The chytrid fungus Blastocladiella emersonii produces spores with swimming tails (zoospores); these cells can sense and swim toward light. Interest in this species stems from ongoing efforts to develop B. emersonii as a model for understanding the evolution of phototaxis and the molecular cell biology of the associated optogenetic circuits. Here, we report a highly contiguous genome assembly and gene annotation of the B. emersonii American Type Culture Collection 22665 strain. We integrate a PacBio long-read library with an Illumina paired-end genomic sequence survey leading to an assembly of 21 contigs totaling 34.27 Mb. Using these data, we assess the diversity of sensory system encoding genes. These analyses identify a rich complement of G-protein-coupled receptors, ion transporters, and nucleotide cyclases, all of which have been diversified by domain recombination and tandem duplication. In many cases, these domain combinations have led to the fusion of a protein domain to a transmembrane domain, tying a putative signaling function to the cell membrane. This pattern is consistent with the diversification of the B. emersonii sensory-signaling systems, which likely plays a varied role in the complex life cycle of this fungus.

Diverse light-sensing organs (i.e., eyes) have evolved across animals. Interestingly, several subcellular analogs have been found in eukaryotic microbes.1 All of these systems have a common "recipe": a light occluding or refractory surface juxtaposed to a membrane-layer enriched in type I rhodopsins.1-4 In the fungi, several lineages have been shown to detect light using a diversity of non-homologous photo-responsive proteins.5-7 However, these systems are not associated with an eyespot-like organelle with one exception found in the zoosporic fungus Blastocladiella emersonii (Be).8Be possesses both elements of this recipe: an eyespot composed of lipid-filled structures (often called the side-body complex [SBC]), co-localized with a membrane enriched with a gene-fusion protein composed of a type I (microbial) rhodopsin and guanylyl cyclase enzyme domain (CyclOp-fusion protein).8,9 Here, we identify homologous pathway components in four Chytridiomycota orders (Chytridiales, Synchytriales, Rhizophydiales, and Monoblepharidiales). To further explore the architecture of the fungal zoospore and its lipid organelles, we reviewed electron microscopy data (e.g., the works of Barr and Hartmann10 and Reichle and Fuller11) and performed fluorescence-microscopy imaging of four CyclOp-carrying zoosporic fungal species, showing the presence of a variety of candidate eyespot-cytoskeletal ultrastructure systems. We then assessed the presence of canonical photoreceptors across the fungi and inferred that the last common fungal ancestor was able to sense light across a range of wavelengths using a variety of systems, including blue-green-light detection. Our data imply, independently of how the fungal tree of life is rooted, that the apparatus for a CyclOp-organelle light perception system was an ancestral feature of the fungi.

One of the effects of systemic corticosteroids is the reduction in the number of eosinophils. Patients with severe asthma require high doses of inhaled corticosteroids (ICS) for long period of time to control disease. The aim of this study was to evaluate the action of ICS on peripheral eosinophils (Eo). Retrospective study of medical records of patients with severe asthma, steps 4/5 (GINA 2019), followed at a tertiary center. Patients using oral corticosteroids were excluded. Demographic data, ICS dose, specific IgE, total serum IgE, serum cortisol and Eo were evaluated. Fifty-eight patients were evaluated, 81% female, mean age of 61 years, disease duration of 43.6 years. The average ICS was 1682.8 mcg/d. Total serum IgE was 398.9 IU/mL and specific IgE was positive in 40 patients (69%). The average cortisol was 5.6 (0-21.8) mcg/dL and the average Eo, 252.1 (0-1380) cell/mm3. No correlation was observed between ICS dose and serum cortisol. However, 41.4% patients had Eo <150 cel/mm3 and there was an inverse correlation between ICS doses and Eo levels (p=0.029, r2=0.1), so the higher the dose of ICS, the lower the level of Eo. GINA 2019 recommends the use of monoclonal antibodies (mAbs) in asthma step 5, directed by phenotype. Some of these mAbs include as treatment criteria Eo 150 or 300 cell/mm3. In this study high-dose ICS was related to lower levels of Eo, patients using high doses of ICS could have reduced Eo masked by high dose of ICS, interfering with the recommendation of some mAbs.

