We investigated genomic diversity of a yeast species that is both an opportunistic pathogen and an important industrial yeast. Under the name Candida krusei, it is responsible for about 2% of yeast infections caused by Candida species in humans. Bloodstream infections with C. krusei are problematic because most isolates are fluconazole-resistant. Under the names Pichia kudriavzevii, Issatchenkia orientalis and Candida glycerinogenes, the same yeast, including genetically modified strains, is used for industrial-scale production of glycerol and succinate. It is also used to make some fermented foods. Here, we sequenced the type strains of C. krusei (CBS573T) and P. kudriavzevii (CBS5147T), as well as 30 other clinical and environmental isolates. Our results show conclusively that they are the same species, with collinear genomes 99.6% identical in DNA sequence. Phylogenetic analysis of SNPs does not segregate clinical and environmental isolates into separate clades, suggesting that C. krusei infections are frequently acquired from the environment. Reduced resistance of strains to fluconazole correlates with the presence of one gene instead of two at the ABC11-ABC1 tandem locus. Most isolates are diploid, but one-quarter are triploid. Loss of heterozygosity is common, including at the mating-type locus. Our PacBio/Illumina assembly of the 10.8 Mb CBS573T genome is resolved into 5 complete chromosomes, and was annotated using RNAseq support. Each of the 5 centromeres is a 35 kb gene desert containing a large inverted repeat. This species is a member of the genus Pichia and family Pichiaceae (the methylotrophic yeasts clade), and so is only distantly related to other pathogenic Candida species.

Only a few genes remain in the mitochondrial genome retained by every eukaryotic organism that carry out essential functions and are implicated in severe diseases. Experimentally relocating these few genes to the nucleus therefore has both therapeutic and evolutionary implications. Numerous unproductive attempts have been made to do so, with a total of only 5 successes across all organisms. We have taken a novel approach to relocating mitochondrial genes that utilizes naturally nuclear versions from other organisms. We demonstrate this approach on subunit 9/c of ATP synthase, successfully relocating this gene for the first time in any organism by expressing the ATP9 genes from Podospora anserina in Saccharomyces cerevisiae. This study substantiates the role of protein structure in mitochondrial gene transfer: expression of chimeric constructs reveals that the P. anserina proteins can be correctly imported into mitochondria due to reduced hydrophobicity of the first transmembrane segment. Nuclear expression of ATP9, while permitting almost fully functional oxidative phosphorylation, perturbs many cellular properties, including cellular morphology, and activates the heat shock response. Altogether, our study establishes a novel strategy for allotopic expression of mitochondrial genes, demonstrates the complex adaptations required to relocate ATP9, and indicates a reason that this gene was only transferred to the nucleus during the evolution of multicellular organisms.

An F(1)F(O) ATP synthase in the inner mitochondrial membrane catalyzes the late steps of ATP production via the process of oxidative phosphorylation. A small protein subunit (subunit c or ATP9) of this enzyme shows a substantial genetic diversity, and its gene can be found in both the mitochondrion and/or nucleus. In a representative set of 26 species of fungi for which the genomes have been entirely sequenced, we found five Atp9 gene repartitions. The phylogenetic distribution of nuclear and mitochondrial Atp9 genes suggests that their evolution has included two independent transfers to the nucleus followed by several independent episodes of the loss of the mitochondrial and/or nuclear gene. Interestingly, we found that in Podospora anserina, subunit c is exclusively produced from two nuclear genes (PaAtp9-5 and PaAtp9-7), which display different expression profiles through the life cycle of the fungus. The PaAtp9-5 gene is specifically and strongly expressed in germinating ascospores, whereas PaAtp9-7 is mostly transcribed during sexual reproduction. Consistent with these observations, deletion of PaAtp9-5 is lethal, whereas PaAtp9-7 deletion strongly impairs ascospore production. The P. anserina PaAtp9-5 and PaAtp9-7 genes are therefore nonredundant. By swapping the 5' and 3' flanking regions between genes we demonstrated, however, that the PaAtp9 coding sequences are functionally interchangeable. These findings show that after transfer to the nucleus, the subunit c gene in Podospora became a key target for the modulation of cellular energy metabolism according to the requirements of the life cycle.