New tools for the new bug Candida auris

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The curious case of nonrepetitive centromeric DNA sequences in Candida auris and related species

Acknowledgements. We thank Adele L. Marston (University of Edinburgh, UK) for critical reading of the manuscript.Peer reviewedPublisher PD

Split-marker-mediated genome editing improves homologous recombination frequency in the CTG clade yeast Candida intermedia

Genome-editing toolboxes are essential for the exploration and exploitation of nonconventional yeast species as cell factories, as they facilitate both genome studies and metabolic engineering. The nonconventional yeast\ua0Candida intermedia\ua0is a biotechnologically interesting species due to its capacity to convert a wide range of carbon sources, including xylose and lactose found in forestry and dairy industry waste and side-streams, into added-value products. However, possibilities of genetic manipulation have so far been limited due to lack of molecular tools for this species. We describe here the development of a genome editing method for\ua0C. intermedia, based on electroporation and gene deletion cassettes containing the\ua0Candida albicans NAT1\ua0dominant selection marker flanked by 1000 base pair sequences homologous to the target loci. Linear deletion cassettes targeting the\ua0ADE2\ua0gene originally resulted in\ua0<1% targeting efficiencies, suggesting that\ua0C. intermedia\ua0mainly uses nonhomologous end joining for integration of foreign DNA fragments. By developing a split-marker based deletion technique for\ua0C. intermedia, we successfully improved the homologous recombination rates, achieving targeting efficiencies up to 70%. For marker-less deletions, we also employed the split-marker cassette in combination with a recombinase system, which enabled the construction of double deletion mutants via marker recycling. Overall, the split-marker technique proved to be a quick and reliable method for generating gene deletions in\ua0C. intermedia, which opens the possibility to uncover and enhance its cell factory potential

Cellular and Subcellular Compartmentation of the 2C-Methyl-D-Erythritol 4-Phosphate Pathway in the Madagascar Periwinkle

The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. In leaves, a complex compartmentation of the MIA biosynthetic pathway occurs at both the cellular and subcellular levels, notably for some gene products of the MEP pathway. To get a complete overview of the pathway organization, we cloned four genes encoding missing enzymes involved in the MEP pathway before conducting a systematic analysis of transcript distribution and protein subcellular localization. RNA in situ hybridization revealed that all MEP pathway genes were coordinately and mainly expressed in internal phloem-associated parenchyma of young leaves, reinforcing the role of this tissue in MIA biosynthesis. At the subcellular level, transient cell transformation and expression of fluorescent protein fusions showed that all MEP pathway enzymes were targeted to plastids. Surprisingly, two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase initially exhibited an artifactual aggregated pattern of localization due to high protein accumulation. Immunogold combined with transmission electron microscopy, transient transformations performed with a low amount of transforming DNA and fusion/deletion experiments established that both enzymes were rather diffuse in stroma and stromules of plastids as also observed for the last six enzymes of the pathway. Taken together, these results provide new insights into a potential role of stromules in enhancing MIA precursor exchange with other cell compartments to favor metabolic fluxes towards the MIA biosynthesis

A single gene encodes isopentenyl diphosphate isomerase isoforms targeted to plastids, mitochondria and peroxisomes in Catharanthus roseus

