A Conserved Mitochondrial ATP-binding Cassette Transporter Exports Glutathione Polysulfide for Cytosolic Metal Cofactor Assembly

An ATP-binding cassette transporter located in the inner mitochondrial membrane is involved in iron-sulfur cluster and molybdenum cofactor assembly in the cytosol, but the transported substrate is unknown. ATM3 (ABCB25) from Arabidopsis thaliana and its functional orthologue Atm1 from Saccharomyces cerevisiae were expressed in Lactococcus lactis and studied in inside-out membrane vesicles and in purified form. Both proteins selectively transported glutathione disulfide (GSSG) but not reduced glutathione in agreement with a 3-fold stimulation of ATPase activity by GSSG. By contrast, Fe²⁺ alone or in combination with glutathione did not stimulate ATPase activity. Arabidopsis atm3 mutants were hypersensitive to an inhibitor of glutathione biosynthesis and accumulated GSSG in the mitochondria. The growth phenotype of atm3-1 was strongly enhanced by depletion of the mitochondrion-localized, GSH-dependent persulfide oxygenase ETHE1, suggesting that the physiological substrate of ATM3 contains persulfide in addition to glutathione. Consistent with this idea, a transportomics approach using mass spectrometry showed that glutathione trisulfide (GS-S-SG) was transported by Atm1. We propose that mitochondria export glutathione polysulfide, containing glutathione and persulfide, for iron-sulfur cluster assembly in the cytosol.     

The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms.

Cyclodextrin glycosyltransferase (CGTase) catalyzes three transglycosylation reactions via a double displacement mechanism involving a covalent enzyme-intermediate complex (substituted-enzyme intermediate). Characterization of the three transglycosylation reactions, however, revealed that they differ in their kinetic mechanisms. Disproportionation (cleavage of an alpha-glycosidic bond of a linear malto-oligosaccharide and transfer of one part to an acceptor substrate) proceeds according to a ping-pong mechanism. Cyclization (cleavage of an alpha-glycosidic bond in amylose or starch and subsequent formation of a cyclodextrin) is a single-substrate reaction with an affinity for the high molecular mass substrate used, which was too high to allow elucidation of the kinetic mechanism. Michaelis-Menten kinetics, however, have been observed using shorter amylose chains. Coupling (cleavage of an alpha-glycosidic bond in a cyclodextrin ring and transfer of the resulting linear malto-oligosaccharide to an acceptor substrate) proceeds according to a random ternary complex mechanism. In view of the different kinetic mechanisms observed for the various reactions, which can be related to differences in substrate binding, it should be possible to mutagenize CGTase in such a manner that a single reaction is affected most strongly. Construction of CGTase mutants that synthesize linear oligosaccharides instead of cyclodextrins thus appears feasible. Furthermore, the rate of interconversion of linear and circular conformations of oligosaccharides in the cyclization and coupling reactions was found to determine the reaction rate. In the cyclization reaction this conversion rate, together with initial binding of the high molecular mass substrate, may determine the product specificity of the enzyme. These new insights will allow rational design of CGTase mutant enzymes synthesizing cyclodextrins of specific sizes.     

The role of transport processes in survival of lactic acid bacteria, Energy transduction and multidrug resistance

Lactic acid bacteria play an essential role in many food fermentation processes. They are anaerobic organisms which obtain their metabolic energy by substrate phosphorylation. In addition three secondary energy transducing processes can contribute to the generation of a proton motive force: proton/substrate symport as in lactic acid excretion, electrogenic precursor/product exchange as in malolactic and citrolactic fermentation and histidine/histamine exchange, and electrogenic uniport as in malate and citrate uptake in Leuconostoc oenos. In several of these processes additional H+ consumption occurs during metabolism leading to the generation of a pH gradient, internally alkaline. Lactic acid bacteria have also developed multidrug resistance systems. In Lactococcus lactis three toxin excretion systems have been characterized: cationic toxins can be excreted by a toxin/proton antiport system and by an ABC-transporter. This cationic ABC-transporter has surprisingly high structural an d functional analogy with the human MDR1-(P-glycoprotein). For anions an ATP-driven ABC-like excretion systems exist.     

Interpretation of UV-survival curves of Aspergillus conidiospores

Semi-logarithmic dose-response curves for survival of UV-irradiated conidiospores of A. nidulans have an initial shoulder (at low doses) followed by a decline which becomes linear. To explain the initial shoulder and the resulting extrapolation number (log S intercept of the linear extrapolation line) a general model is presented, which includes multi-target (n) and multi-hit (h) effects and allows for the effect of initial repair and of a compound parameter k, which stands for inherent sensitivity of the spores and for dose received inside the spores. From experiments on (a) the modification of k (spore wall colour and shelter effects), (b) a repair-deficient strain (shoulderless) and (c) preincubation during which DNA-replication takes place, it is concluded that the shoulder is generated by initial repair rather than by a multi-hit nature of the cell-killing process. In experiments where k takes different values (sub a and c), notably the position of the point of intersection of the linear lines gives conclusive information. In general, the log S intercept of the linear extrapolation line cannot be used to estimate the target number.     

