth concentrations and a greater than 8-fold increase in the minimum biofilm eliminating Ga concentration. Both types of mutants produced Ga resistant biofilms whereas the formation of wild-type biofilms was strongly inhibited by Ga. The gene interrupted in the transposon mutant was hitA, which encodes a periplasmic iron binding protein that delivers Fe³⁺ to the HitB iron permease; complementation of the mutant with the hitA gene restored the Ga sensitivity. This hitA mutant showed a 14-fold decrease in Ga internalization versus the wild-type strain, indicating that the HitAB system is also involved in the Ga uptake. Ga uptake in the spontaneous mutant was also lower, although no mutations were found in the hitAB genes. Instead, this mutant harbored 64 non-silent mutations in several genes including those of the phenazine pyocyanin biosynthesis. The spontaneous mutant produced 2-fold higher pyocyanin basal levels than the wild-type; the addition of this phenazine to wild-type cultures protected them from the Ga bacteriostatic effect. The present data indicate that mutations affecting Ga transport and probably pyocyanin biosynthesis enable cells to develop resistance to Ga., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The role of the multisubunit sodium/proton antiporter (Mrp) of Methanosarcina acetivorans was investigated with a mutant deleted for the gene encoding the MrpA subunit. Antiporter activity was 5-fold greater in acetate-grown versus methanol-grown wild-type cells, consistent with the previously published relative levels of mrp transcript. The rate, final optical density, and dry weight/methane ratio decreased for the mutant versus wild type when cultured with a growth-limiting concentration of acetate. All growth parameters of the mutant or wild type were identical when grown with methanol in medium containing a growth-limiting Na(+) concentration of 1.04 M. The lag phase, growth rate, and final optical density for growth of the mutant were suboptimal compared to the wild type when cultured with acetate in medium containing either 0.54 or 1.04 M Na(+). The addition of 25 mM NaCl to resting cell suspensions stimulated ATP synthesis driven by a potassium diffusion potential. ATP synthesis was greater in wild-type than mutant cells grown with acetate, a trend that held for methanol-grown cells, albeit less pronounced. Both sodium and proton ionophores reduced ATP synthesis in the wild type grown with either substrate. The results indicated that the Mrp complex is essential for efficient ATP synthesis and optimal growth at the low concentrations of acetate encountered in the environment.

A kinetic model of trypanothione [T(SH)(2)] metabolism in Trypanosoma cruzi was constructed based on enzyme kinetic parameters determined under near-physiological conditions (including glutathione synthetase), and the enzyme activities, metabolite concentrations and fluxes determined in the parasite under control and oxidizing conditions. The pathway structure is characterized by a T(SH)(2) synthetic module of low flux and low catalytic capacity, and another more catalytically efficient T(SH)(2) -dependent antioxidant/regenerating module. The model allowed quantification of the contribution of each enzyme to the control of T(SH)(2) synthesis and concentration (flux control and concentration control coefficients, respectively). The main control of flux was exerted by γ-glutamylcysteine synthetase (γECS) and trypanothione synthetase (TryS) (control coefficients of 0.58-0.7 and 0.49-0.58, respectively), followed by spermidine transport (0.24); negligible flux controls by trypantothione reductase (TryR) and the T(SH)(2)-dependent antioxidant machinery were determined. The concentration of reduced T(SH)(2) was controlled by TryR (0.98) and oxidative stress (-0.99); however, γECS and TryS also exerted control on the cellular level of T(SH(2)) when they were inhibited by more than 70%. The model predicted that in order to diminish the T(SH)(2) synthesis flux by 50%, it is necessary to inhibit γECS or TryS by 58 or 63%, respectively, or both by 50%, whereas more than 98% inhibition was required for TryR. Hence, simultaneous and moderate inhibition of γECS and TryS appears to be a promising multi-target therapeutic strategy. In contrast, use of highly potent and specific inhibitors for TryR and the antioxidant machinery is necessary to affect the antioxidant capabilities of the parasites., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Methanosarcina acetivorans was cultured in the presence of CdCl(2) to determine the metal effect on cell growth and biogas production. With methanol as substrate, cell growth and methane synthesis were not altered by cadmium, whereas with acetate, cadmium slightly increased both, growth and methane rate synthesis. In cultures metabolically active, incubations for short-term (minutes) with 10 µM total cadmium increased the methanogenesis rate by 6 and 9 folds in methanol- and acetate-grown cells, respectively. Cobalt and zinc but not copper or iron also activated the methane production rate. Methanogenic carbonic anhydrase and acetate kinase were directly activated by cadmium. Indeed, cells cultured in 100 µM total cadmium removed 41-69% of the heavy metal from the culture and accumulated 231-539 nmol Cd/mg cell protein. This is the first report showing that (i) Cd(2+) has an activating effect on methanogenesis, a biotechnological relevant process in the bio-fuels field; and (ii) a methanogenic archaea is able to remove a heavy metal from aquatic environments.

