Aquatic Fungus Blastocladiella Emersonii Research Articles

Structure and monomer/dimer equilibrium for the guanylyl cyclase domain of the optogenetics protein RhoGC

RhoGC is a fusion protein from the aquatic fungus Blastocladiella emersonii, combining a type I rhodopsin domain with a guanylyl cyclase domain. It has generated excitement as an optogenetics tool for the manipulation of cyclic nucleotide signaling pathways. To investigate the regulation of the cyclase activity, we isolated the guanylyl cyclase domain from Escherichia coli with (GCwCCRho) and without (GCRho) the coiled-coil linker. Both constructs were constitutively active but were monomeric as determined by size-exclusion chromatography and analytical ultracentrifugation, whereas other class III nucleotidyl cyclases are functional dimers. We also observed that crystals of GCRho have only a monomer in an asymmetric unit. Dimers formed when crystals were grown in the presence of the non-cyclizable substrate analog 2',3'-dideoxyguanosine-5'-triphosphate, MnCl2, and tartrate, but their quaternary structure did not conform to the canonical pairing expected for class III enzymes. Moreover, the structure contained a disulfide bond formed with an active-site Cys residue required for activity. We consider it unlikely that the disulfide would form under intracellular reducing conditions, raising the possibility that this unusual dimer might have a biologically relevant role in the regulation of full-length RhoGC. Although we did not observe it with direct methods, a functional dimer was identified as the active state by following the dependence of activity on total enzyme concentration. The low affinity observed for GCRho monomers is unusual for this enzyme class and suggests that dimer formation may contribute to light activation of the full-length protein.

Expression, purification, and spectral tuning of RhoGC, a retinylidene/guanylyl cyclase fusion protein and optogenetics tool from the aquatic fungus Blastocladiella emersonii

RhoGC is a rhodopsin (Rho)-guanylyl cyclase (GC) gene fusion molecule that is central to zoospore phototaxis in the aquatic fungus Blastocladiella emersonii It has generated considerable excitement because of its demonstrated potential as a tool for optogenetic manipulation of cell-signaling pathways involving cyclic nucleotides. However, a reliable method for expressing and purifying RhoGC is currently lacking. We present here an expression and purification system for isolation of the full-length RhoGC protein expressed in HEK293 cells in detergent solution. The protein exhibits robust light-dependent guanylyl cyclase activity, whereas a truncated form lacking the 17- to 20-kDa N-terminal domain is completely inactive under identical conditions. Moreover, we designed several RhoGC mutants to increase the utility of the protein for optogenetic studies. The first class we generated has altered absorption spectra designed for selective activation by different wavelengths of light. Two mutants were created with blue-shifted (E254D, λmax = 390 nm; D380N, λmax = 506 nm) and one with red-shifted (D380E, λmax = 533 nm) absorption maxima relative to the wild-type protein (λmax = 527 nm). We also engineered a double mutant, E497K/C566D, that changes the enzyme to a specific, light-stimulated adenylyl cyclase that catalyzes the formation of cAMP from ATP. We anticipate that this expression/purification system and these RhoGC mutants will facilitate mechanistic and structural exploration of this important enzyme.

Structural and thermodynamic studies of two centrin isoforms from Blastocladiella emersonii upon calcium binding

