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Pseudomonas aeruginosa is the main bacterial model to study cooperative behaviors, since it yields exoproducts such as exoproteases and siderophores that act as public goods and can be exploited by selfish non-producers that behave as social cheaters. Non-producers of the siderophore pyoverdine are typically isolated in media with low free iron, mainly casamino acids medium supplemented with transferrin. Nevertheless, using a protein as the iron chelator could additionally select mutants unable to produce exoproteases that degrade the transferrin to facilitate iron release. Here, we investigated the dynamics of pyoverdine and exoprotease production in media in which iron was limited by using either transferrin or a cation chelating resin. Our experiments show that concomitant loss of pyoverdine and exoprotease production readily develops in media with transferrin whereas only lack of pyoverdine emerges in medium treated with the resin. Genomic characterization of the exoprotease- and pyoverdine-less mutants revealed large deletions (13 to 33 Kb) including Quorum Sensing (lasR, rsal and lasl) and flagellar genes. Complementation experiments, PCR and motility tests confirmed the deletions. 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 observed in our experiment settings can help revisiting or informing the design of similar studies.

The four hydrogenase isozymes; hydrogenase 1 (Hyd-1), hydrogenase 2 (Hyd-2), hydrogenase 3 (Hyd-3) and hydrogenase 4 (Hyd-4) of Escherichia coli have been reported for their crucial functions in the hydrogen metabolism; however, their distinctive roles could not be completely understood. In this study, four ideal hydrogenase operon mutants, Δhyb hyc hyf, Δhya hyc hyf, Δhya hyb hyf, and Δhya hyb hyc, in which only a single hydrogenase is intact in the genome, were constructed as well as one quadruple mutant (Δhya hyb hyc hyf) that all four hydrogenase operons were deleted. First, single operon mutants and single-gene mutants for each hydrogenase showed different hydrogen productivity and growth in the anaerobic fermentation, indicating that bacterial phenotype regarding the hydrogen metabolism via the deletion of each operon is different with that of each single gene. Then, 4 triple hydrogenase operon mutants and one quadruple mutant were investigated to evaluate the hydrogen metabolism (hydrogen production and uptake) using glucose or glycerol as a substrate of hydrogen fermentation. With both the carbon sources, only Hyd-2 and Hyd-3 were able to produce hydrogen. Furthermore, all the hydrogenases showed hydrogen uptake activity. In addition, no hydrogen production and hydrogen uptake were detected in the quadruple mutant which does not have all 4 hydrogenases. Hydrogen production from Hyd-2 and Hyd-3 was further confirmed by complementing their operons in the cloning vector pBR322.

Chagas disease (CD) is recognized as a neglected tropical disease. It is endemic of Latin America, but globalization has led to its spread worldwide. Even though its presence has been tracked at least 9000 years ago, treatment options remain scarce. The purpose of this review is to analyze the evidence for current and future options to combat the disease. CD patients benefit from dietary and exercise recommendations. Benznidazole and nifurtimox continue to be the available treatment options. Besides them, several drugs have been developed, repurposed, and tested in clinical trials, mostly inhibitors of the ergosterol synthesis like albaconazole, itraconazole, posaconazole, ravuconazole/fosravuconazole, voriconazole, and TAK-187; antiarrhythmics like amiodarone/dronedarone; inhibitors of the purine synthesis like allopurinol; nitroimidazoles like fexinidazole; and minerals like selenium. Even though they have shown in vitro and in vivo activity against Trypanosoma cruzi, their effectiveness is inferior to the one from benznidazole. Chronic CD patients who develop the cardio-digestive forms of the disease may eventually need assistive devices and/or surgical procedures to improve their outcome. Benznidazole and nifurtimox are the only trypanosomal drugs available to combat T. cruzi infection, and their effectiveness relies upon the stage of the disease at where the patients are. Their side effects frequently lead to patient’s non-compliance to treatment. Therefore, more effective drugs with a better safety profile are required. Up to date, controversial and promising results have been observed. Further compromise and research must be encouraged to tackle the challenges of developing new effective and safer drugs for CD patients.

Clinical evidence has shown that bacterial infections are more difficult to eradicate when form-ing a biofilm aggregate than when are produced by bacteria in planktonic form. Therefore, com-pounds that inhibit biofilm formation could be used against severe infections. It has been re-ported that bromo 2-(5H) furanones inhibited biofilm formation by their anti-quorum sensing properties. To determine if the 2-(5H) furanone moiety is essential to induce inhibition of biofilm formation, we evaluated ten halogen 2-(5H) furanones derivates previously synthesized. Besides evaluating the inhibition of biofilm formation, we assessed pyocyanin production, swarming motility, and transcription of essential QS genes: rsaL, rhlA, pqsA and phz1 genes. Our results showed that although three bromo-furan-2(5H)-one-type derivatives (A1-A3) and two bromo-4-(phenylamino)-furan-2(5H)-one-type compounds (B2 and B6) inhibited the biofilm formation in both P. aeruginosa PA14 (reference) and PA64 (drug-resistant) strains only the furanones A1-A3 were efficient to inhibit QSS.