In Saccharomyces cerevisiae, the first committed step in the lysine (Lys) biosynthetic pathway is catalysed by the Lys20 and Lys21 homocitrate synthase (HCS) isoforms. Overexpression of Lys20 resulted in eightfold increased Lys, as well as 2-oxoglutarate pools, which were not attained by overexpressing Lys21 or other pathway enzymes (Lys1, Lys9 or Lys12). A metabolic control analysis-based strategy, by gradually and individually manipulating the Lys20 and Lys21 activities demonstrated that the cooperative and strongly feedback-inhibited Lys21 isoform exerted low control of the pathway flux whereas most of the control resided on the non-cooperative and weakly feedback-inhibited Lys20 isoform. Therefore, the higher control of Lys20 over the Lys flux represents an exception to the dogma of higher pathway control by the strongest feedback-inhibited enzyme and points out to multi-site engineering (HCS isoforms and supply of precursors) to increase Lys synthesis., (© 2011 Blackwell Publishing Ltd.)

The removal, uptake and toxicity of chromium in Euglena gracilis cultured in absence and presence of malate with Cr(VI) or Cr(III) was evaluated. The malate extrusion and the extra- and intracellular Cr(VI) reduction capacity were determined and the contents of molecules with thiol group and ascorbate were also evaluated. Absence of malate in the medium decreased cell growth, increased Cr(III) toxicity, induced faster Cr(VI) disappearance from medium, and increased intracellular and intramitochondrial chromium accumulation. Both chromium species induced soluble and particulate ascorbate-dependent chromate reductase activities. Cells also secreted large amounts of malate and increased intracellular contents of thiol-molecules to bind extracellular and intracellular Cr(III), respectively. The former process was supported by significant increase in malate-producing enzyme activities and the assessment of the Cr-complexes indicated the in situ formation with thiol-molecules. The present results establish new paradigms regarding chromium stress on algae-like microorganisms: (i) Cr(III) may be more toxic than Cr(VI), depending on the culture (or environmental) conditions; (ii) several simultaneous mechanisms are turned on to inactivate chromium species and their toxic effects. These mechanisms, now well understood may further optimize, by genetically modifying E. gracilis, and facilitate the development of strategies for using this protist as potential bio-remediator of chromium-polluted water systems., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Ethanol is one of the most efficient carbon sources for Euglena gracilis. Thus, an in-depth investigation of the distribution of ethanol metabolizing enzymes in this organism was conducted. Cellular fractionation indicated localization of the ethanol metabolizing enzymes in both cytosol and mitochondria. Isolated mitochondria were able to generate a transmembrane electrical gradient (Δψ) after the addition of ethanol. However, upon the addition of acetaldehyde no Δψ was formed. Furthermore, acetaldehyde collapsed Δψ generated by ethanol or malate but not by D-lactate. Pyrazole, a specific inhibitor of alcohol dehydrogenase (ADH), abolished the effect of acetaldehyde on Δψ, suggesting that the mitochondrial ADH, by actively consuming NADH to reduce acetaldehyde to ethanol, was able to collapse Δψ. When mitochondria were fractionated, 27% and 60% of ADH and aldehyde dehydrogenase (ALDH) activities were found in the inner membrane fraction. ADH activity showed two kinetic components, suggesting the presence of two isozymes in the membrane fraction, while ALDH kinetics was monotonic. The ADH Km values were 0.64-6.5 mM for ethanol, and 0.16-0.88 mM for NAD+, while the ALDH Km values were 1.7-5.3 μM for acetaldehyde and 33-47 μM for NAD+. These novel enzymes were also able to use aliphatic substrates of different chain length and could be involved in the metabolism of fatty alcohol and aldehydes released from wax esters stored by this microorganism.

