Alex W. Crocker

Budding yeasts present an especially challenging geometry for segregation of chromosomes, which must be delivered across the narrow mother–bud neck into the bud. Studies in the model yeast Saccharomyces cerevisiae have revealed an elaborate set of mechanisms that selectively orient one mitotic spindle pole toward the bud and then drive spindle elongation along the mother–bud axis, ensuring nuclear segregation between mother and bud. It is unclear how these pathways might be adapted to yield similar precision in more complex cell geometries. Here, we provide the first description of the dynamics of mitosis in a multinucleate, multibudding yeast, Aureobasidium pullulans, and identify many unexpected differences from uninucleate yeasts. Mitotic spindles do not orient along the mother–bud axis prior to anaphase, and accurate nuclear segregation often occurs after spindle disassembly. Cortical Num1–dynein forces pull highly mobile nuclei into buds, and once a nucleus enters a bud, it discourages others from entering, ensuring that most daughters inherit only one nucleus.

Individuals with cystic fibrosis (CF) are susceptible to chronic lung infections that lead to inflammation and irreversible lung damage. While most respiratory infections that occur in CF are caused by bacteria, some are dominated by fungi such as the slow-growing black yeast Exophiala dermatitidis . Here, we analyze isolates of E. dermatitidis cultured from two samples, collected from a single subject two years apart. One isolate genome was sequenced using long-read Nanopore technology as an in-population reference to use in comparative single nucleotide polymorphism (SNP) and insertion-deletion (INDEL) variant analyses of twenty-three isolates. We then used population genomics and phylo-genomics to compare the isolates to each other as well as the type strain E. dermatitidis NIH/UT8656. Within the CF lung population, three E. dermatitidis clades were detected, each with varying mutation rates. Overall, the isolates were highly similar suggesting that they were recently diverged. All isolates were MAT 1-1, which was consistent with their high relatedness and the absence of evidence for mating or recombination between isolates. Phylogenetic analysis grouped sets of isolates into clades that contained isolates from both early and late time points indicating there are multiple persistent lineages. Functional assessment of variants unique to each clade identified alleles in genes that encode transporters, cytochrome P450 oxidoreductases, iron acquisition and DNA repair processes. Consistent with the genomic heterogeneity, isolates showed some stable phenotype heterogeneity in melanin production, subtle differences in antifungal minimum inhibitory concentrations, growth on different substrates. The persistent population heterogeneity identified in lung-derived isolates is an important factor to consider in the study of chronic fungal infections, and the analysis of changes in fungal pathogens over time may provide important insights into the physiology of black yeasts and other slow-growing fungi in vivo .

Pseudomonas aeruginosa has a broad metabolic repertoire that facilitates its coexistence with different microbes. Many microbes secrete products that P. aeruginosa can then catabolize, including ethanol, a common fermentation product. Here, we show that under oxygen-limiting conditions P. aeruginosa utilizes AdhA, an NAD-linked alcohol dehydrogenase, as a previously undescribed means for ethanol catabolism. In a rich medium containing ethanol, AdhA, but not the previously described PQQ-linked alcohol dehydrogenase, ExaA, oxidizes ethanol and leads to the accumulation of acetate in culture supernatants. AdhA-dependent acetate accumulation and the accompanying decrease in pH promote P. aeruginosa survival in LB-grown stationary-phase cultures. The transcription of adhA is elevated by hypoxia and under anoxic conditions, and we show that it is regulated by the Anr transcription factor. We have shown that lasR mutants, which lack an important quorum sensing regulator, have higher levels of Anr-regulated transcripts under low-oxygen conditions than their wild-type counterparts. Here, we show that a lasR mutant, when grown with ethanol, has an even larger decrease in pH than the wild type (WT) that is dependent on both anr and adhA. The large increase in AdhA activity is similar to that of a strain expressing a hyperactive Anr-D149A variant. Ethanol catabolism in P. aeruginosa by AdhA supports growth on ethanol as a sole carbon source and electron donor in oxygen-limited settings and in cells growing by denitrification under anoxic conditions. This is the first demonstration of a physiological role for AdhA in ethanol oxidation in P. aeruginosa. IMPORTANCE Ethanol is a common product of microbial fermentation, and the Pseudomonas aeruginosa response to and utilization of ethanol are relevant to our understanding of its role in microbial communities. Here, we report that the putative alcohol dehydrogenase AdhA is responsible for ethanol catabolism and acetate accumulation under low-oxygen conditions and that it is regulated by Anr.

Significance: Drug-resistant subpopulations of microbes or tumor cells are difficult to detect but can confound disease treatment. In this deep characterization of a chronic fungal infection, we report unprecedented heterogeneity in the drug resistance-related gene MRR1 among Clavispora ( Candida ) lusitaniae isolates from a single individual. Cells expressing Mrr1 variants that led to drug resistance, by elevated expression of the MDR1 -encoded efflux protein, were present at low levels in each sample and thus were undetected in standard assays. We provide evidence that these drug-resistant fungi may arise indirectly in response to other factors present in the infection. Our work suggests that alternative methods may be able to identify drug-resistant subpopulations and thus positively impact patient care.

Existing CRISPR-Cas9 genome modification systems for use in Candida albicans , which rely on constructs to endogenously express the Cas9 protein and guide RNA, do not work efficiently in other Candida species due to inefficient promoter activity. Here, we present an expression-free method that uses RNA-protein complexes and demonstrate its use in three Candida species known for their drug resistance profiles. We propose that this system will aid the genetic analysis of fungi that lack established genetic systems.