Several plant extracts exhibit anti-virulence properties due to the interruption of bacterial quorum sensing (QS). However, studies on their effects at the preclinical level are scarce. Here, we used a murine model of abscess/necrosis induced by Pseudomonas aeruginosa to evaluate the anti-pathogenic efficacy of 24 plant extracts at a sub-inhibitory concentration. We analyzed their ability to inhibit QS-regulated virulence factors such as swarming, pyocyanin production, and secretion of the ExoU toxin via the type III secretion system (T3SS). Five of the seven extracts with the best anti-pathogenic activity reduced ExoU secretion, and the extracts of Diphysa americana and Hibiscus sabdariffa were identified as the most active. Therefore, the abscess/necrosis model allows identification of plant extracts that have the capacity to reduce pathogenicity of P. aeruginosa. Furthermore, we evaluated the activity of the plant extracts on Chromobacterium violaceum. T3SS (ΔescU) and QS (ΔcviI) mutant strains were assessed in both the abscess/necrosis and sepsis models. Only the ΔescU strain had lower pathogenicity in the animal models, although no activity of plant extracts was observed. These results demonstrate differences between the anti-virulence activity recorded in vitro and pathogenicity in vivo and between the roles of QS and T3S systems as virulence determinants.

Bdellovibrio bacteriovorus is one of the predatory bacteria; therefore, it can act as a novel “living antibiotic,” unlike the current antibiotics. Here the predation of Escherichia coli by B. bacteriovorus was inhibited in the presence of Pseudomonas aeruginosa. This study investigated whether P. aeruginosa-induced predation inhibition is associated with bacterial quorum sensing (QS). Each las, rhl, or pqs QS mutant in P. aeruginosa was used to check the predatory activity of E. coli cells using B. bacteriovorus. As a result, the predatory activity of B. bacteriovorus increased in a mutant pqs QS system, whereas wild-type PA14 inhibited the predatory activity. Moreover, the addition of 4-hydroxy-2-heptylquinoline (HHQ) or the analog triggered the low predatory activity of B. bacteriovorus and killed B. bacteriovorus cells. Therefore, a defensive action of P. aeruginosa against B. bacteriovorus is activated by the pqs QS system, which produces some quinolone compounds such as HHQ.

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.

Antibiotic failure not only is due to the development of resistance by pathogens but can also often be explained by persistence and tolerance. Persistence and tolerance can be included in the “persistent phenotype,” with high relevance for clinics. Two of the most important molecular mechanisms involved in tolerance and persistence are toxin-antitoxin (TA) modules and signaling via guanosine pentaphosphate/tetraphosphate [(p)ppGpp], also known as “magic spot.” (p)ppGpp is a very important stress alarmone which orchestrates the stringent response in bacteria; hence, (p)ppGpp is produced during amino acid or fatty acid starvation by proteins belonging to the RelA/SpoT homolog family (RSH). However, (p)ppGpp levels can also accumulate in response to a wide range of signals, including oxygen variation, pH downshift, osmotic shock, temperature shift, or even exposure to darkness. Furthermore, the stringent response is not only involved in responses to environmental stresses (starvation for carbon sources, fatty acids, and phosphates or heat shock), but it is also used in bacterial pathogenesis, host invasion, and antibiotic tolerance and persistence. Given the exhaustive and contradictory literature surrounding the role of (p)ppGpp in bacterial persistence, and with the aim of summarizing what is known so far about the magic spot in this bacterial stage, this review provides new insights into the link between the stringent response and persistence. Moreover, we review some of the innovative treatments that have (p)ppGpp as a target, which are in the spotlight of the scientific community as candidates for effective antipersistence agents.

Blocking virulence is a promising alternative to counteract Pseudomonas aeruginosa infections. In this regard, the phenomenon of cell-cell communication by quorum sensing (QS) is an important anti-virulence target. In this field, fatty acids (FA) have gained notoriety for their role as autoinducers, as well as anti-virulence molecules in vitro, like some saturated FA (SAFA). In this study, we analyzed the anti-virulence activity of SAFA with 12 to18 carbon atoms and compared their effect with the putative autoinducer cis-2-decenoic acid (CDA). The effect of SAFA on six QS-regulated virulence factors and on the secretion of the exoenzyme ExoU was evaluated. In addition, a murine cutaneous infection model was used to determine their influence on the establishment and damage caused by P. aeruginosa PA14. Dodecanoic (lauric, C12:0) and tetradecanoic (myristic, C14:0) acids (SAFA C12-14) reduced the production of pyocyanin by 35–58% at 40 and 1,000 µM, while CDA inhibited it 62% at a 3.1 µM concentration. Moreover, the SAFA C12-14 reduced swarming by 90% without affecting biofilm formation. In contrast, CDA reduced the biofilm by 57% at 3 µM but did not affect swarming. Furthermore, lauric and myristic acids abolished ExoU secretion at 100 and 50 µM respectively, while CDA reduced it by ≈ 92% at 100 µM. Remarkably, the coadministration of myristic acid (200 and 1,000 µM) with P. aeruginosa PA14 induced greater damage and reduced survival of the animals up to 50%, whereas CDA to 500 µM reduced the damage without affecting the viability of the PA14 strain. Hence, our results show that SAFA C12-14 and CDA have a role in regulation of P. aeruginosa virulence, although their inhibition/activation molecular mechanisms are different in complex environments such as in vivo systems.