To assess the toxic effect of Cr on energy metabolism, heterotrophic Euglena gracilis was grown in a medium that prompts high yield biomass and in the presence of different Cr(VI) or Cr(III) concentrations. The cell growth IC₅₀ value was 12 and >250μM for Cr(VI) and Cr(III), respectively; in these cells chromium was accumulated and a fraction compartmentalized into mitochondria, and synthesis of cysteine and glutathione was induced. Respiration of control isolated mitochondria was strongly inhibited by added Cr(VI) or Cr(III) with L-lactate or succinate as substrates. In turn, cellular and mitochondrial respiration, respiratory Complexes I, III and IV, glycolysis and cytosolic NAD(+)-alcohol and -lactate dehydrogenases from cells cultured with Cr(VI) were significantly lower than control, whereas AOX and external NADH dehydrogenase activities were unaltered or increased, respectively. Addition of Cr(VI) or Cr(III) to isolated mitochondria or cytosol from control- or Cr(VI)-grown cells induced inhibition of respiration, respiratory Complexes III, IV and AOX, and glycolytic pyruvate kinase; whereas Complex I, external NADH dehydrogenase, and other glycolytic enzymes were unaffected. Protein contents of mitochondrial Complexes I, III, IV and V, and ANT were diminished in Cr(VI)-grown cells. Decreased respiration and glycolysis induced by Cr(VI) resulted in lower cellular ATP content. Results suggested that Cr(VI) cytotoxicity altered gene expression (as widely documented) and hence enzyme content, and induced oxidative stress, but it was also related with direct enzyme inhibition; Cr(III) was also cytotoxic although at higher concentrations. These findings establish new paradigms for chromium toxicity: Cr(VI) direct enzyme inhibition and non-innocuous external Cr(III) toxicity., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Euglena gracilis is a freshwater free-living organism able to grow with ethanol as carbon source; to facilitate this metabolism several alcohol dehydrogenase (ADH) activities have been detected. We report the gene cloning, over-expression, and biochemical characterization of a medium-chain NAD(+)-dependent ADH from E. gracilis (EgADH). The enzyme's amino acid sequence displayed the highest percentages of similarity and identity with ADHs of bacteria and fungi. In the predicted three-dimensional model, all the residues involved in Zn(2+), cofactor, and substrate binding were conserved. A conventional signal peptide for import into mitochondria could not be clearly identified. The protein of 37 kDa was over-expressed, purified to homogeneity, and kinetically characterized. The enzyme's optimal pH was 7.0 for ethanol oxidation displaying a V(m) of 11.7+/-3.6 U/mg protein and a K(m) of 3.2+/-0.7 mM for this substrate. Isopropanol and isopentanol were also utilized, although with less efficiency. It showed specificity for NAD(+) with a K(m) value of 0.39+/-0.1 mM and Mg(2+) or Zn(2+) were essential for activity. The recombinant EgADH reported here may help to elucidate the roles that different ADHs have on the metabolism of short- and long-chain alcohols in this microorganism.

Rhodoquinone (RQ) participates in fumarate reduction under anaerobiosis in some bacteria and some primitive eukaryotes. Euglena gracilis, a facultative anaerobic protist, also possesses significant rhodoquinone-9 (RQ9) content. Growth under low oxygen concentration induced a decrease in cytochromes and ubiquinone-9 (UQ9) content, while RQ9 and fumarate reductase (FR) activity increased. However, in cells cultured under aerobic conditions, a relatively high RQ9 content was also attained together with significant FR activity. In addition, RQ9 purified from E. gracilis mitochondria was able to trigger the activities of cytochrome bc1 complex, bc1-like alternative component and alternative oxidase, although with lower efficiency (higher Km, lower Vm) than UQ9. Moreover, purified E. gracilis mitochondrial NAD+-independent D-lactate dehydrogenase (D-iLDH) showed preference for RQ9 as electron acceptor, whereas L-iLDH and succinate dehydrogenase preferred UQ9. These results indicated a physiological role for RQ9 under aerobiosis and microaerophilia in E. gracilis mitochondria, in which RQ9 mediates electron transfer between D-iLDH and other respiratory chain components, including FR.