Centrins are calcium-binding proteins associated with microtubules organizing centers. Members of two divergent subfamilies of centrins were found in the aquatic fungus Blastocladiella emersonii, contrasting with the occurrence of only one member known for the better explored terrestrial fungi. BeCen1 shows greatest identity with human centrins HsCen1, HsCen2 and green algae centrin CrCenp, while BeCen3 records largest identity with human centrin HsCen3 and yeast centrin Cdc31p. Following the discovery of this unique feature, BeCen1 and BeCen3 centrins were produced to study whether these proteins had distinct features upon calcium binding. Circular dichroism showed opposite calcium binding effects on the α-helix arrangement of the secondary structure. The spectra indicated a decrease in α-helix signal for holo-BeCen1 contrasting with an increase for holo-BeCen3. In addition, only BeCen1 refolds after being de-natured. The fluorescence emission of the hydrophobic probe ANS increases for both proteins likely due to hydrophobic exposure, however, only BeCen1 presents a clear blue shift when calcium is added. ITC experiments identified four calcium binding sites for both proteins. In contrast to calcium binding to BeCen1, which is mainly endothermic, binding to BeCen3 is mainly exothermic. Light-scattering evidenced the formation of large particles in solution for BeCen1 and BeCen3 at temperatures above 30°C and 40°C, respectively. Atomic force microscopy confirmed the presence of supramolecular structures, which differ in the compactness and branching degree. Binding of calcium leads to different structural changes in BeCen1 and BeCen3 and the thermodynamic characteristics of the interaction also differ.

Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii

Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class.

Transcriptional Response to Hypoxia in the Aquatic Fungus Blastocladiella emersonii

Global gene expression analysis was carried out with Blastocladiella emersonii cells subjected to oxygen deprivation (hypoxia) using cDNA microarrays. In experiments of gradual hypoxia (gradual decrease in dissolved oxygen) and direct hypoxia (direct decrease in dissolved oxygen), about 650 differentially expressed genes were observed. A total of 534 genes were affected directly or indirectly by oxygen availability, as they showed recovery to normal expression levels or a tendency to recover when cells were reoxygenated. In addition to modulating many genes with no putative assigned function, B. emersonii cells respond to hypoxia by readjusting the expression levels of genes responsible for energy production and consumption. At least transcriptionally, this fungus seems to favor anaerobic metabolism through the upregulation of genes encoding glycolytic enzymes and lactate dehydrogenase and the downregulation of most genes coding for tricarboxylic acid (TCA) cycle enzymes. Furthermore, genes involved in energy-costly processes, like protein synthesis, amino acid biosynthesis, protein folding, and transport, had their expression profiles predominantly downregulated during oxygen deprivation, indicating an energy-saving effort. Data also revealed similarities between the transcriptional profiles of cells under hypoxia and under iron(II) deprivation, suggesting that Fe(2+) ion could have a role in oxygen sensing and/or response to hypoxia in B. emersonii. Additionally, treatment of fungal cells prior to hypoxia with the antibiotic geldanamycin, which negatively affects the stability of mammalian hypoxia transcription factor HIF-1alpha, caused a significant decrease in the levels of certain upregulated hypoxic genes.

Global Gene Expression Analysis during Sporulation of the Aquatic Fungus Blastocladiella emersonii

The Blastocladiella emersonii life cycle presents a number of drastic biochemical and morphological changes, mainly during two cell differentiation stages: germination and sporulation. To investigate the transcriptional changes taking place during the sporulation phase, which culminates with the production of the zoospores, motile cells responsible for the dispersal of the fungus, microarray experiments were performed. Among the 3,773 distinct genes investigated, a total of 1,207 were classified as differentially expressed, relative to time zero of sporulation, at at least one of the time points analyzed. These results indicate that accurate transcriptional control takes place during sporulation, as well as indicating the necessity for distinct molecular functions throughout this differentiation process. The main functional categories overrepresented among upregulated genes were those involving the microtubule, the cytoskeleton, signal transduction involving Ca(2+), and chromosome organization. On the other hand, protein biosynthesis, central carbon metabolism, and protein degradation were the most represented functional categories among downregulated genes. Gene expression changes were also analyzed in cells sporulating in the presence of subinhibitory concentrations of glucose or tryptophan. Data obtained revealed overexpression of microtubule and cytoskeleton transcripts in the presence of glucose, probably causing the shape and motility problems observed in the zoospores produced under this condition. In contrast, the presence of tryptophan during sporulation led to upregulation of genes involved in oxidative stress, proteolysis, and protein folding. These results indicate that distinct physiological pathways are involved in the inhibition of sporulation due to these two classes of nutrient sources.