Pseudomonas aeruginosa is one of the main models to study social behaviors in bacteria since it synthesizes several exoproducts, including exoproteases and siderophores and release them to the environment. Exoproteases and siderophores are public goods that can be utilized by the individuals that produce them but also by non-producers, that are considered social cheaters. Molecularly exoprotease cheaters are mutants in regulatory genes such as lasR, and are commonly isolated from chronic infections and selected in the laboratory upon serial cultivation in media with protein as a sole carbon source. Despite that the production of exoproteases is exploitable, cooperators have also ways to restrict the growth and selection of social cheaters, for instance by producing toxic metabolites like pyocyanin. In this work, using bacterial competitions, serial cultivation and growth assays, we demonstrated that rhamnolipids which production is regulated by quorum sensing, selectively affect the growth of lasR mutants and are able to restrict social cheating, hence contributing to the maintenance of cooperation in Pseudomonas aeruginosa populations.

Hydrogen production by photofermentation of tequila vinasses (VT) was studied using Rhodopseudomonas pseudopalustris DSM 123. To the best of our knowledge this is the first report on hydrogen production by photofermentation on VT. Hydrogen production was doubled on VT (260 m$props_value{literPattern}/L) as compared to a synthetic medium. Storing VT changed its chemical composition; however, in photofermentations of a sixty-day old stock, growth and hydrogen production were similar to fresh VT. The periodic displacement of hydrogen with nitrogen resulted in a three-fold increase in both hydrogen and growth of R. pseudopalustris (860 mL H2/L and 4.5 g/L respectively) as compared to non-displaced headspace. Hydrogen almost doubled at a light intensity of 270 W/m2 (2249 mL H2) as compared to 68 W/m2. In dark-light cycles, biomass and hydrogen production were highest with continuous illumination. The potential of VT to produce hydrogen in high amounts using R. pseudopalustris has been demonstrated.

Seed oils from oleaginous plants are rich in fatty acids (FAs) that play important roles in the health of the consumers. Recent studies indicate that FA also can play an important role in communication and regulation of virulence in bacteria. Nevertheless, evidence demonstrating protection against bacterial infections mediated by their quorum sensing inhibition (QSI) activity is scarce. In this study, sunflower, chia, and amaranth oils, were assayed for their QSI capacity by inhibiting violacein production and alkaline exoprotease activity of Chromobacterium violaceum. In vitro assays revealed that the oils exhibited QSI activities, whereas in vivo they delayed death of mice inoculated intraperitoneally with the bacterium. Gas chromatography coupled with mass spectrometry analysis of the oils indicated the presence of saturated FA (SAFA) and unsaturated FA as main components. Through a structure-activity relationship study of free FAs, bactericidal effect was identified mainly for polyunsaturated FAs, whereas QSI activity was restricted to SAFA of chains 12-18 carbon atoms in length. These data correlate with a possible interaction suggested by molecular docking analysis of lauric, myristic, and stearic acids with the CviR protein. Our study highlights the antiquorum sensing potential of SAFA, which may be future antivirulence therapeutic agents for the treatment of bacterial infections.

Euglena gracilis is a photosynthetic protist with proved ability to resist and remove toxic single heavy metals under different laboratory conditions. To assess these abilities under more realistic conditions, cells subjected to combinations of heavy metals in a defined culture medium as well as in field polluted enriched medium were analyzed. Cells were cultured in the presence of Cd2+ plus Cu2+ or Cr(VI) plus Fe3+ under aerobiosis or microaerophilia. Under aerobic growth, Cd2+ plus Cu2+ (50 + 80 μM) and Cr(VI) plus Fe3+ (2 + 1 mM) strongly inhibited cell growth by inducing severe oxidative stress. In contrast, under microaerophilia, higher resistance and heavy metal removal were attained. This was indicated by (i) substantial biomass production; (ii) greater activities of glutathione reductase, glutathione peroxidase and ascorbate peroxidase as well as greater amounts of intracellular glutathione and ascorbate; (iii) increased metal removal by biosorption (i.e. greater than 80% for Cd, Cr, and Fe) due to an enhanced biofilm synthesis. The fraction of intracellularly heavy metal accumulated was bound mostly by polyphosphates (polyP). Cells cultured under microaerophilia in heavy metal polluted wastewater and sediments media were also able to grow and remove Fe, Cr, Cu and Cd. It is concluded that microaerophilia favors, the resistance of photosynthetic Euglena gracilis to heavy metal toxicity, and its ability to remove such metals from polluted water and solid samples. This work provides the biochemical basis for designing genetically engineered microorganisms with enhanced efficiency in metal removal. Moreover, it brings forward the combination of physicochemical and biological techniques to profit recovery.

The tryptophan metabolite, quinolinic acid (QUIN), and the mitochondrial toxin 3-nitropropionic acid (3-NP) are two important tools for toxicological research commonly used in neurotoxic models of excitotoxicity, oxidative stress, energy depletion, and neuronal cell death in mammals. However, their toxic properties have yet to be explored in the nematode Caenorhabditis elegans (C. elegans) for the establishment of novel, simpler, complementary, alternative, and predictive neurotoxic model of mammalian neurotoxicity. In this work, the effects of QUIN (1-100 mM) and 3-NP (1-10 mM) were evaluated on various physiological parameters (survival, locomotion, and longevity) in a wild-type (WT) strand of C. elegans (N2). Their effects were also tested in the VC1772 strain (knock out for the antioxidant SKN-1 pathway) and the VP596 strain (worms with a reporter gene for glutathione S-transferase (GST) transcription) in order to establish the role of the SKN-1 pathway in the mode of action of QUIN and 3-NP. In N2, the higher doses of both toxins decreased survival, though only QUIN altered motor activity. Both toxins also reduced longevity in the VC1772 strain (as compared to N2 strain) and augmented GST transcription in the VP596 strain at the highest doses. The changes induced by both toxins require high doses, and therefore appear moderate when compared with other toxic agents. Nevertheless, the alterations produced by QUIN and 3-NP in C. elegans are relevant to mammalian neurotoxicity as they provide novel mechanistic approaches to the assessment of neurotoxic events comprising oxidative stress and excitotoxicity, in the nematode model.

