Table 1.
C. albicans strains used in this study.
Fig 1.
Generation of conditional null PPT2 mutants of C. albicans.
Step 1: Promoter replacement—MET3 promoter introduced 5’ of the start of the PPT2 coding region of one allele with URA3 as a selection marker. Step 2: Remaining wild type PPT2 gene knocked out by replacement of the majority of the coding sequence with the ARG4 gene.
Fig 2.
Alignment of fungal phosphopantetheinyl transferases (A) and acyl carrier proteins (B).
(A) S. cerevisiae Ppt2p (Sc_Ppt2), A. fumigatus PptB (Af_PptB), and C. albicans C1_09480W/Ppt2p (Ca_C1_09480W). The P2 and P3 regions found in PPTase family enzymes are indicated. (B) S. cerevisiae Acp1p (Sc_Acp1), A. fumigatus (Af_AcpA), C. albicans Acp1p and Acp12p (Ca_Acp1 and Ca_Acp12). The conserved pantetheinylated serine residues are indicated by the open triangle. The recombinant versions of the A. fumigatus and C. albicans acyl carrier proteins started at the residue indicated by the closed triangle.
Fig 3.
PPT2 is essential for growth in C. albicans.
Growth phenotypes of PPT2 conditional null mutant (KDP2), conditional heterozygote (KDP1) and SN76 parental strain in presence and absence of 2.5 mM methionine and 2.5 mM cysteine after 4 days incubation at 30°C.
Fig 4.
Expression and purification of recombinant C. albicans Ppt2 and putative acyl carrier protein substrates.
E. coli BL21 DE3 was transformed with (A) pET43.1-Ppt2 (76 kDa), (B) pET30-Acp1 (16 kDa) and (C) pET30-Acp12 (16 kDa) and expression of the recombinant proteins induced with 0.5 mM IPTG. Recombinant proteins were purified by immobilised metal affinity chromatography. Fractions were separated by SDS PAGE. The migration of molecular weight markers is indicated by the figures (kDa) to the left of each panel. Lane 1, E. coli lysate before induction; Lane 2, E. coli lysate after induction with 0.5 mM IPTG and 18h incubation; Lane 3, purified and desalted fraction of recombinant protein.
Fig 5.
Transfer of fluorescent phosphopantetheine from coenzyme A to ACP.
Bodipy TMR-labelled CoA was incubated for 1 hour at room temperature in the presence or absence of 500 ng Ppt2p, 5 μg Acp1p, 5 μg Acp12p and 20 mM EDTA as indicated. The reaction products were then separated by gel electrophoresis and photographed under UV light. (A) Ppt2 transfers a fluorescent phosphopantetheine group from coenzyme A (open arrowhead) to Acp1p (lane 1, closed arrowhead) but not Acp12p (lane 2). (B) Phosphopantetheinylation by Ppt2p requires magnesium—compare the fluorescent product indicated in lane 6 in the presence of 5 mM MgCl2 with lane 7 in the presence of 20 mM EDTA.
Fig 6.
Fluorescence polarisation assay of Ppt2 activity.
Recombinant Ppt2p (0 ng/μl, cross; 2 ng/μl, circle; 4 ng/μl, triangle; 6 ng/ul, square; 8 ng/μl, diamond) was combined with 30 ng/μl recombinant Acp1p and CoA-BTMR. FP values were obtained at various time intervals with 3 biological replicates for each data point and the standard deviations displayed on the graph.