Environmental stresses inhibit splicing in the aquatic fungus Blastocladiella emersonii

BackgroundExposure of cells to environmental stress conditions can lead to the interruption of several intracellular processes, in particular those performed by macromolecular complexes such as the spliceosome.ResultsDuring nucleotide sequencing of cDNA libraries constructed using RNA isolated from B. emersonii cells submitted to heat shock and cadmium stress, a large number of ESTs with retained introns was observed. Among the 6,350 ESTs obtained through sequencing of stress cDNA libraries, 181 ESTs presented putative introns (2.9%), while sequencing of cDNA libraries from unstressed B. emersonii cells revealed only 0.2% of ESTs containing introns. These data indicate an enrichment of ESTs with introns in B. emersonii stress cDNA libraries. Among the 85 genes corresponding to the ESTs that retained introns, 19 showed more than one intron and three showed three introns, with intron length ranging from 55 to 333 nucleotides. Canonical splicing junctions were observed in most of these introns, junction sequences being very similar to those found in introns from genes previously characterized in B. emersonii, suggesting that inhibition of splicing during stress is apparently a random process. Confirming our observations, analyses of gpx3 and hsp70 mRNAs by Northern blot and S1 protection assays revealed a strong inhibition of intron splicing in cells submitted to cadmium stress.ConclusionIn conclusion, data indicate that environmental stresses, particularly cadmium treatment, inhibit intron processing in B. emersonii, revealing a new adaptive response to cellular exposure to this heavy metal.

Evidence of a Ca 2+- [rad]NO-cGMP signaling pathway controlling zoospore biogenesis in the aquatic fungus Blastocladiella emersonii

The sporulation stage of the aquatic fungus Blastocladiella emersonii culminates with the formation and release to the medium of a number of zoospores, which are motile cells responsible for the dispersal of the fungus. The presence in the sporulation solution of 1 H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a potent and selective inhibitor of nitric oxide-sensitive guanylyl cyclases, completely prevented biogenesis of the zoospores. In addition, this compound was able to significantly reduce cGMP levels, which increase drastically during late sporulation, suggesting the existence of a nitric oxide-dependent mechanism for cGMP synthesis. Furthermore, increased levels of nitric oxide-derived products were detected during sporulation by fluorescence assays using DAF-2 DA, whose signal was drastically reduced in the presence of the nitric oxide synthase inhibitor Nω-Nitro- l-arginine methyl ester ( l-NAME). These results were confirmed by quantitative chemiluminescent determination of the intracellular levels of nitric oxide-derived products. A putative nitric oxide synthase (NOS) activity was detected throughout sporulation, and this enzyme activity decreased significantly when l-NAME and 1-[2-(Trifluoromethyl)phenyl]imidazole (TRIM) were added to the assays. NOS assays carried out in the presence of EGTA showed decreased enzyme activity, suggesting the involvement of calcium ions in enzyme activation. Additionally, expressed sequence tags (ESTs) encoding putative guanylyl cyclases and a cGMP-phosphodiesterase were found in B. emersonii EST database ( http://blasto.iq.usp.br), and the mRNA levels of the corresponding genes were observed to increase during sporulation. Altogether, data presented here revealed the presence and expression of guanylyl cyclase and cGMP phosphodiesterase genes in B. emersonii and provided evidence of a Ca 2+- NO-cGMP signaling pathway playing a role in zoospore biogenesis.

