Table 1.
Strains and plasmids used and/or constructed in this work.
Fig 1.
RP-HPLC analysis of sterols from the wild-type and crtR- mutant strain.
Chromatograms (at 280 nm) correspond to sterols extracted after 72 h of culture from the (A) wild-type (CBS 6938) and (B) CBSTr strain (crtR-). The corresponding spectra were displayed beside each peak (peaks N° 1 to 3). Peak 1 corresponds to ergosterol, which was confirmed by co-injecting the sample with standard ergosterol.
Fig 2.
X. dendrorhous CBR.1, CBR.2 and CYB5 protein amino acid sequence alignment.
Amino acid sequence alignment of conserved protein motives: (A) CBR and (B) CYB5. The X. dendrorhous CBR.1, CBR.2 and CYB5 are included. The UniProtKB or GenBank accession number is indicated in brackets beside the organism name and above each alignment; a scheme of the deduced X. dendrorhous protein structure is included. Tm: trans-membrane segment.
Fig 3.
Phylogenetic analysis of the CBR genes from X. dendrorhous.
The accession numbers for each of the amino acid sequences are: Cryptococcus gattii CBR1 [E6REZ8] and CBR2 [E6REQ5], C. neoformans CBR1 [P0CP14] and CBR2 [J9VWT4], P. chrysosporium CBR1 [E2QDB5], T. versicolor CBR1 [XP_008033072], U. maydis CBR1 [Q4PGW7] and CBR2 [Q4P7Y8], P. stipitis CBR1 [A3GF86] and CBR2 [A3LT66], C. albicans CBR1 [Q59P03] and CBR2 [Q59M70], N. crassa CBR1 [Q7RXL1] and CBR2 [Q7SFY2], A. niger CBR1 [A2QCV4] and CBR2 [A2Q898], A. fumigatus CBR1 [Q4X0B5] and CBR2 [A1CRK9], S. pombe CBR1 [O74557], S. cerevisiae CBR1 [P38626] and CBR2 [P36060], V. vinifera CBR1 [F6HIY1], T. urartu CBR1 [M7YU08], A. thaliana CBR1 [Q9ZNT1], H. armigera CBR1 [F1CZW3], D. rerio CBR1 [Q7ZVF8], S. scrofa CBR1 [F1S4N2], H. sapiens CBR1 [Q9UHQ9], M. musculus CBR1 [Q9DB73], and R. norvegicus CBR1 [G3V9S0]. The UniProtKB or GenBank accession number is indicated in brackets. The value at each node indicates the Bootstrap after 1000 iterations using MEGA v.6.06.
Fig 4.
PCR-based analysis of the CBS-cbr.1- and CBS-cbr.2- strains.
(A) Each gel shows the PCR reaction products obtained with different primers pairs numbered according to S1 Table and, as template, genomic DNA from the following strains: CBS-cbr.1- (line 1), CBS 6938 (line 2), negative primer control (line 3), CBS-cbr.2- (line 4), CBS 6938 (line 5), and negative primer control (line 6). Under each gel, a general scheme of the PCR analyses is included representing the resistance cassettes (green), the corresponding CBR gene (red) and the CBR flanking DNA (pink). A 1 kb plus DNA ladder (20.0, 10.0, 7.0, 5.0, 4.0, 3.0, 2.0, 1.5, 1.0, 0.7, and 0.5 Kbp) was used as the molecular weight standard in the right lane of each gel. (B) Color phenotype of the strains CBS 6938 (WT), CBS-cbr.1-, CBS-cbr.2- and CBSTr after 5 days of culture on YM medium incubated at 22°C with constant agitation.
Table 2.
Carotenoids and sterols produced by the X. dendrorhous strains cultured in parallel after 36 and 72 h of cultivation.
Fig 5.
Cytochrome c reductase activity of the wild-type and mutant strains.
Assays were performed with NADPH (black bars) or NADH (grey bars) as cofactor using microsomal fractions extracted after (A) 36 h and (B) 72 h of culture. Values are the mean ± standard deviation of two technical replicates from three independent cultures. Statistical significant differences between wild-type and mutant strain compared to the same assay are indicated (Student’s t test, *P<0.05, **P<0.01 with α = 0.05).
Fig 6.
RT-qPCR for CBR.1. CBR.2, CYB5 and crtR transcript levels in the wild-type and mutant strains.
(A) CBR.1 (B) CBR.2, (C) CYB5 and (D) crtR genes. The transcript level of each gene was normalized to actin mRNA. Black and grey bars represent the results using RNA extracted after 36 and 72 h of culture, respectively. Values are the mean ± standard deviation of two technical replicates from three independent cultures (Student’s t-test was performed to compare each mutant to the parental strain. * P <0.05 and ** P <0.01).