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. 2022 Oct 6;11:e80604. doi: 10.7554/eLife.80604

Figure 1. FRP1 is essential for hemoglobin-iron acquisition, whereas FRP2 contributes to growth on hemoglobin at alkaline pH.

(A) Fivefold dilutions of cultures of the indicated strains were spotted on YPD or YPD pH 8.5, with the indicated supplements, and incubated for 3 days (Hb and BPS plates) or 2 days (YPD plates) at 30°C. WT = KC2, FRP1+/-=KC859, frp1-/-=KC870, frp1-/-<FRP1 >= KC1024, FRP2+/-=KC901, frp2-/-=KC912, frp2-/-<FRP2 >= KC1379. (B) The strains with indicated genotypes in the ccc2-/- background were grown in triplicates in YPD or YPD pH 8.5 media supplemented with 1 mM ferrozine and the indicated amounts of hemoglobin, and incubated at 30°C for 3 days. Each result is the average of three cultures. Standard deviations are indicated by vertical bars. WT = KC811, frp1-/-=KC1146, frp2-/-=KC1414, frp1-/- frp2-/-=KC1412, frp1-/-<FRP1 >= KC1146, frp2-/-<FRP2>=KC1411. (C) The frp1-/- and frp2-/- heme-iron utilization phenotype was compared to that of the CFEM protein mutants rbt5-/- and pga7-/-. The strains were grown in YPD or YPD pH 8.5, with 1 mM ferrozine and the indicated concentrations of hemoglobin or hemin, and grown and measured as in B. Wild type = KC68, rbt5-/-=KC139, pga7-/-=KC485, frp1-/-=KC923, frp2-/-=KC913. All strains in B and C carry a deletion of the CCC2 gene, which causes a defect in high-affinity iron import and prevents growth in the presence of ferrozine.

Figure 1—source data 1. Excel file with data used to make Figure 1B.
Figure 1—source data 2. Excel file with data used to make Figure 1C.

Figure 1.

Figure 1—figure supplement 1. Proximity tree of Ascomycete CFEM protein sequences.

Figure 1—figure supplement 1.

The genomes of ascomycete species were screened for sequences similar to Candida albicans Rbt5 by BLAST. Eighty-six sequences were aligned using the MAFFT G-INS-i algorithm (Katoh et al., 2005) and a tree was built on this alignment using the NJ method. The 46 Candiaceae CFEM proteins from 14 species (green lines) cluster in three groups, with homology to Rbt5, Csa2, and Pga7, respectively. Species prefixes of proteins in the three clusters most similar to C. albicans Rbt5, Csa2, and Pga7: no prefix – C. albicans; Cp – Candida parapsilosis; Ct – Candida tropicalis; Dh – Debaryomyces hansenii; Lelon – Lodderomyces elongisporus; Pstip – Pichia stipitis; Ctan – Candida tanzawaensis; Spas – Spathaspora passalidarum; Ctenuis – Candida tenuis; Clus – Candida lusitaniae; Mbicus – Metschnikowia bicuspidate; Milfar – Millerozyma farinosa; Cmal – Candida maltosa; Hburt – Hypopichia burtonii. Eight species show one apparent ortholog each of Rbt5, Csa2, and Pga7, 4 species show orthologs of two of the three at least, and two species show orthologs of only one. Additional CFEM proteins (bottom part of the figure) are from more distantly related species and may not participate in heme acquisition.
Figure 1—figure supplement 2. Similarity between the Frp1 and Frp2 sequences and the predicted C. albicans ferric reductases sequences.

Figure 1—figure supplement 2.

(A) Proximity tree of Candida albicans ferric reductase-related protein sequences. Seventeen protein sequences most related to Fre10 were aligned using the MAFFT G-INS-i algorithm (Katoh et al., 2005) and a tree was built on this alignment using the NJ method with bootstrap resampling to estimate the confidence of each branching (indicated as percentages near each node). (B) Schematic alignment of the 17 C. albicans ferric reductase-like protein sequences. The level of sequence conservation is color-coded red-blue-gray from highest to lowest. The alignment was obtained using NCBI’s ‘Cobalt’ multiple alignment tool. The conserved domains are indicated on top. They were assigned using NCBI’s conserved domains database. The standard orf19 names of the named proteins are: FRP1 – orf19.5634; FRP2 – orf19.7112; CFL4 – orf19.1932; FRE10 – orf19.1415; CFL1 – orf19.1263; FRE3 – orf19.1270; FRE6 – orf19.6138; CFL5 – orf19.1930; FRE7 – orf19.7077; CFL11 – orf19.701; CFL2 – orf19.1264; FRE9 – orf19.3538; FRE4 – orf19.1844; FRE30-7 is the combined reading frames of orf19.6139 and orf19.6140, which were alternatively assigned a single or two distinct ORFs in different versions of the C. albicans genome database. Our re-sequencing indicates that they in fact form a single ORF.
Figure 1—figure supplement 3. Synteny of the FRP1-PGA7 (left) and CSA1-FRP2 (right) genomic regions across Saccharomycetales species listed in the Candida Gene Order Browser database (cgob3.ucd.ie; Fitzpatrick et al., 2010; Maguire et al., 2013).

Figure 1—figure supplement 3.

Both regions are syntenic in Candida albicans, Candida dubliniensis, Candida parapsilosis, Candida orthopsilosis, Lodderomyces elongisporus and Scheffersomyces stipitis, and only FRP1-PGA7 in Candida lusitaniae. FRP1 is absent in Spathaspora passalidarum, FRP2 is absent in Debaryomyces hansenii, Candida tenuis, and Candida auris. Both are absent in Meyerozyma guilliermondii. Overall, the FRP1-PGA7 synteny is conserved in 75% and the CSA1-FRP2 synteny in 70% of genomes where they are present. Note that RBT5, encoding another CFEM protein that participates in heme acquisition, is also part of the FRP1-PGA7 region.
Figure 1—figure supplement 4. FRP2 under the control of the FRP1 promoter is unable to complement the frp1-/- mutant but does complement the frp2-/- mutant.

Figure 1—figure supplement 4.

(Top) The frp1-/- mutant strain KC870 was transformed either with the vector plasmid BES116, with the FRP1-containing plasmid KB2546 or with an FRP2 open reading frame fused to the FRP1 promoter, KB2575. The cells were spotted on YPD, on YPD with 1 mM BPS, with or without 1 μM hemoglobin, as indicated. (Bottom) The frp2-/- mutant strain KC912 was transformed either with the vector plasmid BES116, with the FRP2-containing plasmid KB2576, or with an FRP2 open reading frame fused to the FRP1 promoter, KB2575. The cells were spotted on YPD, on YPD with 1 mM BPS, with or without 1 μM hemoglobin, all at pH 8.5, as indicated.