Blastocladiella emersonii is an aquatic fungus of the phylum Blastocladiomycota, localized near the base of the fungal tree. Previous studies have shown that B. emersonii responds to heat shock and cadmium exposure inducing the transcription of a high number of genes. EST sequencing from heat shocked and cadmium exposed B. emersonii cells has shown that exposure to cadmium causes strong splicing inhibition. Despite the knowledge about splicing inhibition by cadmium, it is still unclear if other metal contaminants can cause the same response. In the present study, we have demonstrated that the effect of cadmium exposure on splicing inhibition is much stronger than that of other divalent metals such as cobalt and manganese. Data presented here also indicate that intron retention occurs randomly among the fungal transcripts, as verified by analyzing differently affected transcripts. In addition, we identified in the genome of B. emersonii the genes encoding the snRNA splicing components U1, U2, U4, U5 and U6 and observed that spliceosome snRNAs are upregulated in the presence of metals, in particular snRNA U1 in cells under cadmium exposure. This observation suggests that snRNA upregulation might be a defense of the fungal cell against the metal stress condition.

Small heat shock proteins (sHsps) are molecular chaperones of low molecular weight involved in an early association with misfolded proteins. In response to heat shock, B. emersonii induces the synthesis of a number of proteins. As sHsps are still poorly studied in B. emersonii and in fungi overall, the aim of this work was to carry out a in-depth characterization of sHsps during B. emersonni life cycle, as well as in response to thermal stress. We verified a strong induction of the hsp17 gene in cells exposed to heat shock both in germination and sporulation stages, and that Hsp17 protein levels show the same pattern of variation of its mRNA. Unlike hsp17 and hsp30, hsp16 gene is not significantly induced during heat shock, in germination or sporulation cells. However, at normal temperatures, the hsp16 gene presents high mRNA levels in sporulation cells, whereas the hsp30 gene presents high mRNA levels in germination cells. Interestingly, heat shock mRNA levels for hsp17 and hsp30 genes are 10 times higher in germination cells than in sporulation cells. Thus, our data show that the expression of these sHsp genes is quite distinct, both under normal temperature as during heat shock.

The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.

The model yeast Saccharomyces cerevisiae elicits a transcriptional response to phosphate (Pi) depletion. To determine the origins of the phosphate response (PHO) system, we bioinformatically identified putative PHO components in the predicted proteomes of diverse fungi. Our results suggest that the PHO system is ancient; however, components have been expanded or lost in different fungal lineages. To show that a similar physiological response is present in deeply-diverging fungi we examined the transcriptional and physiological response of PHO genes to Pi depletion in the blastocladiomycete Blastocladiella emersonii. Our physiological experiments indicate that B. emersonii relies solely on high-affinity Na+-independent Pho84-like transporters. In response to Pi depletion, BePho84 paralogues were 4-8-fold transcriptionally upregulated, whereas several other PHO homologues like phosphatases and vacuolar transporter chaperone (VTC) complex components show 2-3-fold transcriptional upregulation. Since Pi has been shown to be important during the development of B. emersonii, we sought to determine if PHO genes are differentially regulated at different lifecycle stages. We demonstrate that a similar set of PHO transporters and phosphatases are upregulated at key points during B. emersonii development. Surprisingly, some genes upregulated during Pi depletion, including VTC components, are repressed at these key stages of development indicating that PHO genes are regulated by different pathways in different developmental and environmental situations. Overall, our findings indicate that a complex PHO network existed in the ancient branches of the fungi, persists in diverse extant fungi, and that this ancient network is likely to be involved in development and cell cycle regulation.