Characterization and expression of two genes encoding isoforms of a putative Na, K-ATPase in the chytridiomycete Blastocladiella emersonii

A P-type ATPase gene (BePAT1) from the aquatic fungus Blastocladiella emersonii, which surprisingly showed high similarity with the α-subunit of Na, K-ATPases from animal cells, has been reported recently [Biochim. Biophys. Acta 1383 (1998) 183]. In the present study, we describe the characterization of a second gene, denominated BePAT2, and show that these two genes have a different intron–exon structure but encode putative proteins with greater than 90% amino acid identity. Northern blot and multiplex reverse transcription and polymerase chain reaction (RT-PCR) assays have revealed that BePAT1 and BePAT2 genes have a non-coordinate, developmentally regulated expression during B. emersonii life cycle. Phosphoenzyme formation experiments using the immunopurified enzymes have indicated the presence of a Na, K-ATPase-like activity. Furthermore, immunofluorescence studies using B. emersonii zoospores localized the ATPases on the plasma membrane of these cells.

PEST sequences in cAMP-dependent protein kinase subunits of the aquatic fungus Blastocladiella emersonii are necessary for in vitro degradation by endogenous proteases.

During Blastocladiella emersonii germination, the regulatory (R) and the catalytic (C) subunits of the cAMP-dependent protein kinase (PKA) are rapidly and concurrently degraded, after PKA activation in response to a transient increase in intracellular cAMP levels. The possibility that PEST sequences could be acting as proteolytic recognition signals in this process was investigated, and high score PEST sequences were found in both B. emersonii R and C subunits. Deletions in the PEST sequences were obtained by site-directed mutagenesis and the different PKA subunits were independently expressed in Escherichia coli. Proteolysis assays of the various R and C recombinant forms, using B. emersonii cell extracts as the source of proteases, showed a strong correlation between the presence of high score PEST sequences and susceptibility to degradation. Furthermore, the amino-terminal sequence of the proteolytic fragments indicated that the cleavage sites in both subunits are located at or near the PEST regions. The PEST sequence in B. emersonii C subunit, which when deleted or disrupted leads to resistance to proteolysis, is entirely contained in the 72-amino-acid extension located in the N-terminus of the protein. C subunit mutants carrying deletions in this region displayed little difference in their kinetic properties or enzyme thermostability. These results suggest that the N-terminal extension may only play a role in C subunit degradation.

The induction of sporulation in the aquatic fungus Blastocladiella emersonii is dependent on extracellular calcium

Induction of sporulation in Blastocladiella emersonii is absolutely dependent on extracellular calcium. Vegetative cells grown in media with or without calcium do not sporulate in media devoid of calcium or in CaCl 2 with EGTA. Calcium channel blockers, CoCl 2 and nifedipine, and ionophore A23187 inhibited the induction of sporulation. The calmodulin antagonists trifluoperazine and chlorpromazine inhibited the sporulation when present in the cultures at least 60 min after induction. So, calcium that is accumulated during growth is not sufficient or is not mobilized to initiate sporulation, and a calcium influx is likely to occur by type II calcium channel functions, essential for the response to nutritional starvation. A calmodulin-like protein has been suggested to mediate calcium events in sporulation.

The induction of sporulation in the aquatic fungus blastocladiella emersonii is dependent on extracellular calcium

Induction of sporulation in Blastocladiella emersonii is absolutely dependent on extracellular calcium. Vegetative cells grown in media with or without calcium do not sporulate in media devoid of calcium or in CaCl(2) with EGTA. Calcium channel blockers, CoCl(2) and nifedipine, and ionophore A23187 inhibited the induction of sporulation. The calmodulin antagonists trifluoperazine and chlorpromazine inhibited the sporulation when present in the cultures at least 60 min after induction. So, calcium that is accumulated during growth is not sufficient or is not mobilized to initiate sporulation, and a calcium influx is likely to occur by type II calcium channel functions, essential for the response to nutritional starvation. A calmodulin-like protein has been suggested to mediate calcium events in sporulation.

Characterization and submitochondrial localization of the alpha subunit of the mitochondrial processing peptidase from the aquatic fungus Blastocladiella emersonii.