Characteristics of insulin binding to H35 hepatoma cells

Well differentiated hepatoma cells in culture exhibit insulin binding and insulin effects. We have studied insulin binding in control and in H35 hepatoma cells down-regulated with insulin. H35 cells were grown in monolayers in alpha MEM. Insulin binding was measured with A14 mono 125I labelled insulin 72 h after seeding. Binding was time, temperature and pH-dependent. Receptor down-regulation was studied by exposing cells to increasing concentrations of unlabelled insulin. Monolayers preincubated with 10 micrograms/ml unlabelled insulin for 24 h showed a decrease of 65% in the number of insulin binding sites. There was no change in affinity.     

Transmission of molecularly undetectable circulating parasite clones leads to high infection complexity in mosquitoes post feeding

Plasmodium falciparum malaria infections often comprise multiple distinct parasite clones. Few datasets have directly assessed infection complexity in humans and mosquitoes they infect. Examining parasites using molecular tools may provide insights into the selective transmissibility of isolates. Using capillary electrophoresis genotyping and next generation amplicon sequencing, we analysed complexity of parasite infections in human blood and in the midguts of mosquitoes that became infected in membrane feeding experiments using the same blood material in two West African settings. Median numbers of clones in humans and mosquitoes were higher in samples from Burkina Faso (4.5, interquartile range 2–8 for humans; and 2, interquartile range 1–3 for mosquitoes) than in The Gambia (2, interquartile range 1–3 and 1, interquartile range 1–3, for humans and mosquitoes, respectively). Whilst the median number of clones was commonly higher in human blood samples, not all transmitted alleles were detectable in the human peripheral blood. In both study sample sets, additional parasite alleles were identified in mosquitoes compared with the matched human samples (10–88.9% of all clones/feeding assay, n?=?73 feeding assays). The results are likely due to preferential amplification of the most abundant clones in peripheral blood but confirm the presence of low density clones that produce transmissible sexual stage parasites.     

The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

In vertebrates, the presence of viral RNA in the cytosol is sensed by members of the RIG‐I‐like receptor (RLR) family, which signal to induce production of type I interferons (IFN). These key antiviral cytokines act in a paracrine and autocrine manner to induce hundreds of interferon‐stimulated genes (ISGs), whose protein products restrict viral entry, replication and budding. ISGs include the RLRs themselves: RIG‐I, MDA5 and, the least‐studied family member, LGP2. In contrast, the IFN system is absent in plants and invertebrates, which defend themselves from viral intruders using RNA interference (RNAi). In RNAi, the endoribonuclease Dicer cleaves virus‐derived double‐stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that target complementary viral RNA for cleavage. Interestingly, the RNAi machinery is conserved in mammals, and we have recently demonstrated that it is able to participate in mammalian antiviral defence in conditions in which the IFN system is suppressed. In contrast, when the IFN system is active, one or more ISGs act to mask or suppress antiviral RNAi. Here, we demonstrate that LGP2 constitutes one of the ISGs that can inhibit antiviral RNAi in mammals. We show that LGP2 associates with Dicer and inhibits cleavage of dsRNA into siRNAs both in vitro and in cells. Further, we show that in differentiated cells lacking components of the IFN response, ectopic expression of LGP2 interferes with RNAi‐dependent suppression of gene expression. Conversely, genetic loss of LGP2 uncovers dsRNA‐mediated RNAi albeit less strongly than complete loss of the IFN system. Thus, the inefficiency of RNAi as a mechanism of antiviral defence in mammalian somatic cells can be in part attributed to Dicer inhibition by LGP2 induced by type I IFNs. LGP2‐mediated antagonism of dsRNA‐mediated RNAi may help ensure that viral dsRNA substrates are preserved in order to serve as targets of antiviral ISG proteins.     

Ecological selection of siderophore‐producing microbial taxa in response to heavy metal contamination

Some microbial public goods can provide both individual and community‐wide benefits, and are open to exploitation by non‐producing species. One such example is the production of metal‐detoxifying siderophores. Here, we investigate whether conflicting selection pressures on siderophore production by heavy metals – a detoxifying effect of siderophores, and exploitation of this detoxifying effect – result in a net increase or decrease. We show that the proportion of siderophore‐producing taxa increases along a natural heavy metal gradient. A causal link between metal contamination and siderophore production was subsequently demonstrated in a microcosm experiment in compost, in which we observed changes in community composition towards taxa that produce relatively more siderophores following copper contamination. We confirmed the selective benefit of siderophores by showing that taxa producing large amounts of siderophore suffered less growth inhibition in toxic copper. Our results suggest that ecological selection will favour siderophore‐mediated decontamination, with important consequences for potential remediation strategies.