Caffeic acid (CA) is a hydroxycinnamic acid derivative and polyphenol with antioxidant and anti-inflammatory activities. The neuroprotective properties of CA still need detailed characterization in different biological models. Here, the antioxidant and neuroprotective effects of CA were compared in in vitro and in vivo neurotoxic models. Biochemical outcomes of cell dysfunction, oxidative damage, and transcriptional regulation were assessed in rat cortical slices, whereas endpoints of physiological stress and motor alterations were characterized in Caenorhabditis elegans (C. elegans). In rat cortical slices, CA (100 mu M) prevented, in a differential manner, the loss of reductive capacity, the cell damage, and the oxidative damage induced by the excitotoxin quinolinic acid (QUIN, 100 mu M), the pro-oxidant ferrous sulfate (FeSO4, 25 mu M), and the dopaminergic toxin 6-hydroxydopamine (6-OHDA, 100 mu M). CA also restored the levels of nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE; a master antioxidant regulatory pathway) binding activity affected by the three toxins. In wild-type (N2) of C. elegans, but not in the skn-1 KO mutant strain (worms lacking the orthologue of mammalian Nrf2), CA (25 mM) attenuated the loss of survival induced by QUIN (100 mM), FeSO4 (15 mM), and 6-OHDA (25 mM). Motor alterations induced by the three toxic models in N2 and skn-1 KO strains were prevented by CA in a differential manner. Our results suggest that (1) CA affords partial protection against different toxic insults in mammalian brain tissue and in C. elegans specimens; (2) the Nrf2/ARE binding activity participates in the protective mechanisms evoked by CA in the mammalian cortical tissue; (3) the presence of the orthologous skn-1 pathway is required in the worms for CA to exert protective effects; and (4) CA exerts antioxidant and neuroprotective effects through homologous mechanisms in different species.

Pseudomonas aeruginosa is an opportunistic bacterium associated with healthcare infections in intensive care units (ICUs), ventilator-associated pneumonia (VAP), surgical site infections, and burns. This bacterium causes 75% of death in burned patients, since it can develop a persistent biofilm associated with infections, express several virulence factors, and antibiotic-resistance mechanisms. Some of these virulence factors are proteases such as elastase and alkaline protease, or toxic metabolites such as pyocyanin and is one of the few microorganisms able to produce cyanide, which inhibits the cytochrome oxidase of host cells. These virulence factors are controlled by quorum sensing (QS). In this work, 30 P. aeruginosa clinical strains isolated from burned patients from a tertiary hospital in Mexico City were studied. Antibiotic susceptibility tests were done, and virulence factors (elastase, alkaline protease, HCN, and pyocyanin) were determined in presence of an N-acylhomoserine lactonase, AiiM able to hydrolyze a wide range of acyl homoserine lactones. The treatment reduced significantly the activities of elastase and alkaline protease, and the production of pyocyanin and HCN in all producer strains but not the secretion of toxins through the type III secretion system. Our work suggests that AiiM treatment may be an effective therapy to combat P. aeruginosa infection in burn patients.

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.

Acinetobacter baumannii is well adapted to hospital environments, and the persistence of its chronic infections is mainly due to its ability to form biofilms resistant to conventional antibiotics and host immune systems. Hence, the inhibitions of biofilm formation and virulence characteristics provide other means of addressing infections. In this study, the antibiofilm activities of twelve flavonoids were initially investigated. Three most active flavonoids, namely, fisetin, phloretin, and curcumin, dose-dependently inhibited biofilm formation by a reference A. baumannii strain and by several clinical isolates, including four multidrug-resistant isolates. Furthermore, the antibiofilm activity of curcumin (the most active flavonoid) was greater than that of the well-known biofilm inhibitor gallium nitrate. Curcumin inhibited pellicle formation and the surface motility of A. baumannii. Interestingly, curcumin also showed antibiofilm activity against Candida albicans and mixed cultures of C. albicans and A. baumannii. In silico molecular docking of the biofilm response regulator BfmR showed that the binding efficacy of flavonoids with BfmR was correlated with antibiofilm efficacy. In addition, curcumin treatment diminished A. baumannii virulence in an in vivo Caenorhabditis elegans model without cytotoxicity. The study shows curcumin and other flavonoids have potential for controlling biofilm formation by and the virulence of A. baumannii.

