Transcriptome and proteome profiling reveals complex adaptations of Candida parapsilosis cells assimilating hydroxyaromatic carbon sources.

Many fungal species utilize hydroxyderivatives of benzene and benzoic acid as carbon sources. The yeast Candida parapsilosis metabolizes these compounds via the 3-oxoadipate and gentisate pathways, whose components are encoded by two metabolic gene clusters. In this study, we determine the chromosome level assembly of the C. parapsilosis strain CLIB214 and use it for transcriptomic and proteomic investigation of cells cultivated on hydroxyaromatic substrates. We demonstrate that the genes coding for enzymes and plasma membrane transporters involved in the 3-oxoadipate and gentisate pathways are highly upregulated and their expression is controlled in a substrate-specific manner. However, regulatory proteins involved in this process are not known. Using the knockout mutants, we show that putative transcriptional factors encoded by the genes OTF1 and GTF1 located within these gene clusters function as transcriptional activators of the 3-oxoadipate and gentisate pathway, respectively. We also show that the activation of both pathways is accompanied by upregulation of genes for the enzymes involved in ß-oxidation of fatty acids, glyoxylate cycle, amino acid metabolism, and peroxisome biogenesis. Transcriptome and proteome profiles of the cells grown on 4-hydroxybenzoate and 3-hydroxybenzoate, which are metabolized via the 3-oxoadipate and gentisate pathway, respectively, reflect their different connection to central metabolism. Yet we find that the expression profiles differ also in the cells assimilating 4-hydroxybenzoate and hydroquinone, which are both metabolized in the same pathway. This finding is consistent with the phenotype of the Otf1p-lacking mutant, which exhibits impaired growth on hydroxybenzoates, but still utilizes hydroxybenzenes, thus indicating that additional, yet unidentified transcription factor could be involved in the 3-oxoadipate pathway regulation. Moreover, we propose that bicarbonate ions resulting from decarboxylation of hydroxybenzoates also contribute to differences in the cell responses to hydroxybenzoates and hydroxybenzenes. Finally, our phylogenetic analysis highlights evolutionary paths leading to metabolic adaptations of yeast cells assimilating hydroxyaromatic substrates.

A SARS-CoV-2 mutant from B.1.258 lineage with ∆H69/∆V70 deletion in the Spike protein circulating in Central Europe in the fall 2020.

SARS-CoV-2 mutants carrying the ∆H69/∆V70 deletion in the amino-terminal domain of the Spike protein emerged independently in at least six lineages of the virus (namely, B.1.1.7, B.1.1.298, B.1.160, B.1.177, B.1.258, B.1.375). We analyzed SARS-CoV-2 samples collected from various regions of Slovakia between November and December 2020 that were presumed to contain B.1.1.7 variant due to drop-out of the Spike gene target in an RT-qPCR test caused by this deletion. Sequencing of these samples revealed that although in some cases the samples were indeed confirmed as B.1.1.7, a substantial fraction of samples contained another ∆H69/∆V70 carrying mutant belonging to the lineage B.1.258, which has been circulating in Central Europe since August 2020, long before the import of B.1.1.7. Phylogenetic analysis shows that the early sublineage of B.1.258 acquired the N439K substitution in the receptor-binding domain (RBD) of the Spike protein and, later on, also the deletion ∆H69/∆V70 in the Spike N-terminal domain (NTD). This variant was particularly common in several European countries including the Czech Republic and Slovakia but has been quickly replaced by B.1.1.7 early in 2021.

Genome sequence of the opportunistic human pathogen Magnusiomyces capitatus.

The yeast Magnusiomyces capitatus is an opportunistic human pathogen causing rare yet severe infections, especially in patients with hematological malignancies. Here, we report the 20.2 megabase genome sequence of an environmental strain of this species as well as the genome sequences of eight additional isolates from human and animal sources providing an insight into intraspecies variation. The distribution of single-nucleotide variants is indicative of genetic recombination events, supporting evidence for sexual reproduction in this heterothallic yeast. Using RNAseq-aided annotation, we identified genes for 6518 proteins including several expanded families such as kexin proteases and Hsp70 molecular chaperones. Several of these families are potentially associated with the ability of M. capitatus to infect and colonize humans. For the purpose of comparative analysis, we also determined the genome sequence of a closely related yeast, Magnusiomyces ingens. The genome sequences of M. capitatus and M. ingens exhibit many distinct features and represent a basis for further comparative and functional studies.

