Global analysis of mutations driving microevolution of a heterozygous diploid fungal pathogen

Data deposition: The sequence reported in this paper has been deposited in the NCBI Sequence Read Archive, https://www.ncbi.nlm.nih.gov/bioproject (BioProject ID PRJNA345600). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1806002115/-/DCSupplemental.Peer reviewedPublisher PD

Prospects for utilizing microbial consortia for lignin conversion

Naturally occurring microbial communities are able to decompose lignocellulosic biomass through the concerted production of a myriad of enzymes that degrade its polymeric components and assimilate the resulting breakdown compounds by members of the community. This process includes the conversion of lignin, the most recalcitrant component of lignocellulosic biomass and historically the most difficult to valorize in the context of a biorefinery. Although several fundamental questions on microbial conversion of lignin remain unanswered, it is known that some fungi and bacteria produce enzymes to break, internalize, and assimilate lignin-derived molecules. The interest in developing efficient biological lignin conversion approaches has led to a better understanding of the types of enzymes and organisms that can act on different types of lignin structures, the depolymerized compounds that can be released, and the products that can be generated through microbial biosynthetic pathways. It has become clear that the discovery and implementation of native or engineered microbial consortia could be a powerful tool to facilitate conversion and valorization of this underutilized polymer. Here we review recent approaches that employ isolated or synthetic microbial communities for lignin conversion to bioproducts, including the development of methods for tracking and predicting the behavior of these consortia, the most significant challenges that have been identified, and the possibilities that remain to be explored in this field

Novel Structural Components of the Ventral Disc and Lateral Crest in Giardia intestinalis

Giardia intestinalis is a ubiquitous parasitic protist that is the causative agent of giardiasis, one of the most common protozoan diarrheal diseases in the world. Giardia trophozoites attach to the intestinal epithelium using a specialized and elaborate microtubule structure, the ventral disc. Surrounding the ventral disc is a less characterized putatively contractile structure, the lateral crest, which forms a continuous perimeter seal with the substrate. A better understanding of ventral disc and lateral crest structure, conformational dynamics, and biogenesis is critical for understanding the mechanism of giardial attachment to the host. To determine the components comprising the ventral disc and lateral crest, we used shotgun proteomics to identify proteins in a preparation of isolated ventral discs. Candidate disc-associated proteins, or DAPs, were GFP-tagged using a ligation-independent high-throughput cloning method. Based on disc localization, we identified eighteen novel DAPs, which more than doubles the number of known disc-associated proteins. Ten of the novel DAPs are associated with the lateral crest or outer edge of the disc, and are the first confirmed components of this structure. Using Fluorescence Recovery After Photobleaching (FRAP) with representative novel DAP::GFP strains we found that the newly identified DAPs tested did not recover after photobleaching and are therefore structural components of the ventral disc or lateral crest. Functional analyses of the novel DAPs will be central toward understanding the mechanism of ventral disc-mediated attachment and the mechanism of disc biogenesis during cell division. Since attachment of Giardia to the intestine via the ventral disc is essential for pathogenesis, it is possible that some proteins comprising the disc could be potential drug targets if their loss or disruption interfered with disc biogenesis or function, preventing attachment

Genome-Wide Functional Profiling Reveals Genes Required for Tolerance to Benzene Metabolites in Yeast

Benzene is a ubiquitous environmental contaminant and is widely used in industry. Exposure to benzene causes a number of serious health problems, including blood disorders and leukemia. Benzene undergoes complex metabolism in humans, making mechanistic determination of benzene toxicity difficult. We used a functional genomics approach to identify the genes that modulate the cellular toxicity of three of the phenolic metabolites of benzene, hydroquinone (HQ), catechol (CAT) and 1,2,4-benzenetriol (BT), in the model eukaryote Saccharomyces cerevisiae. Benzene metabolites generate oxidative and cytoskeletal stress, and tolerance requires correct regulation of iron homeostasis and the vacuolar ATPase. We have identified a conserved bZIP transcription factor, Yap3p, as important for a HQ-specific response pathway, as well as two genes that encode putative NAD(P)H:quinone oxidoreductases, PST2 and YCP4. Many of the yeast genes identified have human orthologs that may modulate human benzene toxicity in a similar manner and could play a role in benzene exposure-related disease

<em>Candida albicans</em> White and Opaque Cells Undergo Distinct Programs of Filamentous Growth