The assessment of phenotypic variation across populations can help to select genotypes that are potentially resistant to the ongoing and predicted climatic variations. Phenotypic variation is expressed as morphological, physiological, or metabolic changes in response to environmental factors. Agave mapisaga and A. salmiana populations exhibit morphological variation regarding their geographical distribution and due to intense anthropogenic influence. This study aimed to identify metabolic traits in young plants of A. mapisaga and A. salmiana, involved in plant resistance to temperature stress, which can help to understand differences between provenances and between species. Agave plants propagated from four seed origins, collected from three “pulque” (a fermented beverage) production areas in the state of Hidalgo, Mexico, were assessed. Four-month-old plants of each seed origin (species*provenance) were maintained during one month in growth chambers, at day/night temperature (T) regimes for 12/12 h: (1) 25/25T, (2) 25/15T, and (3) 25/15 plus 45 °C T for 4 h between 11 am and 3 pm (45T). The plant dry biomass, maximum quantum efficiency of PSII photochemistry (Fv/Fm), total free amino acids (TFAA), total free phenol (TFP), and free soluble sugar concentrations were measured. Minimum values for dry biomass, TFP, glucose, and sucrose concentrations occurred in the 25/25T regime. Plant biomass, and TFAA and glucose concentrations were differently affected by seed provenances; but Fv/Fm rate, or the TFP, fructose, and sucrose concentrations had no relation to seed provenance. Moderate high night temperatures drastically affected the growth and metabolism of A. mapisaga and A. salmiana. In conclusion, the response to different temperature stress treatments depends partly on species, but less on seed provenance.

Protocols for seed germination in the Opuntia genus are different and unsuitable for all their species. Dormancy of Opuntia seeds can be modified by the combination of scarification and an oxidizing agent such as O3, which could induce antioxidant and DNA-repair mechanisms or dormancy-breaking effects in hydrated seeds. The objective of this study was to evaluate the effects that the combination of mechanical and chemical scarification with exposition to sub-lethal O3 doses have on seed germination and seedling growth of O. streptacantha Lem., O. megacantha Salm-Dyck, and O. ficus-indica (L.) Mill. Our hypothesis was that O3 favors germination on scarified seeds and that the magnitude of the effect is species-dependent. Water uptake and germination were quantified in seeds every 48 h, until their roots reached a 5-mm length. The results were analyzed with an analysis of variance and multiple means comparisons with the Tukey test. Accelerated water uptake was observed during the first 48 h; the max...

Caffeic acid (CA) is a hydroxycinnamic acid derivative and polyphenol with antioxidant and anti-inflammatory activities. The neuroprotective properties of CA still need detailed characterization in different biological models. Here, the antioxidant and neuroprotective effects of CA were compared in in vitro and in vivo neurotoxic models. Biochemical outcomes of cell dysfunction, oxidative damage and transcriptional regulation were assessed in rat cortical slices, whereas endpoints of physiological stress and motor alterations were characterized in Caenorhabditis elegans (C. elegans). In rat cortical slices, CA (100 μM) prevented, in a differential manner, the loss of reductive capacity, the cell damage, and the oxidative damage induced by the excitotoxin quinolinic acid (QUIN, 100 μM), the pro-oxidant ferrous sulfate (FeSO(4), 25 μM), and the dopaminergic toxin 6-hydroxydopamine (6-OHDA, 100 μM). CA also restored the levels of nuclear factor erythroid 2-related factor 2/Antioxidant Response Element (Nrf2/ARE; a master antioxidant regulatory pathway) binding activity affected by the three toxins. In wild-type (N2) of C. elegans, but not in the skn-1 KO mutant strain (worms lacking the orthologue of mammalian Nrf2), CA (25 mM) attenuated the loss of survival induced by QUIN (100 mM), FeSO(4) (15 mM) and 6-OHDA (25 mM). Motor alterations induced by the three toxic models in N2 and skn-1 KO strains were prevented by CA in a differential manner. Our results suggest that: (1) CA affords partial protection against different toxic insults in mammalian brain tissue and in C. elegans specimens; (2) the Nrf2/ARE binding activity participates in the protective mechanisms evoked by CA in the mammalian cortical tissue; (3) the presence of the orthologous skn-1 pathway is required in the worms for CA to exert protective effects; and (4) CA exerts antioxidant and neuroprotective effects through homologous mechanisms in different species.

Quorum sensing (QS) plays a key role in activating bacterial functions through small molecules called autoinducers. In this study, the QS of Gram-negative bacteria in waste sewage sludge (WSS) was downregulated by adding the quorum quenching enzyme, AiiM lactonase, which cleaved the acyl-homoserine lactone (AHL) autoinducer signals from Gram-negative bacteria, and subsequently methane production was inhibited by over 400%. The pH was lowered after 2 days in the anaerobic fermentation whereas protease activity at the hydrolysis step was almost the same with or without AiiM. The production of acetic acid significantly increased during the fermentation in the presence of AiiM. The bacterial community at day 2 indicated that the population of Gram-positive bacteria increased in the presence of AiiM, and the percentage of Gram-negative bacteria decreased in the WSS containing AiiM. The change in the bacterial community in the presence of AiiM may be due to the different antimicrobial agents produced in the WSS because some of the Gram-positive bacteria were killed by adding the solid-phase extraction (SPE) fraction from the WSS without AiiM. In contrast, the SPE fraction with AiiM had reduced bactericidal activity against Gram-negative bacteria. Thus, bacterial signaling between Gram-negative bacteria is critical for methane production by the microbial consortia.

