amdS as a dominant recyclable marker in Cryptococcus neoformans

Abstract

While the fungal pathogen Cryptoccocus neoformans is a leading cause of death in immunocompromised individuals, the molecular toolkit currently available to study this important pathogen is extremely limited. To enable an unprecedented level of control over manipulation of the genome, we have developed a dominant recyclable marker by expanding on the classic studies of the amdS gene by Michael J. Hynes and John Pateman. The ascomycete Aspergillus nidulans employs the acetamidase AmdS to hydrolyse acetamide to ammonium and acetate, which serve as a nitrogen and carbon source, respectively. Acetamidase activity has never been reported in the Basidiomycota. Here we have successfully demonstrated that acetamide can be utilized as a good nitrogen source in C. neoformans heterologously expressing amdS and that this activity does not influence virulence, enabling it to be used as a basic dominant selectable marker. The expression of this gene in C. neoformans also causes sensitivity to fluoroacetamide, permitting counterselection. Taking advantage of this toxicity we have modified our basic marker to create a comprehensive series of powerful and reliable tools to successfully delete multiple genes in the one strain, generate markerless strains with modifications such as fluorescent protein fusions at native genomic loci, and establish whether a gene is essential in C. neoformans.

Bub1 kinase acts as a signalling hub for the entire Cryptococcus neoformans spindle assembly checkpoint pathway

“…Our expectation was that the Cnbub3Δ phenotype would be as equally severe as bub1Δ , so first we introduced a second copy of the CnBUB3 gene that was N-terminally tagged with the HA-epitope, regulated by the galactose-inducible promoter ( P GAL7 ) 29 and integrated at the safe haven locus on Chr3. Next, we transformed this strain with a bub3 deletion blaster cassette 30 replacing the endogenous BUB3 gene with the amdS (acetamide) marker, and maintained cells on galactose until knock-out transformants were confirmed by PCR (SuppFig.1b). As expected, switching these cells from galactose to glucose media led to gradual depletion of HA-tagged Bub3 (Fig.1d) and cells with a very similar phenotype to bub1Δ .…”

“…d) Fixed time points were analysed(30,60, 90, 120 and 180 mins after 2.5µg nocodazole addition) for bright GFP-Bub1 foci (SAC arrested cells). The bub1-kd cells delay, but can't maintain the mitotic arrest.preprint…”

Cryptococcus neoformans Database in Synthetic Biology Open Language

“…The promoters were acquired from genes encoding actin (ACT1) ( 9 ), antiphagocytic protein (App1) ( 17 ), galactose kinase (GAL1) ( 18 ), galactose transferase (GAL7) ( 18 , 19 ), UDP-glucose epimerase (UGE2) ( 18 ), glyceraldehyde-3-phosphate dehydrogenase (GPD) ( 20 ), U6 (CnU6) ( 10 ), orotidine monophosphate pyrophosphorylase (URA5) ( 21 ), and elongation factor 1 (TEF1) ( 9 ). The coding sequences were acetamidase (AmdS) ( 22 ), Cas9 ( 9 , 23 ), the fluorescent protein mCherry ( 24 ), green fluorescent protein (GFP) ( 24 ), URA5 ( 21 ), hygromycin (HYG) ( 22 , 25 ), neomycin (NEO/G418) ( 22 , 26 ), and nourseothricin (NAT/NrsR) ( 24 , 27 ) resistance genes. The terminators belonged to genes encoding phosphoribosylanthranilate isomerase (TRP1) ( 22 ), HOG1 (HOG1t) ( 24 ), TEF1 ( 9 ), CnU6 ( 10 ), and URA5 ( 21 ).…”

“…The coding sequences were acetamidase (AmdS) ( 22 ), Cas9 ( 9 , 23 ), the fluorescent protein mCherry ( 24 ), green fluorescent protein (GFP) ( 24 ), URA5 ( 21 ), hygromycin (HYG) ( 22 , 25 ), neomycin (NEO/G418) ( 22 , 26 ), and nourseothricin (NAT/NrsR) ( 24 , 27 ) resistance genes. The terminators belonged to genes encoding phosphoribosylanthranilate isomerase (TRP1) ( 22 ), HOG1 (HOG1t) ( 24 ), TEF1 ( 9 ), CnU6 ( 10 ), and URA5 ( 21 ). In conclusion, the database is ready and accessible to all the community in the Synthetic Biology area.…”

Genetic Transformation in Cryptococcus Species

“…In addition to URA5 and ADE2, the AMDS gene encoding the acetamidase and the NMT487D gene encoding the myristoyl-CoA:protein N-myristoyl transferase were used as selection markers in the transformation of Cryptococcus [25,26]. Moreover, a mutation in the gene encoding the 60S ribosomal protein L41 resulting in increased sensitivity to cycloheximide was also explored as a selectable marker [6].…”