Expression Plasmids for Use inCandida glabrata

Abstract

We describe a series of CEN/ARS episomal plasmids containing different Candida glabrata promoters, allowing for a range of constitutive or regulated expression of proteins in C. glabrata. The set of promoters includes three constitutive promoters (EGD2pr, HHT2pr, PDC1pr), two macrophage/phagocytosis-induced promoters (ACO2pr, LYS21pr), and one nutritionally regulated promoter (MET3pr). Each promoter was cloned into two plasmid backbones that differ in their selectable marker, URA3, or the dominant-selectable NAT1 gene, which confers resistance to the drug nourseothricin. Expression from the 12 resulting plasmids was assessed using GFP as a reporter and flow cytometry or quantitative reverse-transcription polymerase chain reaction to assess expression levels. Together this set of plasmids expands the toolkit of expression vectors available for use with C. glabrata.

Expression vectors for C-terminal fusions with fluorescent proteins and epitope tags in Candida glabrata

“…(Zordan et al, 2013(Zordan et al, : 1675 have recently developed a group of replicative vectors that allow evaluating genes of interest under constitutive or inducible promoters. However, expression vectors that allow fluorescent and epitope tagging in a systematic way such as those described here are still needed.…”

“…glabrata is an accessible organism to study and the close phylogenetic relationship with the model yeast Saccharomyces cerevisiae has allowed the use in C. glabrata of most of the molecular biology methods designed for S. cerevisiae. The rate of homologous recombination is relatively high so that constructs in plasmids can easily be introduced in the homologous site in the genome to generate mutants in the corresponding locus (Cormack and Falkow, 1999: 979-87) some vectors have also been designed to express genes under different promoters in C. glabrata (Zordan et al, 2013(Zordan et al, : 1675. However, there are not enough vectors available to generate a range of protein fusions to study transcriptional and translational regulation of any gene, proteinprotein interactions and the intracellular localization of the corresponding proteins in C. glabrata.…”

CgPDR1 gain‐of‐function mutations lead to azole‐resistance and increased adhesion in clinical Candida glabrata strains

“…Because uracil auxotrophy might affect adherence, CgURA3 was added back to the CgPDR1‐ disrupted strains. A CEN/ARS episomal plasmid pCN‐PDC1‐GFP was used for ectopic expression of CgPDR1 in the △ pdr1 strains . Whole PDR1 genes (including the promoter and terminator regions) from wild‐types strains and CBS138 strains were PCR‐amplified and ligated to Sac I/ Kpn I‐digested pCN‐PDC1‐GFP to generate plasmids containing CgPDR1 alleles with different GOF mutations.…”

The superoxide dismutases of Candida glabrata protect against oxidative damage and are required for lysine biosynthesis, DNA integrity and chronological life survival

“…The SOD complementation plasmids were constructed by amplifying the SOD genes with their own promoters from the genomic DNA of BG14 strain. These fragments were cloned into pGRB2.0 (Zordan et al, 2013). To construct SOD-GFP fusions, a Cterminal segment of SOD1 or SOD2 without a stop codon was placed in phase with GFP.…”