Molecules exhibiting antioxidant, neuroprotective, and regulatory properties inherent to natural products consumed by humans are gaining attention in biomedical research. Ferulic acid (FA) is a phenolic compound possessing antioxidant and cytoprotective properties. It is found in several vegetables, including sugarcane, where it serves as the main antioxidant component. Here, we compared the antioxidant and cytoprotective effects of FA with those of the total sugarcane aqueous extract (SCAE). Specifically, we assessed biochemical markers of cell dysfunction in rat cortical brain slices and markers of physiological stress in Caenorhabditis elegans upon exposure to toxins evoking different mechanisms of neurotoxicity, including direct oxidative stress and/or excitotoxicity. In rat cortical slices, FA (250 and 500 μM), but not SCAE (~ 270 μM of total polyphenols), prevented the loss of reductive capacity induced by the excitotoxin quinolinic acid (QUIN, 100 μM), the pro-oxidant agent ferrous sulfate (FeSO4, 25 μM), and the dopaminergic pro-oxidant 6-hydroxydopamine (6-OHDA, 100 μM). In wild-type (N2) C. elegans, FA (38 mM) exerted protective effects on decreased survival induced by FeSO4 (15 mM) and 6-OHDA (25 mM), and the motor alterations induced by QUIN (100 mM), FeSO4, and 6-OHDA. In contrast, SCAE (~ 13.5 mM of total polyphenols) evoked protective effects on the decreased survival induced by the three toxic agents, the motor alterations induced by FeSO4, and the reproductive deficit induced by FeSO4. In addition, FA was unable to reverse the decreased survival induced by all these toxins in the skn-1-/- strain (VC1772), which lacks the homolog of mammalian Nrf2, a master antioxidant gene. Altogether, our results suggest that (1) both FA and SCAE afford protection against toxic conditions, (2) not all the effects inherent to SCAE are due to FA, and (3) FA requires the skn-1 pathway to exert its protective effects in C. elegans.

Although bacteria are unicellular organisms, they use a sophisticated mechanism to cooperate with other bacteria which allows them to carry out multicellular-like processes, such as swarming, biofilm formation, induce infections, luminescence, etc. This mechanism is known as quorum sensing (QS) and it functions through the individual release, diffusion and collective sensing of small molecules, called autoinducers, which under their accumulation lead to changes in gene regulation. A range of approaches have been used to study the complex way in which QS works, these tools combine biology, physics, and mathematics. We discuss here how a multidisciplinary perspective is ideal to study QS and present an example of QS in Pseudomonas aeruginosa, a human pathogen. In this bacterial species, QS coordinates the expression of virulence factors, that are public goods utilized by the whole population of bacteria regardless if they invested or not in their production. Hence, public good production is a cooperative behavior, susceptible to be exploited by non-public good producers, known as social cheaters. These individuals, which are QS-deficient, are also less able to tolerate oxidative stress. Our group has shown that pyocyanin (which promotes the generation of reactive oxygen species) produced by the wild-type population may work as a policing mechanism to select functional QS systems in this bacterium. Using an agent-based model (ABM) we are able to confirm that indeed this compound increases the fitness of the cooperative QS proficient individuals. We further explore, with the ABM, how the bacterial environment diffusion properties may be contributing to stabilize QS in certain growth conditions.

UNLABELLED Quorum quenching decreases Pseudomonas aeruginosa virulence factors and biofilm formation, alleviating infections in animal models. Nevertheless, it is usually performed in laboratory strains such as PAO1 and PA14, and studies involving clinical or environmental isolates are scarce. In this work, the effects of ZnO nanoparticles, a potent quorum and virulence quencher for the PAO1 strain, were tested in six clinical strains from cystic fibrosis patients, a furanone C-30 resistant clinical strain from urine, two PA14 gallium resistant mutants, a PA14 C-30 resistant mutant and four environmental isolates. ZnO nanoparticles effectively decreased elastase, pyocyanin, and biofilm formation for most of the strains; regardless their origin or their resistance against the canonical quorum quencher C-30 or the novel antimicrobial gallium. The data indicate ZnO nanoparticles may have a broad spectrum for the quorum quenching of relevant strains and that may be an alternative to treat Ps. aeruginosa recalcitrant infections. SIGNIFICANCE AND IMPACT OF THE STUDY Virulence inhibition by quorum quenchers in Pseudomonas aeruginosa is usually tested in laboratory strains and studies of their effects in relevant clinical and environmental strains are scarce. This study is significant as the effects of ZnO nanoparticles in QS-dependent virulence factor production were tested in six clinical strains from cystic fibrosis patients, a C-30 resistant clinical strain from urine, two PA14 gallium resistant mutants, a PA14 C-30 resistant mutant, and four environmental isolates. ZnO nanoparticles decreased elastase, pyocyanin, and biofilms for most of the strains; indicating they have broad spectrum and may be an alternative to treat Ps. aeruginosa infections.