Isopentenyl diphosphate isomerases (IDI) catalyze the interconversion of the two isoprenoid universal C5 units, isopentenyl diphosphate and dimethylally diphosphate, to allow the biosynthesis of the large variety of isoprenoids including both primary and specialized metabolites. This isomerisation is usually performed by two distinct IDI isoforms located either in plastids/peroxisomes or mitochondria/peroxisomes as recently established in Arabidopsis thaliana mainly accumulating primary isoprenoids. By contrast, almost nothing is known in plants accumulating specialized isoprenoids. Here we report the cloning and functional validation of an IDI encoding cDNA (CrIDI1) from Catharanthus roseus that produces high amount of monoterpenoid indole alkaloids. The corresponding gene is expressed in all organs including roots, flowers and young leaves where transcripts have been detected in internal phloem parenchyma and epidermis. The CrIDI1 gene also produces long and short transcripts giving rise to corresponding proteins with and without a N-terminal transit peptide (TP), respectively. Expression of green fluorescent protein fusions revealed that the long isoform is targeted to both plastids and mitochondria with an apparent similar efficiency. Deletion/fusion experiments established that the first 18-residues of the N-terminal TP are solely responsible of the mitochondria targeting while the entire 77-residue long TP is needed for an additional plastid localization. The short isoform is targeted to peroxisomes in agreement with the presence of peroxisome targeting sequence at its C-terminal end. This complex plastid/mitochondria/peroxisomes triple targeting occurring in C. roseus producing specialized isoprenoid secondary metabolites is somehow different from the situation observed in A. thaliana mainly producing housekeeping isoprenoid metabolites.This work was financially supported by the “Ministère de l’Enseignement Supérieur et de la Recherche” (MESR) and by a grant from the University of Tours. Grégory Guirimand and Anthony Guihur were financed by MESR fellowships.Peer reviewe

Triple subcellular targeting of isopentenyl diphosphate isomerases encoded by a single gene

Isopentenyl diphosphate isomerase (IDI) is a key enzyme of the isoprenoid pathway, catalyzing the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, the universal precursors of all isoprenoids. In plants, several subcellular compartments, including cytosol/ER, peroxisomes, mitochondria and plastids, are involved in isoprenoid biosynthesis. Here, we report on the unique triple targeting of two Catharanthus roseus IDI isoforms encoded by a single gene (CrIDI1). The triple localization of CrIDI1 in mitochondria, plastids and peroxisomes is explained by alternative transcription initiation of CrIDI1, by the specificity of a bifunctional N-terminal mitochondria/plastid transit peptide and by the presence of a C-terminal peroxisomal targeting signal. Moreover, bimolecular fluorescence complementation assays revealed self-interactions suggesting that the IDI likely acts as a multimer in vivo.Peer reviewe

Diversity and Evolution of Sensor Histidine Kinases in Eukaryotes

Histidine kinases (HKs) are primary sensor proteins that act in cell signaling pathways generically referred to as "two component systems" (TCSs). TCSs are among the most widely distributed transduction systems used by both prokaryotic and eukaryotic organisms to detect and respond to a broad range of environmental cues. The structure and distribution of HK proteins are now well documented in prokaryotes but information is still fragmentary for eukaryotes. Here, we have taken advantage of recent genomic resources to explore the structural diversity and the phylogenetic distribution of HKs in the prominent eukaryotic supergroups. Searches of the genomes of 67 eukaryotic species spread evenly throughout the phylogenetic tree of life identified 748 predicted HK proteins. Independent phylogenetic analyses of predicted HK proteins were carried out for each of the major eukaryotic supergroups. This allowed most of the compiled sequences to be categorised into previously described HK groups. Beyond the phylogenetic analysis of eukaryotic HKs, this study revealed some interesting findings: (i) characterisation of some previously undescribed eukaryotic HK groups with predicted functions putatively related to physiological traits; (ii) discovery of HK groups that were previously believed to be restricted to a single kingdom in additional supergroups and (iii) indications that some evolutionary paths have led to the appearance, transfer, duplication, and loss of HK genes in some phylogenetic lineages. This study provides an unprecedented overview of the structure and distribution of HKs in the Eukaryota and represents a first step towards deciphering the evolution of TCS signaling in living organisms

Combination of different molecular mechanisms leading to fluconazole resistance in a Candida lusitaniae clinical isolate.

International audienceWe report on the underlying molecular mechanisms likely responsible for the high-level fluconazole resistance in a Candida lusitaniae clinical isolate. Fluconazole resistance correlated with overexpression of ERG11 and of several efflux pump genes, in particular, the orthologs of the Candida albicans MDR1, PDR16, CDR1, CDR2, and YOR1

Cytokinin receptor turns 20

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Social amoebae as a new chassis for drug production

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