Exploring the genomic diversity of black yeasts and relatives (Chaetothyriales, Ascomycota).

The order Chaetothyriales (Pezizomycotina, Ascomycetes) harbours obligatorily melanised fungi and includes numerous etiologic agents of chromoblastomycosis, phaeohyphomycosis and other diseases of vertebrate hosts. Diseases range from mild cutaneous to fatal cerebral or disseminated infections and affect humans and cold-blooded animals globally. In addition, Chaetothyriales comprise species with aquatic, rock-inhabiting, ant-associated, and mycoparasitic life-styles, as well as species that tolerate toxic compounds, suggesting a high degree of versatile extremotolerance. To understand their biology and divergent niche occupation, we sequenced and annotated a set of 23 genomes of main the human opportunists within the Chaetothyriales as well as related environmental species. Our analyses included fungi with diverse life-styles, namely opportunistic pathogens and closely related saprobes, to identify genomic adaptations related to pathogenesis. Furthermore, ecological preferences of Chaetothyriales were analysed, in conjuncture with the order-level phylogeny based on conserved ribosomal genes. General characteristics, phylogenomic relationships, transposable elements, sex-related genes, protein family evolution, genes related to protein degradation (MEROPS), carbohydrate-active enzymes (CAZymes), melanin synthesis and secondary metabolism were investigated and compared between species. Genome assemblies varied from 25.81 Mb (Capronia coronata) to 43.03 Mb (Cladophialophora immunda). The bantiana-clade contained the highest number of predicted genes (12 817 on average) as well as larger genomes. We found a low content of mobile elements, with DNA transposons from Tc1/Mariner superfamily being the most abundant across analysed species. Additionally, we identified a reduction of carbohydrate degrading enzymes, specifically many of the Glycosyl Hydrolase (GH) class, while most of the Pectin Lyase (PL) genes were lost in etiological agents of chromoblastomycosis and phaeohyphomycosis. An expansion was found in protein degrading peptidase enzyme families S12 (serine-type D-Ala-D-Ala carboxypeptidases) and M38 (isoaspartyl dipeptidases). Based on genomic information, a wide range of abilities of melanin biosynthesis was revealed; genes related to metabolically distinct DHN, DOPA and pyomelanin pathways were identified. The MAT (MAting Type) locus and other sex-related genes were recognized in all 23 black fungi. Members of the asexual genera Fonsecaea and Cladophialophora appear to be heterothallic with a single copy of either MAT-1-1 or MAT-1-2 in each individual. All Capronia species are homothallic as both MAT1-1 and MAT1-2 genes were found in each single genome. The genomic synteny of the MAT-locus flanking genes (SLA2-APN2-COX13) is not conserved in black fungi as is commonly observed in Eurotiomycetes, indicating a unique genomic context for MAT in those species. The heterokaryon (het) genes expansion associated with the low selective pressure at the MAT-locus suggests that a parasexual cycle may play an important role in generating diversity among those fungi.

Genome variation in Cryptococcus gattii, an emerging pathogen of immunocompetent hosts.

Cryptococcus gattii recently emerged as the causative agent of cryptococcosis in healthy individuals in western North America, despite previous characterization of the fungus as a pathogen in tropical or subtropical regions. As a foundation to study the genetics of virulence in this pathogen, we sequenced the genomes of a strain (WM276) representing the predominant global molecular type (VGI) and a clinical strain (R265) of the major genotype (VGIIa) causing disease in North America. We compared these C. gattii genomes with each other and with the genomes of representative strains of the two varieties of Cryptococcus neoformans that generally cause disease in immunocompromised people. Our comparisons included chromosome alignments, analysis of gene content and gene family evolution, and comparative genome hybridization (CGH). These studies revealed that the genomes of the two representative C. gattii strains (genotypes VGI and VGIIa) are colinear for the majority of chromosomes, with some minor rearrangements. However, multiortholog phylogenetic analysis and an evaluation of gene/sequence conservation support the existence of speciation within the C. gattii complex. More extensive chromosome rearrangements were observed upon comparison of the C. gattii and the C. neoformans genomes. Finally, CGH revealed considerable variation in clinical and environmental isolates as well as changes in chromosome copy numbers in C. gattii isolates displaying fluconazole heteroresistance.

