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The ability to grow on media containing certain D-amino acids as a sole nitrogen source is widely utilized to differentiate  Cryptococcus gattii  from  C .  neoformans . We used the  C .  neoformans  H99 and  C .  gattii  R265 strains to dissect the mechanisms of D-amino acids utilization. We identified three putative D-amino acid oxidase ( DAO ) genes in both strains and showed that each  DAO  gene plays different roles in D-amino acid utilization in each strain. Deletion of  DAO2  retarded growth of R265 on eleven D-amino acids suggesting its prominent role on D-amino acid assimilation in R265. All three R265  DAO  genes contributed to growth on D-Asn and D-Asp.  DAO3  was required for growth and detoxification of D-Glu by both R265 and H99. Although growth of H99 on most D-amino acids was poor, deletion of  DAO1  or  DAO3  further exacerbated it on four D-amino acids. Overexpression of  DAO2  or  DAO3  enabled H99 to grow robustly on several D-amino acids suggesting that expression levels of the native  DAO  genes in H99 were insufficient for growth on D-amino acids. Replacing the H99  DAO2  gene with a single copy of the R265  DAO2  gene also enabled its utilization of several D-amino acids. Results of gene and promoter swaps of the  DAO2  genes suggested that enzymatic activity of Dao2 in H99 might be lower compared to the R265 strain. A reduction in virulence was only observed when all  DAO  genes were deleted in R265 but not in H99 indicating a pathobiologically exclusive role of the  DAO  genes in R265. These results suggest that  C .  neoformans  and  C .  gattii  divergently evolved in D-amino acid utilization influenced by their major ecological niches.

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Differences between Cryptococcus neoformans and Cryptococcus gattii in the Molecular Mechanisms Governing Utilization of D-Amino Acids as the Sole Nitrogen Source