A gradient of domestication based on morphological characteristics has been observed in species of Opuntia genus; but, a biophysical and physiological gradient have been not described. This study aimed to quantify biophysical and physiological characteristics in nopalitos (edible young cladodes of flat-stemmed spiny cacti) of 15 variants of five Opuntia species in a domestication gradient, including wild (O. streptacantha Lem.), semi-wild (O. hyptiacantha F.A.C. Weber), semi-domesticated (O. megacantha Salm-Dyck), more domesticated (O. albicarpa Scheinvar), and that recognized with the highest degree of domestication [O. ficus-indica (L.) Miller]. We hypothesized that biophysical and physiological characteristics of nopalitos are modified throughout the domestication process and that such changes will be evident when variants with different degree of domestication are grown under drought conditions. Tissue firmness, water potential (ψW), osmotic potential (ψπ), content of total soluble solids (TSS), content of malic acid, and membranes permeability were evaluated in nopalitos harvested from plants after 60 days without watering. A randomized design, with six replications and a plant as experimental unit by treatment, was used. The data were analysed by ANOVA, multiple comparisons of means by Tukey test (P ≤ 0.05) among variants and species, and multivariate analysis of principal components. Firmness (from 4.6 ± 0.082 to 6.8 ± 0.053 kgf/cm2), TSS (from 4.86 ± 0.105 to 6.33 ± 0.142 °Brix), membrane permeability (from 26 ± 0.712 to 31 ± 0.474 (µS cm−1)/g−1) and malic acid content (from 0.61 ± 0.008 to 0.82 ± 0.014 g 100 g−1 wet matter) increased during domestication, and simultaneously ψW and ψS decreased (from −0.85 ± 0.034 to −1.1 ± 0.030 MPa and −1.01 ± 0.011 to −1.15 ± 0.011 MPa). The firmness, SST and ψS are the main characteristics that contributed to separate the Opuntia species on the gradient of domestication. Opuntia species on a domestication gradient based on morphometric characteristics of plants (cladodes, nopalitos and cactus pear fruits) correlate to biophysical and physiological characteristics of nopalitos.

Low soil water availability is the major environmental factor limiting plant growth and yield. The objective of this study was to elucidate the mechanisms underlying photosynthesis inhibition during water stress and recovery in Capsicum annuum L. cv. Cannon by evaluating soil and plant water relations, gas exchange and the prompt fluorescence rise OJIP. The soil ( Ψ S ) and leaf ( Ψ L ) water potential decreased from −0.16 and −0.53 to −1.1 and −1.7 MPa, respectively, and recovered after re-watering. The stomatal conductance ( g s ) decreased to 114 and 13 mmol m −2 s −1 under moderate and severe water stress, respectively. Similarly, the CO 2 assimilation ( A ) and transpiration ( Tr ) rates decreased during water stress but recovered after re-watering. During severe water stress, photosynthesis decreased due to stomatal closure and to both slower maximum carboxylation rate ( V cmax ) and ribulose 1,5-bisphosphate (RuBP) regeneration capacity mediated by maximum electron transport rate ( J max ). In fact, the fluorescence parameters reflecting the electron flow from the intersystem carriers to final reduction of photosystem I (PSI) end electron acceptors declined throughout water deficit development. In conclusion, water stress mainly damaged the electron transfer from the plastoquinone (PQ) pool to the PSI terminal acceptors; this, along with constraints to both stomatal and non-stomatal components of photosynthesis, limited carbon assimilation. Photosynthesis recovery after re-watering was mainly restricted by both stomatal conductance and the gradual recovery of the electron transport chain. Finally, JIP-test parameters that quantifying electron transfer from the PQ pool to the PSI end acceptors are effectives for monitoring water stress in crop plants.

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.

Background and Aims Quorum sensing (QS) is a process of bacterial cell-cell communication that controls a large number of systems affecting pathogenicity. Interrupting this communication system can provide nonvirulent pathogenic bacteria. The aim of this study was to evaluate the anti-quorum sensing (anti-QS) potential of an anacardic acids mixture isolated from Amphipterygium adstringens, a medicinal plant known as "cuachalalate", to prevent the onset of bacterial infections as an alternate to antibiotics. Methods Initially we investigated the anti-QS activity of A. adstringens hexane extract (HE) by the inhibition of violacein production in Chromobacterium violaceum . From the active HE, an anacardic acid mixture (AAM) was obtained. The anti-quorum sensing activity of AAM was investigated by the rhamnolipid and pyocyanin production constraint as well as decrease of elastase activity, all being quorum sensing-controlled virulence factors expressed in the pathogenic bacteria Pseudomonas aeruginosa . Results HE induced a 91.6% of inhibition of the violecin production at 55 μg/mL concentration, whereas AAM showed 94% of inhibition at 166 μg/mL. In both cases, inhibition of violacein production did not affect the viability of the bacterium. AAM inhibited pyocyanin (86% at 200 μg/mL) and rhamnolipid (91% at 500 μg/mL) production in a dose/response form and decrease the elastase (75% at 500 μg/mL) activity in P. aeruginosa without affecting its development. Conclusions Because an anacardic acids mixture isolated from A. adstringens demonstrated anti-QS, it could be further exploited for novel molecules to treat the emerging infections of antibiotic-resistant bacterial pathogens.

Summary To assess what defence mechanisms are triggered by Cd2+ 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 Cd2+ 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) Cd2+ 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.

The quorum-quenching compounds brominated furanone C-30 and 5-fluorouracil inhibit the pathogenicity of the Pseudomonas aeruginosa laboratory strains PA01 and PA14; however, there is no report studying the effectiveness of these compounds for clinical isolates. Therefore, the effect of both quorum quenchers on the production of pyocyanin, elastase and alkaline protease of eight clinical strains from children was evaluated. Although both compounds were in general effective for the attenuation of these factors, three strains resistant to C-30 were found. For 5-fluorouracil, PA01 and some clinical isolates showed resistance for at least one phenotype.

