Three enzymes - One substrate Regulation of carbon flux through a "non-canonical" metabolic branchpoint

Pseudomonas aeruginosa is a common opportunistic pathogen. Recent work indicates that in many infection scenarios, P. aeruginosa exhibits an exquisite predilection for metabolizing fatty acids to yield acetyl-CoA. In most higher organisms, acetyl-CoA cannot be used for biomass production because the two carbon atoms which enter the TCA cycle are lost as CO2 . However, many bacteria are able to bypass these oxidative decarboxylation steps, allowing them to conserve carbon for gluconeogenesis. They perform this by using the “glyoxylate shunt”. Here, isocitrate is cleaved by isocitrate lyase (ICL) to yield succinate and glyoxylate (which, in a subsequent reaction, is combined with a further acetyl-CoA unit to yield the gluconeogenic precursor, malate). However, ICL has to compete with the TCA cycle enzyme, isocitrate dehydrogenase (ICD), for the available isocitrate, and it is the outcome of this “metabolic tussle” which dictates the flux of carbon through the glyoxylate shunt. In E. coli, ICD is inactivated by AceK-dependent phosphorylation, allowing flux through the glyoxylate shunt. However, P. aeruginosa is “wired up” differently because it employs not one, but two highly-expressed isocitrate dehydrogenases (ICD and IDH). For this PhD project, I focused on these three enzymes (ICD, IDH and ICL). I cloned, overexpressed and purified them at high yield to perform a thorough investigation of their kinetics, regulation and more interestingly crystal structures. I found that only one of these (the E. coli-like ICD) is regulated by AceK-mediated phosphorylation. The other, IDH, is allosterically regulated, as is the isocitrate lyase. These findings demonstrate that in P. aeruginosa the rerouting of the carbon flux through the glyoxylate shunt is delicately regulated via allostery mainly. The conditions in which the cells grow and access to either poor or rich carbon sources heavily influence the partitioning of the central metabolism. In P. aeruginosa, the TCA cycle remains more active (than in E. coli for example) even during growth on poor nutrient and this is probably an important aspect to manage oxidative stress accompanying growth. Finally, I have solved the x-ray crystal structures of ICD, IDH and ICL. These are entirely novel structures that have not been defined previously and are new entries to the Protein Data Bank. The structure solving work highlighted very interesting peculiarities to these enzymes when compared with other bacterial pathogens. This emphasizes the growing idea that Pseudomonas aeruginosa is a unique bacterium that cannot be modelled by the well-studied Escherichia coli. All this work crystallizes the knowledge to build up a picture of how flux is likely to be regulated at this “non- canonical” metabolic branchpoint and features new interesting directions for downstream applications such as drug-design.Cambridge European Scholarship Cambridge Philosophical Society Research Studentshi

