Metallothioneins may not be enough - the role of phytochelatins in invertebrate metal detoxification

'Viewpoint' article

Translational arrest due to cytoplasmic redox stress delays adaptation to growth on methanol and heterologous protein expression in a typical fed-batch culture of Pichia pastoris

Results We have followed a typical fed-batch induction regime for heterologous protein production under the control of the AOX1 promoter using both microarray and metabolomic analysis. The genetic constructs involved 1 and 3 copies of the TRY1 gene, encoding human trypsinogen. In small-scale laboratory cultures, expression of the 3 copy-number construct induced the unfolded protein response (UPR) sufficiently that titres of extracellular trypsinogen were lower in the 3-copy construct than with the 1-copy construct. In the fed-batch-culture, a similar pattern was observed, with higher expression from the 1-copy construct, but in this case there was no significant induction of UPR with the 3-copy strain. Analysis of the microarray and metabolomic information indicates that the 3-copy strain was undergoing cytoplasmic redox stress at the point of induction with methanol. In this Crabtree-negative yeast, this redox stress appeared to delay the adaptation to growth on methanol and supressed heterologous protein production, probably due to a block in translation. Conclusion Although redox imbalance as a result of artificially imposed hypoxia has previously been described, this is the first time that it has been characterised as a result of a transient metabolic imbalance and shown to involve a stress response which can lead to translational arrest. Without detailed analysis of the underlying processes it could easily have been mis-interpreted as secretion stress, transmitted through the UPR

A metabolic signature of long life in Caenorhabditis elegans

<p>Abstract</p> <p>Background</p> <p>Many <it>Caenorhabditis elegans </it>mutations increase longevity and much evidence suggests that they do so at least partly via changes in metabolism. However, up until now there has been no systematic investigation of how the metabolic networks of long-lived mutants differ from those of normal worms. Metabolomic technologies, that permit the analysis of many untargeted metabolites in parallel, now make this possible. Here we use one of these, ¹H nuclear magnetic resonance spectroscopy, to investigate what makes long-lived worms metabolically distinctive.</p> <p>Results</p> <p>We examined three classes of long-lived worms: dauer larvae, adult Insulin/IGF-1 signalling (IIS)-defective mutants, and a translation-defective mutant. Surprisingly, these ostensibly different long-lived worms share a common metabolic signature, dominated by shifts in carbohydrate and amino acid metabolism. In addition the dauer larvae, uniquely, had elevated levels of modified amino acids (hydroxyproline and phosphoserine). We interrogated existing gene expression data in order to integrate functional (metabolite-level) changes with transcriptional changes at a pathway level.</p> <p>Conclusions</p> <p>The observed metabolic responses could be explained to a large degree by upregulation of gluconeogenesis and the glyoxylate shunt as well as changes in amino acid catabolism. These responses point to new possible mechanisms of longevity assurance in worms. The metabolic changes observed in dauer larvae can be explained by the existence of high levels of autophagy leading to recycling of cellular components.</p> <p>See associated minireview: <url>http://jbiol.com/content/9/1/7</url></p

Rapid screening of cellular stress responses in recombinant Pichia pastoris strains using metabolite profiling

Heterologous protein production in the yeast Pichia pastoris can be limited by biological responses to high expression levels; the unfolded protein response (UPR) is a key determinant of the success of protein production in this organism. Here, we used untargeted NMR metabolic profiling (metabolomics) of a number of different recombinant strains, carried out in a miniaturized format suitable for screening-level experiments. We identified a number of metabolites (from both cell extracts and supernatants) which correlated well with UPR-relevant gene transcripts, and so could be potential biomarkers for future high-throughput screening of large numbers of P. pastoris clones.This study was wholly funded by the Bioprocessing Research Industry Club (BRIC), a partnership between BBSRC, EPSRC and a consortium of leading companies (http://www. bbsrc.ac.uk/business/collaborative-research/industry-clubs/bric/background.aspx, Grant reference BBF0049071)

Hypertonic saline therapy in cystic fibrosis: do population shifts caused by the osmotic sensitivity of infecting bacteria explain the effectiveness of this treatment?

Cystic fibrosis (CF) is caused by a defect in the CF transmembrane regulator that leads to depletion and dehydration of the airway surface liquid (ASL) of the lung epithelium, providing an environment that can be infected by bacteria leading to increased morbidity and mortality. Pseudomonas aeruginosa chronically infects more than 80% of CF patients and one hallmark of infection is the emergence of a mucoid phenotype associated with a worsening prognosis and more rapid decline in lung function. Hypertonic saline (HS) is a clinically proven treatment that improves mucociliary clearance through partial rehydration of the ASL of the lung. Strikingly, while HS therapy does not alter the prevalence of P. aeruginosa in the CF lung it does decrease the frequency of episodes of acute, severe illness known as infective exacerbations among CF patients. In this article, we propose a hypothesis whereby the positive clinical effects of HS treatment are explained by the osmotic sensitivity of the mucoid sub-population of P. aeruginosa in the CF lung leading to selection against this group in favor of the osmotically resistant non-mucoid variants

Ecological drivers influence the distributions of two cryptic lineages in an earthworm morphospecies