Blastocladiella emersonii is an early diverging fungus of the phylum Blastocladiomycota. During the life cycle of the fungus, mitochondrial morphology changes significantly, from a fragmented form in sessile vegetative cells to a fused network in motile zoospores. In this study, we visualize these morphological changes using a mitochondrial fluorescent probe and show that the respiratory capacity in zoospores is much higher than in vegetative cells, suggesting that mitochondrial morphology could be related to the differences in oxygen consumption. While studying the respiratory chain of the fungus, we observed an antimycin A and cyanide-insensitive, salicylhydroxamic (SHAM)-sensitive respiratory activity, indicative of a mitochondrial alternative oxidase (AOX) activity. The presence of AOX was confirmed by the finding of a B. emersonii cDNA encoding a putative AOX, and by detection of AOX protein in immunoblots. Inhibition of AOX activity by SHAM was found to significantly alter the capacity of the fungus to grow and sporulate, indicating that AOX participates in life cycle control in B. emersonii.

Dissection of the subcellular eye of microorganisms called warnowiid dinoflagellates reveals that this structure is composed of elements of two cellular organelles - the plastid and the mitochondrion. See Letter p.204

ABSTRACT Phototaxis in flagellated zoospores of the aquatic fungus Blastocladiella emersonii depends on a novel photosensor, Blastocladiella emersonii GC1 (BeGC1), comprising a type I (microbial) rhodopsin fused to a guanylyl cyclase catalytic domain, that produces the conserved second messenger cyclic GMP (cGMP). The rapid and transient increase in cGMP levels during the exposure of zoospores to green light was shown to be necessary for phototaxis and dependent on both rhodopsin function and guanylyl cyclase activity. It is noteworthy that BeGC1 was localized to the zoospore eyespot apparatus, in agreement with its role in the phototactic response. A putative cyclic nucleotide-gated channel (BeCNG1) was also identified in the genome of the fungus and was implicated in flagellar beating via the action of a specific inhibitor ( l - cis -diltiazem) that compromised zoospore motility. Here we show that B. emersonii expresses a K + channel that is activated by cGMP. The use of specific channel inhibitors confirmed the activation of the channel by cGMP and its K + selectivity. These characteristics are consistent with the function of an ion channel encoded by the BeCNG1 gene. Other blastocladiomycete fungi, such as Allomyces macrogynus and Catenaria anguillulae , possess genes encoding a similar K + channel and the rhodopsin–guanylyl cyclase fusion protein, while the genes encoding both these proteins are absent in nonflagellated fungi. The presence of these genes as a pair seems to be an exclusive feature of blastocladiomycete fungi. Taken together, these data demonstrate that the B. emersonii cGMP-activated K + channel is involved in the control of zoospore motility, most probably participating in the cGMP-signaling pathway for the phototactic response of the fungus.

Background: With the aim of remaining viable, bacteria must deal with changes in environmental conditions, including increases in external osmolarity. While studies concerning bacterial response to this stress condition have focused on soil, marine and enteric species, this report is about Caulobacter crescentus, a species inhabiting freshwater oligotrophic habitats.Results: A genomic analysis reported in this study shows that most of the classical genes known to be involved in intracellular solute accumulation under osmotic adaptation are missing in C. crescentus. Consistent with this observation, growth assays revealed a restricted capability of the bacterium to propagate under hyperosmotic stress, and addition of the compatible solute glycine betaine did not improve bacterial resistance. A combination of transcriptomic and proteomic analyses indicated quite similar changes triggered by the presence of either salt or sucrose, including down-regulation of many housekeeping processes and up-regulation of functions related to environmental adaptation. Furthermore, a GC-MS analysis revealed some metabolites at slightly increased levels in stressed cells, but none of them corresponding to well-established compatible solutes.Conclusion: Despite a clear response to hyperosmotic stress, it seems that the restricted capability of C. crescentus to tolerate this unfavorable condition is probably a consequence of the inability to accumulate intracellular solutes. This finding is consistent with the ecology of the bacterium, which inhabits aquatic environments with low nutrient concentration.