La domestication de l’abeille par le territoire

En cherchant à requalifier la domestication des abeilles à partir de pratiques plus holistes et plus ancrées dans le territoire, le propos de cet article est d’interroger la manière dont les apiculteurs du Sud-Ouest marocain (D’Essaouira à Tan Tan) intègrent idéologiquement et techniquement les abeilles jaunes sahariennes dans leur monde. Les savoir-faire des apiculteurs de ces régions reposent en partie sur la manipulation de différents types d’espaces agraires, et l’apiculture est partie intégrante de l’agro-écosystème global. Bien que sédentaire, l’apiculture dans le sud du Maroc implique une grande diversité d’espaces, comprenant les falaises à chutes d’eau, les champs de céréales et d’arganiers, les zones arboricoles fermées, les zones de monoculture de figuiers de barbarie, les parcours dominés par les euphorbes et les arganiers. Chaque unité spatiale du territoire rural est le support d’un certain degré de domestication des abeilles : capture des essaims sauvages, acclimatation des ruches, production de miel, reproduction des essaims. Les savoir-faire des apiculteurs s’illustrent par la connaissance sur le comportement des abeilles (comportement alimentaire, temporalité des préférences de matières apicoles), leur métabolisme (vertus des plantes sur la vie des abeilles, leur capacité à essaimer, leur capacité à nettoyer la ruche), et la pollinisation qu’elles permettent. Les apiculteurs du Sud-Ouest marocain jouent ainsi sur la pluralité des espaces de cultures et des espèces qui y poussent pour intégrer l’abeille dans le territoire rural de manière holiste. Plus encore, par l’emplacement dans l’espace de la ruche, l’apiculteur exerce un contrôle direct sur la reproduction de l’animal et la maîtrise des rythmes physiologiques liés à son alimentation, condition sine qua non à sa domestication. La domestication se réalise ainsi par l’intermédiaire du territoire, car l’apiculteur joue sur la multifonctionnalité des espaces en termes de cortège végétal pour maîtriser le cycle de vie et de production de l’abeille. La domestication de l’abeille saharienne résulte donc autant de gestes techniques appréciables sur le moment, que de longs processus d’aménagement du territoire et de sélection des espèces en tenant compte de la variable apicole.By searching to redefine the domestication of bees from more holistic practices, the purpose of this article is to examine how beekeepers of South West Morocco (From Essaouira to Tan Tan) incorporate ideologically and technically yellow saharian bees in their territory. In south Morocco, beekeeping is a result of the co-evolution between the Berber groups and a unique agro-forestry system, the argan tree (Argania spinosa L.). This agro-ecosystem, shaped during innumerable generations by Berber peasants, covers some 850 000 hectares in the semi-arid regions of southwest Morocco. The know-hows of beekeepers in these areas are partly based on the manipulation of various types of agrarian areas, and beekeeping is part of the global agro-ecosystem. Although sedentary, beekeeping in southern Morocco involves a wide variety of areas, including the cliffs waterfalls, agricultural fields with argan trees, arboricultural closed areas, monoculture areas of prickly pairs, pastoral areas dominated by euphorbias and argan trees. Each spatial unit of rural territory is the support of some degree of domestication of bees. Depending on the season and his needs, the beekeeper decides to place his hives in one habitat or another in order to collect new colonies, favor honey production, the health of his colonies, their rapid multiplication, or their protection in the event of extended drought. Beekeepers of South Western Morocco play on the plurality of areas of crops and species that grow there to integrate the bee in rural territory in a holistic manner. Even more, thanks to the moving location in space of the hive, the beekeeper has a direct control on the reproduction of animals and the physiological rhythms associated with its food, prerequisite to its domestication. The domestication is realized through territory because the beekeeper plays on multi functionality of spaces in terms of biodiversity to control the cycle of life and production of bees

Which microbial factors really are important in Pseudomonas aeruginosa infections?

Over the last two decades, tens of millions of dollars have been invested in understanding virulence in the human pathogen, Pseudomonas aeruginosa. However, the top 'hits' obtained in a recent TnSeq analysis aimed at identifying those genes that are conditionally essential for infection did not include most of the known virulence factors identified in these earlier studies. Instead, it seems that P. aeruginosa faces metabolic challenges in vivo, and unless it can overcome these, it fails to thrive and is cleared from the host. In this review, we look at the kinds of metabolic pathways that the pathogen seems to find essential, and comment on how this knowledge might be therapeutically exploited.Work in the MW laboratory is funded by the BBSRC (grant BB/M019411/1) and the EU (Marie Curie Educational Training Network “INTEGRATE”). AC is supported by the Cambridge Trusts. EM is funded by a studentship from the MRC. SB is supported by a Hershel Smith studentship. E-FU is a clinical research fellow funded by the CF Trust (UK), Papworth Hospital NHS Trust and the Wellcome Trust. YA is supported by a scholarship from the Yosef Jameel Foundation. YB is an EPSRC-funded PhD student. Work in the laboratory of AF is supported by the Wellcome Trust. Work in the DRS laboratory is supported by the EPSRC.This is the author accepted manuscript. The final version is available from Future Science Group via http://dx.doi.org/10.2217/fmb.15.10

Titin domains with reduced core hydrophobicity cause dilated cardiomyopathy.