Substantial genetic diversity exists within earthworm morphotypes, such that traditional species designations may be incomplete. It is, however, currently not known whether these different genetic variants show ubiquity or specialty in their distribution across separated sites subject to different climatic, biotic or soil physicochemical factors. Here we report on the results of a survey in which individuals of the Lumbricus rubellus morphotype, a species known to comprise two deeply divergent genetic lineages in England and Wales, were sampled from 26 plots. Sequences from the mitochondrial cytochrome oxidase I gene were used to distinguish lineages for 787 individuals. In conjunction, a range of geographic, climatic, biotic and soil physiochemical variables were also collected for each locality. Genotyping indicated that Lineage A was more common than Lineage B, comprising 58% of the collected L. rubellus. Six site populations comprised only Lineage A, while only a single site comprised entirely Lineage B. The remaining 20 sites contained both lineages. A multivariate ordination of site variables identified major difference between sites were associated with low pH, organic-rich soils in Western wet upland areas and pollutant levels associated with sites in the South. Earthworm genotype (as proportion of Lineage A) was not correlated with either of these major environmental axes. When individual variables of soil pH and the percentage of soil organic matter, which are known to be key driver of soil species distributions, were investigated as single variables significant relationship with lineage frequency were found. Soil organic matter content was significantly negatively correlated with Lineage A proportion, while pH was significantly positively correlated. This lineage preference may be related to lineage metabolism and/or behavioral differences. Measurement of tissue metal concentrations in worms from 17 sites identified a significant site effect in all cases, but a lineage effect only for arsenic (higher Lineage B). Tissue arsenic concentrations varied between lineages, supporting previous observations that there are differences in the way the two lineages have adapted to manage exposure to this metalloid

Aromatic metabolites from the coelomic fluid of Eisenia earthworm species

Earthworms from the genus Eisenia express coelomic fluid when under severe stress. This coelomic fluid contains a complex mixture of small-molecule metabolites, including aromatic metabolites which are known to be species-specific, yet their actual identities remain unknown. We have aimed to characterize selected high-concentration coelomic fluid metabolites. The major aromatic compound in Eisenia veneta coelomic fluid is the rare metabolite a-nicotinamide riboside; and the major aromatic compound for Eisenia fetida is closely related to the (already characterized) metabolite of Eisenia andrei, which consists of two aromatic quinazoline-2,4-dione ring structures linked by N-acetylspermine. The biological function(s) of these metabolites in earthworms is unknown, but we hypothesize that they represent remnants of larger molecules, possibly bacterial in origin, that are recalcitrant to metabolism by earthworm enzymes. (C) 2016 The Authors. Published by Elsevier Masson SAS

Translational arrest due to cytoplasmic redox stress delays adaptation to growth on methanol and heterologous protein expression in a typical fed-batch culture of <i>Pichia pastoris</i>

<div><p>Results</p><p>We have followed a typical fed-batch induction regime for heterologous protein production under the control of the <i>AOX1</i> promoter using both microarray and metabolomic analysis. The genetic constructs involved 1 and 3 copies of the <i>TRY1</i> gene, encoding human trypsinogen. In small-scale laboratory cultures, expression of the 3 copy-number construct induced the unfolded protein response (UPR) sufficiently that titres of extracellular trypsinogen were lower in the 3-copy construct than with the 1-copy construct. In the fed-batch-culture, a similar pattern was observed, with higher expression from the 1-copy construct, but in this case there was no significant induction of UPR with the 3-copy strain. Analysis of the microarray and metabolomic information indicates that the 3-copy strain was undergoing cytoplasmic redox stress at the point of induction with methanol. In this Crabtree-negative yeast, this redox stress appeared to delay the adaptation to growth on methanol and supressed heterologous protein production, probably due to a block in translation.</p><p>Conclusion</p><p>Although redox imbalance as a result of artificially imposed hypoxia has previously been described, this is the first time that it has been characterised as a result of a transient metabolic imbalance and shown to involve a stress response which can lead to translational arrest. Without detailed analysis of the underlying processes it could easily have been mis-interpreted as secretion stress, transmitted through the UPR.</p></div

Unique metabolites protect earthworms against plant polyphenols

All higher plants produce polyphenols, for defence against above-ground herbivory. These polyphenols also influence the soil micro- and macro-fauna that break down plant leaf litter. Polyphenols therefore indirectly affect the fluxes of soil nutrients and, ultimately, carbon turnover and ecosystem functioning in soils. It is unknown how earthworms, the major component of animal biomass in many soils, cope with high-polyphenol diets. Here, we show that earthworms possess a class of unique surface-active metabolites in their gut, which we term ‘drilodefensins’. These compounds counteract the inhibitory effects of polyphenols on earthworm gut enzymes, and high-polyphenol diets increase drilodefensin concentrations in both laboratory and field populations. This shows that drilodefensins protect earthworms from the harmful effects of ingested polyphenols. We have identified the key mechanism for adaptation to a dietary challenge in an animal group that has a major role in organic matter recycling in soils worldwide

Mapping an atlas of tissue-specific drosophila melanogaster metabolomes by high resolution mass spectrometry

Metabolomics can provide exciting insights into organismal function, but most work on simple models has focussed on the whole organism metabolome, so missing the contributions of individual tissues. Comprehensive metabolite profiles for ten tissues from adult Drosophila melanogaster were obtained here by two chromatographic methods, a hydrophilic interaction (HILIC) method for polar metabolites and a lipid profiling method also based on HILIC, in combination with an Orbitrap Exactive instrument. Two hundred and forty two polar metabolites were putatively identified in the various tissues, and 251 lipids were observed in positive ion mode and 61 in negative ion mode. Although many metabolites were detected in all tissues, every tissue showed characteristically abundant metabolites which could be rationalised against specific tissue functions. For example, the cuticle contained high levels of glutathione, reflecting a role in oxidative defence; the alimentary canal (like vertebrate gut) had high levels of acylcarnitines for fatty acid metabolism, and the head contained high levels of ether lipids. The male accessory gland uniquely contained decarboxylated S-adenosylmethionine. These data thus both provide valuable insights into tissue function, and a reference baseline, compatible with the FlyAtlas.org transcriptomic resource, for further metabolomic analysis of this important model organism, for example in the modelling of human inborn errors of metabolism, aging or metabolic imbalances such as diabetes