The structural and kinetic analyses of the components of the lactate shuttle from heterotrophic Euglena gracilis were carried out. Mitochondrial membrane-bound, NAD(+)-independent d-lactate dehydrogenase (d-iLDH) was purified by solubilization with CHAPS and heat treatment. The active enzyme was a 62-kDa monomer containing non-covalently bound FAD as cofactor. d-iLDH was specific for d-lactate and it was able to reduce quinones of different redox potential values. Oxalate and l-lactate were mixed-type inhibitors of d-iLDH. Mitochondrial l-iLDH also catalyzed the reduction of quinones, but it was inactivated during the extraction with detergents. Both l-iLDH and d-iLDH were inhibited by the specific flavoprotein-inhibitor diphenyleneiodonium, suggesting that l-iLDH was also a flavoprotein. Affinity chromatography revealed that the E. gracilis cytosolic fraction contained two types of NAD(+)-dependent LDH specific for the generation of d- and l-lactate (d-nLDH and l-nLDH, respectively). These two enzymes were tetramers of 126-132 kDa and showed an ordered bi-bi kinetic mechanism. Kinetic properties were different in both enzymes. Pyruvate reduction by d-nLDH was inhibited by its two products; the d-lactate oxidation was 40-fold lower than forward reaction. l-lactate oxidation by l-nLDH was not detected, whereas pyruvate reduction was activated by fructose-1, 6-bisphosphate, K(+) or NH(4)(+). Interestingly, membrane-bound l- and d-lactate dehydrogenases with quinone reductase activity have been only detected in bacteria, whereas the activity of soluble d-nLDH has been identified in bacteria and some yeast. Also, FBP-activated l-nLDH has been found solely in lactic bacteria. Based on their similar kinetic and structural characteristics, a possible common origin among bacterial and E. gracilis lactic dehydrogenase enzymes is discussed.

The synthesis of ATP in the human parasite Entamoeba histolytica is carried out solely by the glycolytic pathway. Little kinetic and structural information is available for most of the pathway enzymes. We report here the gene cloning, overexpression and purification of hexokinase, hexose-6-phosphate isomerase, inorganic pyrophosphate-dependent phosphofructokinase, fructose-1,6 bisphosphate aldolase (ALDO), triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase, phosphoglycerate mutase (PGAM), enolase, and pyruvate phosphate dikinase (PPDK) enzymes from E. histolytica. Kinetic characterization of these 10 recombinant enzymes was made, establishing the kinetic constants at optimal and physiological pH values, analyzing the effect of activators and inhibitors, and investigating the storage stability and oligomeric state. Determination of the catalytic efficiencies at the pH optimum and at pH values that resemble those of the amoebal trophozoites was performed for each enzyme to identify possible controlling steps. This analysis suggested that PGAM, ALDO, GAPDH, and PPDK might be flux control steps, as they showed the lowest catalytic efficiencies. An in vitro reconstruction of the final stages of glycolysis was made to determine their flux control coefficients. Our results indicate that PGAM and PPDK exhibit high control coefficient values at physiological pH.

To assess the expression and physiological role of the mitochondrial NAD(+)-independent lactate dehydrogenase (iLDH) in Euglena gracilis, cells were grown with different carbon sources, and the d- and l-iLDH activities and several key metabolic intermediates were examined. iLDH activity was significant throughout the growth period, increasing by three- to fourfold from latency to the stationary phase. Intracellular levels of D- and L-lactate were high (5-40 mm) from the start of the culture and increased (20-80 mm) when the stationary phase was entered. All external carbon sources were actively consumed, reaching a minimum upon entering the stationary phase, when degradation of paramylon started. The level of ATP was essentially unchanged under all experimental conditions. Oxalate, an inhibitor of iLDH, strongly inhibited oligomycin-sensitive respiration and growth, whereas rotenone, an inhibitor of respiratory complex I, only slightly affected these parameters in lactate-grown cells. Isolated mitochondria exhibited external NADH-supported respiration, which was sensitive to rotenone and flavone, and an inability to oxidize pyruvate. Addition of cytosol, NADH and pyruvate to mitochondria incubated with rotenone and flavone prompted significant O2 uptake, which was blocked by oxalate. The data suggested that iLDH expression in Euglena is independent of substrate availability and that iLDHs play a key role in the transfer of reducing equivalents from the cytosol to the respiratory chain (lactate shuttle).