DNA sequence and structure requirements for cleavage of V(D)J recombination signal sequences.

Purified RAG1 and RAG2 proteins can cleave DNA at V(D)J recombination signals. In dissecting the DNA sequence and structural requirements for cleavage, we find that the heptamer and nonamer motifs of the recombination signal sequence can independently direct both steps of the cleavage reaction. Proper helical spacing between these two elements greatly enhances the efficiency of cleavage, whereas improper spacing can lead to interference between the two elements. The signal sequences are surprisingly tolerant of structural variation and function efficiently when nicks, gaps, and mismatched bases are introduced or even when the signal sequence is completely single stranded. Sequence alterations that facilitate unpairing of the bases at the signal/coding border activate the cleavage reaction, suggesting that DNA distortion is critical for V(D)J recombination.

Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps.

Formation of double-strand breaks at recombination signal sequences is an early step in V(D)J recombination. Here we show that purified RAG1 and RAG2 proteins are sufficient to carry out this reaction. The cleavage reaction can be divided into two distinct steps. First, a nick is introduced at the 5' end of the signal sequence. The other strand is then broken, resulting in a hairpin structure at the coding end and a blunt, 5'-phosphorylated signal end. The hairpin is made as a direct consequence of the cleavage mechanism. Nicking and hairpin formation each require the presence of a signal sequence and both RAG proteins.

DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect.

Severe combined immunodeficient (SCID) mice are deficient in a recombination process utilized in both DNA double-strand break repair and in V(D)J recombination. The phenotype of these mice involves both cellular hypersensitivity to ionizing radiation and a lack of B and T cell immunity. The catalytic subunit of DNA-dependent protein kinase, p350, was identified as a strong candidate for the murine gene SCID. Both p350 and a gene complementing the SCID defect colocalize to human chromosome 8q11. Chromosomal fragments expressing p350 complement the SCID phenotype, and p350 protein levels are greatly reduced in cells derived from SCID mice compared to cells from wild-type mice.

Rch1, a protein that specifically interacts with the RAG-1 recombination-activating protein.

RAG1 and RAG2 are lymphoid-specific genes that together induce V(D)J recombinase activity in a variety of nonlymphoid cell types. While no other lymphoid-specific factors are required to induce recombination, other factors with more widespread expression patterns have been implicated in the reaction. However, none of these factors have been cloned, and their relationship to the RAG proteins is unclear. Using the yeast two-hybrid assay, we have identified RCH1, a gene encoding a protein of molecular weight 58,000 that interacts specifically with RAG-1. The predicted Rch1 protein sequence is 47% identical to yeast SRP1, a protein associated with the nuclear envelope. A truncated form of Rch1, which retains the ability to interact with RAG-1, reduces V(D)J recombination activity in HeLa cells.

Analysis of regions of RAG-2 important for V(D)J recombination.

The recombinase activating genes RAG-1 and RAG-2 operate together to activate V(D)J recombination, and thus play an essential role in the generation of immune system diversity. As a first step in understanding the function of the RAG-2 protein, we have tested a series of deletion and insertion mutations for their ability to induce V(D)J joining of a variety of model substrates. Mutants were assayed for their ability to induce deletional and inversional V(D)J joining, thereby testing their proficiency at forming both signal and coding joints, and, in some cases, for their ability to carry out recombination of both extrachromosomal and integrated recombination substrates. All these reactions were affected similarly by any one mutation. Although the RAG-2 protein shows extensive evolutionary conservation across its length, we found that the carboxy-terminal portion of RAG-2, including an acidic region, is dispensable for all forms of recombination tested. In contrast, all mutations we created in the N-terminal region severely decreased recombination. Thus, the core active region required for V(D)J recombination is confined to the first three-quarters of the RAG-2 protein.