A factor limiting the availability of onion seed is its accelerated postharvest deterioration. The research aim was to evaluate the recovery of seed germination in aged onion after application of chemical solutions. The onion seeds were artificially aged for 48, 96 and 144 h. Next the seeds were placed in solutions: gibberellic acid (3.46 mg•L-1), (BAP (2.25 mg•L-1), calcium chloride (55.50 mg•L-1) and potassium nitra¬te (50.50 mg•L-1), 8 to 12 h according to cultivar. The imbibed seeds were placed in standard germination conditions. The CaCl2 treatment in aged seeds (48 h) increased the percentage of normal seedlings in the cultivar Contessa. All other seed treatments increased the time to reach T50 and negatively affected germination. The use of BAP caused the higher reduction in the percentage of normal seedlings, lower seedlings biomass and the consequent loss of vigor. Toro and Contessa were the cultivars with greatest vigor, and Crystal white wax and Cojumatlan presented intermediate vigor, while Red burgundy showed the lowest vigor.

A novel protease secreted by Brevibacillus sp. KH3 isolated from excess sludge at 50 °C and used as a sludge-lysing strain was investigated in this study. Sludge reduction was minimized by protease inhibitors and a 40-kDa protease, which significantly contributed to this sludge-reducing activity, was purified as the target protein. The final purified protease demonstrated 92-fold higher specific activity than the initial crude extracts. The sludge-reducing efficiency deteriorated relative to decreased protease activity triggered by EDTA; thus, the purified protease was a causative agent in reducing excess sludge. The 40-kDa protease was a serine metalloprotease and showed the highest activity at 50 °C and pH 8.0, and the activity was enhanced in the presence of calcium ions, indicating that the purified protease contained calcium ion. Furthermore, this 40-kDa protease inhibited biofilm formation in excess sludge. These results imply that sludge reduction is because of reduction of biofilm formation in excess sludge.

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.

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.

a b s t r a c t Pseudomonas aeruginosa PA14 cells resistant to the novel antimicrobial gallium nitrate (Ga) were devel- oped 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 Fe 3+ 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 muta- tions 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.

Endemic populations of Hechtia perotensis have been described in Puebla and Veracruz, Mexico. Good quality seed collections can be used in conservation, research and ecological restoration. To evaluate seed quality of wild and endemic species, some compounds are used as effective promoters of germination, such as potassium nitrate (KNO3) and gibberellic acid (AG3), because they increase seed germination capacity and reduce latency. The triphenyl tetrazolium chloride (tetrazolium) test correlates seed viability because it is based on the activity of dehydrogenases in live tissues that catalyze mitochondrial respiration. The objective of this study was to obtain information on size and weight of capsules and seeds and seed germination and viability of H. perotensis, collected in Veracruz in the year 2012 and 2015. The hypotheses were 1) that seed germination and viability are independent of the year of collection, 2) that there is a tetrazolium concentration that can identify seed viability better than others, and 3) that pretreatment with KNO3 or AG3 improves seed germination. Seed germination was assessed using a completely randomized design with three treatments (control and the germination promoters 0.2 % KNO3 and 500 mg/L AG3), four treatments for the viability test (control, 0.2, 0.5 and 1.0 % of tetrazolium) and six replicates for each treatment. A total of one hundred seeds for germination experiments, and 25 seeds for the viability test were used. The results between and within years were analyzed with ANOVA and multiple comparison with the Tukey test. The proportion of non-germinated seeds was quantified along with the number of normal and abnormal seedlings, seeds with viable embryo, seeds without embryo, and seeds with low or no viability. On average, for the 2012 collected sample, 36 % had viable embryos, 7 % had low viability, 24 % were not viable and 33 % had no embryo. This result was significantly different from the 2015 sample, for which 87 % of seed showed viable embryos, 10 % had low viability, 0 % was not viable and 3 % had no embryo. Seed germination was also significantly different between years (22 and 92 %) Pregerminative treatments did not improve germination. Seed germination and viability of H. perotensis significantly varied between years of seed collection.

The physiological role of the membrane-bound pyrophosphatase of Rhodospirillum rubrum was investigated by the characterization of a mutant strain. Comparisons of growth levels between the wild type and the mutant under different low-potential conditions and during transitions between different metabolisms indicate that this enzyme provides R. rubrum with an alternative energy source that is important for growth in low-energy states.

The cytoplasmic pyrophosphatase of the photosynthetic bacterium Rhodospirillum rubrum was purified to electrophoretic homogeneity. The enzyme is a homohexamer of 20-kDa monomers. The gene was cloned and sequenced. Alignment of the deduced 179-amino-acid protein with known bacterial pyrophosphatases revealed conservation of all residues in the active site. Attempts to obtain an insertion mutant of the cytoplasmic pyrophosphatase gene did not yield any cell completely devoid of cytoplasmic pyrophosphatase activity. The mutants obtained showed 50% of the enzymatic activity and grew in twice the generation time of wild-type cells. This suggests that the membrane-bound pyrophosphatase of Rsp. rubrum is not sufficient for a normal growth rate, whereas the cytoplasmic enzyme is essential for growth. The characteristics of the gene and the encoded protein fit those of prokaryotic family I pyrophosphatases.

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.

The nature of the ammonium import into prokaryotes has been controversial. A systems biological approach makes us hypothesize that AmtB-mediated import must be active for intracellular NH(4)(+) concentrations to sustain growth. Revisiting experimental evidence, we find the permeability assays reporting passive NH(3) import inconclusive. As an inevitable consequence of the proposed NH(4)(+) transport, outward permeation of NH(3) constitutes a futile cycle. We hypothesize that the regulatory protein GlnK is required to fine-tune the active transport of ammonium in order to limit futile cycling whilst enabling an intracellular ammonium level sufficient for the cell's nitrogen requirements.