The underlying genetic defect in most cases of dilated cardiomyopathy (DCM), a common inherited heart disease, remains unknown. Intriguingly, many patients carry single missense variants of uncertain pathogenicity targeting the giant protein titin, a fundamental sarcomere component. To explore the deleterious potential of these variants, we first solved the wild-type and mutant crystal structures of I21, the titin domain targeted by pathogenic variant p.C3575S. Although both structures are remarkably similar, the reduced hydrophobicity of deeply buried position 3575 strongly destabilizes the mutant domain, a scenario supported by molecular dynamics simulations and by biochemical assays that show no disulfide involving C3575. Prompted by these observations, we have found that thousands of similar hydrophobicity-reducing variants associate specifically with DCM. Hence, our results imply that titin domain destabilization causes DCM, a conceptual framework that not only informs pathogenicity assessment of gene variants but also points to therapeutic strategies counterbalancing protein destabilization.S

Usages, propriétés antibactériennes et physicochimie de miels marocains

MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF

La domestication de l’abeille par le territoire : un exemple d’apiculture holiste dans le Sud ouest marocain

International audienc

La domestication de l’abeille par le territoire : un exemple d’apiculture holiste dans le Sud ouest marocain

International audienc

Gluconeogenic precursor availability regulates flux through the glyoxylate shunt in Pseudomonas aeruginosa.

The glyoxylate shunt bypasses the oxidative decarboxylation steps of the tricarboxylic acid (TCA) cycle, thereby conserving carbon skeletons for gluconeogenesis and biomass production. In Escherichia coli, carbon flux is redirected through the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), following phosphorylation and inactivation of the TCA cycle enzyme, isocitrate dehydrogenase (ICD), by the kinase/phosphatase, AceK. In contrast, mycobacterial species lack AceK and employ a phosphorylation-insensitive isocitrate dehydrogenase (IDH), which is allosterically activated by the product of ICL activity, glyoxylate. However, Pseudomonas aeruginosa expresses IDH, ICD, ICL, and AceK, raising the question of how these enzymes are regulated to ensure proper flux distribution between the competing pathways. Here, we present the structure, kinetics, and regulation of ICL, IDH, and ICD from P. aeruginosa We found that flux partitioning is coordinated through reciprocal regulation of these enzymes, linking distribution of carbon flux to the availability of the key gluconeogenic precursors, oxaloacetate and pyruvate. Specifically, a greater abundance of these metabolites activated IDH and inhibited ICL, leading to increased TCA cycle flux. Regulation was also exerted through AceK-dependent phosphorylation of ICD; high levels of acetyl-CoA (which would be expected to accumulate when oxaloacetate is limiting) stimulated the kinase activity of AceK, whereas high levels of oxaloacetate stimulated its phosphatase activity. In summary, the TCA cycle-glyoxylate shunt branch point in P. aeruginosa has a complex enzymology that is profoundly different from those in other species characterized to date. Presumably, this reflects its predilection for consuming fatty acids, especially during infection scenarios

Pâturages

« Nourrir le Monde ! », « Respecter l’environnement et le bien être animal ! » « Se préoccuper des goûts et de la santé du consommateur ! » L’élevage est l’objet d’injonctions sociales et politiques parfois paradoxales que la mondialisation accentue chaque jour davantage, et qui visent particulièrement les manières d’élever et de nourrir les bêtes. Au nord comme au sud, dans des territoires souvent marginalisés, les pâturages jouent un rôle crucial. « Pâturer, c’est naturel ». Non bien au contraire. Loin de l’image bucolique, l’anthropologue, le géographe, le zootechnicien montrent dans ce Thema de Techniques&Culture en quoi la plus ancienne façon de nourrir les animaux, que les éleveurs réinterprètent sans cesse, est aussi probablement l’une des plus complexes et des plus vives en termes social, économique et politique. De la Laponie à la Patagonie en passant par les montagnes françaises, le pâturage nous permet d’interroger à nouveaux frais la place de l’élevage et des éleveurs dans le monde contemporain