Chromosome-level genome assembly of the yeast Lodderomyces beijingensis reveals the genetic nature of metabolic adaptations and identifies subtelomeres as hotspots for amplification of mating type loci.

Lodderomyces beijingensis is an ascosporic ascomycetous yeast. In contrast to related species Lodderomyces elongisporus, which is a recently emerging human pathogen, L. beijingensis is associated with insects. To provide an insight into its genetic makeup, we investigated the genome of its type strain, CBS 14171. We demonstrate that this yeast is diploid and describe the high contiguity nuclear genome assembly consisting of eight chromosome-sized contigs with a total size of about 15.1 Mbp. We find that the genome sequence contains multiple copies of the mating type loci and codes for essential components of the mating pheromone response pathway, however, the missing orthologs of several genes involved in the meiotic program raise questions about the mode of sexual reproduction. We also show that L. beijingensis genome codes for the 3-oxoadipate pathway enzymes, which allow the assimilation of protocatechuate. In contrast, the GAL gene cluster underwent a decay resulting in an inability of L. beijingensis to utilize galactose. Moreover, we find that the 56.5 kbp long mitochondrial DNA is structurally similar to known linear mitochondrial genomes terminating on both sides with covalently closed single-stranded hairpins. Finally, we discovered a new double-stranded RNA mycovirus from the Totiviridae family and characterized its genome sequence.

Chromosome-Level Genome Assembly of the Yeast Candida verbasci.

Candida verbasci is an anamorphic ascomycetous yeast. We report the genome sequence of its type strain, 11-1055 (CBS 12699). The nuclear genome assembly consists of seven chromosome-sized contigs with a total size of 12.1 Mbp and has a relatively low G+C content (28.1%).

Nanopore sequencing of SARS-CoV-2: Comparison of short and long PCR-tiling amplicon protocols.

Surveillance of the SARS-CoV-2 variants including the quickly spreading mutants by rapid and near real-time sequencing of the viral genome provides an important tool for effective health policy decision making in the ongoing COVID-19 pandemic. Here we evaluated PCR-tiling of short (~400-bp) and long (~2 and ~2.5-kb) amplicons combined with nanopore sequencing on a MinION device for analysis of the SARS-CoV-2 genome sequences. Analysis of several sequencing runs demonstrated that using the long amplicon schemes outperforms the original protocol based on the 400-bp amplicons. It also illustrated common artefacts and problems associated with PCR-tiling approach, such as uneven genome coverage, variable fraction of discarded sequencing reads, including human and bacterial contamination, as well as the presence of reads derived from the viral sub-genomic RNAs.

Genome Sequence of an Arthroconidial Yeast, Saprochaete fungicola CBS 625.85.

Saprochaete fungicola is an arthroconidial yeast classified in the Magnusiomyces/Saprochaete clade of the subphylum Saccharomycotina. Here, we report the genome sequence of holotype strain CBS 625.85, assembled to five putative chromosomes. The genome sequence is 20.2 Mbp long and codes for 6,138 predicted proteins.

Genome Sequence of the Yeast Saprochaete ingens CBS 517.90.

Chromosome-scale genome assembly of the yeast Saprochaete ingens CBS 517.90 was determined by a combination of technologies producing short (HiSeq X; Illumina) and long (MinION; Oxford Nanopore Technologies) reads. The 21.2-Mbp genome sequence has a GC content of 36.9% and codes for 6,475 predicted proteins.

Genome Sequence of Flavor-Producing Yeast Saprochaete suaveolens NRRL Y-17571.

Saprochaete suaveolens is an ascomycetous yeast that produces a range of fruity flavors and fragrances. Here, we report the high-contiguity genome sequence of the ex-holotype strain, NRRL Y-17571 (CBS 152.25). The nuclear genome sequence contains 24.4 Mbp and codes for 8,119 predicted proteins.