Fluorescent protein fusions in Candida guilliermondii

EA2106, Biomolécules et Biotechnologies Végétales, Université François-Rabelais de Tours, France.
Fungal Genetics and Biology (Impact Factor: 2.59). 07/2011; 48(11):1004-11. DOI: 10.1016/j.fgb.2011.07.004
Source: PubMed


Candida guilliermondii is an emerging fungal agent of candidiasis often associated with oncology patients. This yeast also remains a promising biotechnological model for the industrial production of value-added metabolites. In the present study, we developed a recipient strain as well as a set of plasmids for construction of fluorescent protein (FP) fusions in this species. We demonstrated that C. guilliermondii phosphoglycerate kinase transcription-regulating sequences allow a constitutive expression of codon-optimized green, cyan, yellow and mCherry FP genes in C. guilliermondii cells and the fluorescence signal could be directly observed at the colony and blastospore level by epifluorescence microcopy. To illustrate differential targeting of the FPs into specified cellular compartments, we studied and validated the expected subcellular localization of various C. guilliermondii predicted proteins fused to FPs. Furthermore, co-expression experiments of various couples of FP-tagged C. guilliermondii predicted proteins in the same cell showed that the fluorescence of each FP could be detected independently, providing firm evidences that YFP/CFP and GFP/mCherry pairs can be used for dual labeling in C. guilliermondii cells. This technical advance will facilitate future studies of protein co-expression and co-localization in C. guilliermondii and will give precious help for elucidating new molecular events supporting pathogenicity, antifungal resistance and for exploring the potential of yeast metabolic engineering.

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    • "The recent sequencing of the C. guilliermondii ATCC 6260 reference strain whole genome (Butler et al., 2009) clearly represents a major step that should provide crucial information for elucidating new molecular events supporting pathobiology and for exploring the potential of yeast metabolic engineering. On the continuation of our efforts to develop molecular tools in C. guilliermondii (Courdavault et al., 2011; Millerioux et al., 2011a, b; Foureau et al., 2012a, b), the present work was focused on the generation of a phleomycin-resistant cassette to evaluate its applicability for efficient genetic transformation of various C. guilliermondii wild-type strains. Among Saccharomycotina, it is currently accepted that most species belonging to the Candida genus have adopted a specific codon usage, whereby the CTG codon encodes a serine instead of leucine (Kawaguchi et al., 1989). "
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    ABSTRACT: We designed an efficient transformation system for C. guilliermondii wild-type strains. We demonstrated that the Staphylococcus aureus MRSA 252 ble coding sequence placed under the control of the yeast phosphoglycerate kinase gene transcription-regulating regions confers phleomycin resistance to transformed C. guilliermondii cells. To illustrate the potential of this drug-resistant cassette, we carried out the disruption of the C. guilliermondii ADE2 gene. This new dominant selectable marker represents a powerful tool to study the function of various genes in this yeast of clinical and biotechnological interest. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
    FEMS Yeast Research 02/2013; 13(3):354-358. DOI:10.1111/1567-1364.12034 · 2.82 Impact Factor
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    • "Bar 2 lm. For further details, see Courdavault et al. (2011) Curr Genet promoting research programs aiming at identifying new molecular events supporting pathogenicity and antifungal resistance in this emerging yeast. Finally, some C. guilliermondii isolates have been shown to have a high potential for use as biological control agents. "
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    ABSTRACT: Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the Saccharomycotina CTG clade which has been studied over the last forty years due to its biotechnological interest, biological control potential and clinical importance. Such a wide range of applications in various areas of fundamental and applied scientific research has progressively made C. guilliermondii an attractive model for exploring the potential of yeast metabolic engineering as well as for elucidating new molecular events supporting pathogenicity and antifungal resistance. All these research fields now take advantage of the establishment of a useful molecular toolbox specifically dedicated to C. guilliermondii genetics including the construction of recipient strains, the development of selectable markers and reporter genes and optimization of transformation protocols. This area of study is further supported by the availability of the complete genome sequence of the reference strain ATCC 6260 and the creation of numerous databases dedicated to gene ontology anotation (metabolic pathways, virulence, morphogenesis). These genetic tools and genomic resources represent essential prerequisites for further successful development of C. guilliermondii research in medical mycology and in biological control by facilitating the identification of the multiple factors that contribute to its pathogenic potential. These genetic and genomic advances should also expedite future practical uses of C. guilliermondii strains of biotechnological interest by opening a window into a better understanding of the biosynthetic pathways of valuable metabolites.
    Current Genetics 01/2013; · 2.68 Impact Factor
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    • "To illustrate the capacity of the TRP5 blaster system to achieve a second round of gene disruption, we targeted the C. guilliermondii LEU2 gene encoding -isopropylmalate dehydrogenase (Courdavault et al. 2011) in the fcy1 R background . For this purpose, we inserted the RT5R sequence into the central region of the cloned LEU2 gene to produce the plasmid pG-LEU2-RT5R containing the 5ЈLEU2- RT5R-3ЈLEU2 cassette. "
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    ABSTRACT: Candida guilliermondii is an interesting biotechnological model for the industrial production of value-added metabolites and also remains an opportunistic emerging fungal agent of candidiasis often associated with oncology patients. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both an ATCC 6260-derived recipient strain and a recyclable selection marker. We first disrupted the TRP5 gene in the wild-type strain and demonstrated that trp5 mutants were tryptophan auxotroph as well as being resistant to the antimetabolite 5-fluoroanthranilic acid (FAA). Following an FAA selection of spontaneous mutants derived from the ATCC 6260 strain and complementation analysis, we demonstrated that trp5 genotypes could be directly recovered on FAA-containing medium. The TRP5 wild-type allele, flanked by two short repeated sequences of its 3′UTR, was then used to disrupt the FCY1 gene in C. guilliermondii trp5 recipient strains. The resulting fcy1 mutants displayed strong flucytosine resistance and a counter-selection on FAA allowed us to pop-out the TRP5 allele from the FCY1 locus. To illustrate the capacity of this blaster system to achieve a second round of gene disruption, we knocked out both the LEU2 and the HOG1 genes in the trp5, fcy1 background. Although all previously described yeast “TRP blaster” disruption systems used TRP1 as counter-selectable marker, this study demonstrated the potential of the TRP5 gene in such strategies. This newly created “TRP5 blaster” disruption system thus represents a powerful genetic tool to study the function of a large pallet of genes in C. guilliermondii.
    Current Genetics 05/2012; 58(4):245-54. DOI:10.1007/s00294-012-0377-3 · 2.68 Impact Factor
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