Some kinetic and thermodynamic properties of the plasma membrane adenylyl cyclase (AC) from the protist Euglena gracilis were examined. The AC kinetics for Mg-ATP was hyperbolic with a K(m) value of 0.33-0.43 mM, whereas the inhibition exerted by 2('),5(')-dideoxyadenosine was of the mixed type with a K(i) of 80-147 microM. The V(m) value (0.9 or 1.8 nmol(mg protein)(-1)min(-1)) changed, depending upon the carbon source in the growth medium (lactic acid or glutamate plus malate). Lactic acid membrane AC was slightly more thermolabile (from 28 to 40 degrees C) and showed higher activation energy (range 15-25 degrees C). With lactate, the total and saturated fatty acid percentage content in the plasma membrane was significantly greater than with glutamate plus malate, whereas the percentage content of polyunsaturated (n-3) fatty acids was lower. The data suggest that the fatty acid composition, as changed by the carbon source in the growth medium, may modulate the AC activity in Euglena.

The activity of the pyridine nucleotide-independent lactate dehydrogenase (iLDH) was characterized in mitochondria isolated from the protist Euglena gracilis. The dissociation constants for L- and D-lactate were similar, but the V(max) was higher with the d isomer. A ping-pong kinetic mechanism was displayed with 2,4-dichlorophenol-indolphenol (DCPIP), or coenzyme Q(1), reacting as the second substrate with the modified, reduced enzyme. Oxamate was a competitive inhibitor against both L- and D-lactate. Oxalate exerted a mixed-type inhibition regarding L- or D-lactate and also against DCPIP. The rate of L-lactate uptake was partially inhibited by mersalyl and lower than the rate of dehydrogenation, which was mersalyl-insensitive. These data suggested that the active site of L-iLDH was orientated toward the intermembrane space. The following observations indicated the existence of two stereo-specific iLDH enzymes in the inner membrane of Euglena mitochondria: a greater affinity of the D-iLDH for both inhibitors, D-iLDH thermo-stability at 70 degrees C and denaturation of L-iLDH, opposite signs in the enthalpy change for the association reaction of the isomers to the enzyme, differential solubilization of both activities with detergents, and different molecular mass., (Copyright 2001 Academic Press.)

The effect of antimycin, myxothiazol, 2-heptyl-4-hydroxyquinoline-N-oxide, stigmatellin and cyanide on respiration, ATP synthesis, cytochrome c reductase, and membrane potential in mitochondria isolated from dark-grown Euglena cells was determined. With L-lactate as substrate, ATP synthesis was partially inhibited by antimycin, but the other four inhibitors completely abolished the process. Cyanide also inhibited the antimycin-resistant ATP synthesis. Membrane potential was collapsed (<60 mV) by cyanide and stigmatellin. However, in the presence of antimycin, a H(+)60 mV) that sufficed to drive ATP synthesis remained. Cytochrome c reductase, with L-lactate as donor, was diminished by antimycin and myxothiazol. Cytochrome bc(1) complex activity was fully inhibited by antimycin, but it was resistant to myxothiazol. Stigmatellin inhibited both L-lactate-dependent cytochrome c reductase and cytochrome bc(1) complex activities. Respiration was partially inhibited by the five inhibitors. The cyanide-resistant respiration was strongly inhibited by diphenylamine, n-propyl-gallate, salicylhydroxamic acid and disulfiram. Based on these results, a model of the respiratory chain of Euglena mitochondria is proposed, in which a quinol-cytochrome c oxidoreductase resistant to antimycin, and a quinol oxidase resistant to antimycin and cyanide are included.