<div><p>The ability to switch between yeast and filamentous forms is central to <i>Candida albicans</i> biology. The yeast-hyphal transition is implicated in adherence, tissue invasion, biofilm formation, phagocyte escape, and pathogenesis. A second form of morphological plasticity in <i>C. albicans</i> involves epigenetic switching between white and opaque forms, and these two states exhibit marked differences in their ability to undergo filamentation. In particular, filamentous growth in white cells occurs in response to a number of environmental conditions, including serum, high temperature, neutral pH, and nutrient starvation, whereas none of these stimuli induce opaque filamentation. Significantly, however, we demonstrate that opaque cells can undergo efficient filamentation but do so in response to distinct environmental cues from those that elicit filamentous growth in white cells. Growth of opaque cells in several environments, including low phosphate medium and sorbitol medium, induced extensive filamentous growth, while white cells did not form filaments under these conditions. Furthermore, while white cell filamentation is often enhanced at elevated temperatures such as 37°C, opaque cell filamentation was optimal at 25°C and was inhibited by higher temperatures. Genetic dissection of the opaque filamentation pathway revealed overlapping regulation with the filamentous program in white cells, including key roles for the transcription factors <i>EFG1</i>, <i>UME6</i>, <i>NRG1</i> and <i>RFG1</i>. Gene expression profiles of filamentous white and opaque cells were also compared and revealed only limited overlap between these programs, although <i>UME6</i> was induced in both white and opaque cells consistent with its role as master regulator of filamentation. Taken together, these studies establish that a program of filamentation exists in opaque cells. Furthermore, this program regulates a distinct set of genes and is under different environmental controls from those operating in white cells.</p> </div

<em>MTL</em>–Independent Phenotypic Switching in <em>Candida tropicalis</em> and a Dual Role for Wor1 in Regulating Switching and Filamentation

<div><p>Phenotypic switching allows for rapid transitions between alternative cell states and is important in pathogenic fungi for colonization and infection of different host niches. In <i>Candida albicans</i>, the white-opaque phenotypic switch plays a central role in regulating the program of sexual mating as well as interactions with the mammalian host. White-opaque switching is controlled by genes encoded at the <i>MTL</i> (mating-type-like) locus that ensures that only <b>a</b> or α cells can switch from the white state to the mating-competent opaque state, while <b>a</b>/α cells are refractory to switching. Here, we show that the related pathogen <i>C. tropicalis</i> undergoes white-opaque switching in all three cell types (<b>a</b>, α, and <b>a</b>/α), and thus switching is independent of <i>MTL</i> control. We also demonstrate that <i>C. tropicalis</i> white cells are themselves mating-competent, albeit at a lower efficiency than opaque cells. Transcriptional profiling of <i>C. tropicalis</i> white and opaque cells reveals significant overlap between switch-regulated genes in <i>MTL</i> homozygous and <i>MTL</i> heterozygous cells, although twice as many genes are white-opaque regulated in <b>a</b>/α cells as in <b>a</b> cells. In <i>C. albicans</i>, the transcription factor Wor1 is the master regulator of the white-opaque switch, and we show that Wor1 also regulates switching in <i>C. tropicalis</i>; deletion of <i>WOR1</i> locks <b>a</b>, α, and <b>a</b>/α cells in the white state, while <i>WOR1</i> overexpression induces these cells to adopt the opaque state. Furthermore, we show that <i>WOR1</i> overexpression promotes both filamentous growth and biofilm formation in <i>C. tropicalis</i>, independent of the white-opaque switch. These results demonstrate an expanded role for <i>C. tropicalis</i> Wor1, including the regulation of processes necessary for infection of the mammalian host. We discuss these findings in light of the ancestral role of Wor1 as a transcriptional regulator of the transition between yeast form and filamentous growth.</p> </div

<i>WOR1</i> is the master regulator of the white-opaque switch in <i>C. tropicalis</i>.

<p>(A) Cell morphologies of <i>Δwor1</i>, white, opaque, and <i>pTDH3-WOR1</i> (<i>WOR1</i>-overexpressing) strains. Cells were grown in Spider medium at room temperature to 0.8–1.0 OD₆₀₀. Scale bars = 5 µm. (B) Gene expression in <b>a</b>/α white cells (CAY1511), <i>Δwor1</i> cells (CAY4043), opaque cells (CAY4048), and <i>pTDH3-WOR1</i> cells (CAY4045), relative to white cells (CAY1511). Expression profiles for each state were divided by white expression values and filtered for those genes with an expression change greater than 4-fold in 4 or more experiments. (C) <i>WOR1</i> expression in <i>Δwor1</i>, white, opaque, and <i>pTDH3-WOR1</i> strains derived from <b>a</b>, α, and <b>a</b>/α cells. Expression levels measured by qRT-PCR. Error bars indicate SEM for replicate experiments from 3 different biological replicates.</p