In an effort to investigate the molecular mechanisms responsible for the drastic morphological changes the mitochondria go through during the life cycle of the aquatic fungus Blastocladiella emersonii, the gene encoding the alpha subunit of the mitochondrial processing peptidase (alpha-MPP) was isolated. Nucleotide sequence analysis revealed that the predicted alpha-MPP polypeptide comprises 474 amino acids with a calculated molecular mass of 51,900 Da, presenting a characteristic mitochondrial signal sequence. Northern blot analysis indicated a single 1.4-kb transcript encoding the B. emersonii alpha-MPP, whose levels decrease during sporulation, becoming very low in the zoospore, and increase again during germination. Despite these variations in mRNA concentration, B. emersonii alpha-MPP protein levels do not change significantly during the life cycle of the fungus, as observed in Western blots. Experiments to investigate the submitochondrial localization of B. emersonii alpha-MPP and beta-MPP were also carried out, and the results indicated that both subunits are associated with the mitochondrial inner membrane, possibly as part of the bc1 complex, as described for plants.

A P-type ATPase from the aquatic fungus Blastocladiella emersonii similar to animal Na,K-ATPases

We have cloned a P-type ATPase gene from the aquatic fungus Blastocladiella emersonii (BePAT1) using a probe obtained with degenerate oligonucleotides, corresponding to two amino acid sequences highly conserved among all P-type ATPase isoforms, and the polymerase chain reaction technique. Nucleotide sequence analysis revealed a 3.4 kb open reading frame encoding a putative peptide of 1080 amino acid residues with a calculated molecular mass of 119 kDa, which presents all diagnostic features of P-type transporting ATPases. Comparison to other members of the family and phylogenetic analyses have shown that the BePAT1 protein belongs to the subfamily of Na,K- and H,K-ATPases, indicating that the divergence between the α-subunit of the Na,K-ATPase and other members of the P-type ATPase family has occurred before the divergence between the animal and fungal lineages in evolution.

Identification of a developmentally regulated Gα protein in Blastocladiella emersonii

We have previously demonstrated the stimulation of adenylyl cyclase by guanine nucleotides in the plasma membranes of the aquatic fungus Blastocladiella emersonii. The existence of a G-protein in these membranes similar to Gs, the G-protein responsible for hormonal stimulation of adenylyl cyclase in mammals, was shown. In this report the crude membranes were further analyzed and a polypeptide of ~ 48 kDa reacted with the G α common GA/1 antibody, which recognises the GTP binding site of G-proteins. A polypeptide of similar molecular mass is ADP-ribosylated with pertussis toxin on a cysteine residue, like the counterparts in mammals (Gi, Gt and Go). The appearance of this Gi protein is developmentally controlled, as judged from pertussis toxin labelling and immunoblotting. We suggest the involvement of this G-protein in the control of adenylyl cyclase activity, or ion channels, thus regulating the rapid transition between zoospores and vegetative cells.

Identification of a developmentally regulated Gα protein inBlastocladiella emersonii

We have previously demonstrated the stimulation of adenylyl cyclase by guanine nucleotides in the plasma membranes of the aquatic fungus Blastocladiella emersonii . The existence of a G-protein in these membranes similar to Gs, the G-protein responsible for hormonal stimulation of adenylyl cyclase in mammals, was shown. In this report the crude membranes were further analyzed and a polypeptide of ~ 48 kDa reacted with the Gαcommon GA/1 antibody, which recognises the GTP binding site of G-proteins. A polypeptide of similar molecular mass is ADP-ribosylated with pertussis toxin on a cysteine residue, like the counterparts in mammals (Gi, Gt and Go). The appearance of this Gi protein is developmentally controlled, as judged from pertussis toxin labelling and immunoblotting. We suggest the involvement of this G-protein in the control of adenylyl cyclase activity, or ion channels, thus regulating the rapid transition between zoospores and vegetative cells. * GTP[S] : guanosine 5′-[γ-thio]triphosphate GDP[S] : guanosine 5′-[β-thio]diphosphate Gpp(NH)p : 5″-guanylylimidodiphosphate

A unique intron-containing hsp70 gene induced by heat shock and during sporulation in the aquatic fungus Blastocladiella emersonii

We have isolated and characterized cDNA and genomic DNA clones encoding the 70-kDa heat-shock protein (Hsp70) from the aquatic fungus Blastocladiella emersonii (Be). Nucleotide (nt) sequence analysis predicts an acidic protein containing 650 amino acids, with a calculated molecular mass of 70.8 kDa. The Be hsp70 gene is induced by heat shock (HS), as well as during sporulation of the fungus, and its coding region is interrupted by a single intron. All the evidence seems to indicate that this is the only hsp70 in Be. S1 nuclease protection assays revealed that splicing of the hsp70 intron is highly thermoresistant; at the lethal temperature of 42°C, only 30% of the hsp70 mRNAs have not been processed. A single transcription start point ( tsp), localized about 30 nt downstream from a putative TATA box, was determined both during HS and at normal temperatures. The promoter region presented several NGAAN repeats (where N is any nucleotide) characteristic of HS elements, as well as putative binding sites for ATF, Spl and two metal-responsive elements.

Cloning and structural analysis of the gene for the regulatory subunit of cAMP-dependent protein kinase in Blastocladiella emersonii.

We have isolated and characterized cDNA and genomic DNA clones encoding the regulatory subunit of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii. Nucleotide sequence analysis has shown that the predicted protein comprises 403 amino acids with a calculated molecular mass of 44,263 Da and an overall 40% identity to mammalian RII subunits, including a serine in the phosphorylation site, which confirms the Blastocladiella protein as a type II regulatory subunit. The B. emersonii R gene presents two introns, one located in the 5'-noncoding region, whereas the other interrupts the coding region, just after the dimerization domain of the protein. The promoter region does not contain recognizable TATA or CCAAT sequences and is very GC rich, a characteristic shared by mammalian cAMP-dependent protein kinase subunit genes previously analyzed. S1 mapping and primer extension experiments revealed multiple transcription initiation sites. Several sequence motifs were identified in the 5'-flanking region which could be responsible for the regulation of this gene.

Coordinate pretranslational control of cAMP-dependent protein kinase subunit expression during development in the water mold Blastocladiella emersonii

The aquatic fungus Blastocladiella emersonii provides a system for studying the regulation of expression of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA). Blastocladiella cells contain a single PKA with properties very similar to type II kinases of mammalian tissues. During development cAMP-dependent protein kinase activity and its associated cAMP-binding activity change drastically. We have previously shown that the increase in cAMP-binding activity during sporulation is due to de novo synthesis of R subunit and to an increase in the translatable mRNA coding for R (Marques et al., Eur. J. Biochem. 178, 803, 1989). In the present work we have continued these studies to investigate the mechanism by which the changes in the level of kinase activity take place. The C subunit of Blastocladiella has been purified; antiserum has been raised against it and used to determine the relative amounts of C subunit throughout the fungus' life cycle. A sharp increase in C subunit content occurs during sporulation and peaks at the zoospore stage. Northern blot analyses, using Blastocladiella C and R cDNA probes, have shown that the levels of C and R mRNAs parallel their intracellular protein concentrations. These results indicate a coordinate pretranslational control for C and R subunit expression during differentiation in Blastocladiella.

The mitochondrial genome of the aquatic phycomycete Blastocladiella emersonii

Mitochondrial DNA from the aquatic fungus Blastocladiella emersonii Cantino and Hyatt has been isolated and characterized. By restriction enzyme analysis the size of the mitochondrial genome was found to be 35.5 kb pairs. A restriction site map was constructed using the cleavage data for 6 endonucleases which showed the mitochondrial genome to be circular. The genes for the small and large ribosomal RNA, the ATPase subunits 6 and 9, the cytochrome c oxidase subunits 1, 2, and 3, and the apocytochrome b were located in the mitochonridal genome of B. emersonii by hybridizations with mitochondrial DNA probes from Saccharomyces cerevisiae and Neurospora crassa