Allomyces Macrogynus Research Articles

Mitochondrial genome characterization and phylogenetic analysis of Blastocladiella sp. (Blastocladiales: Blastocladiaceae)

In the present study, the complete mitochondrial genome of an early diverging fungus Blastocladiella sp. was assembled by the next-generation sequencing. The complete mitochondrial genome of Blastocladiella sp. is 33, 800 bp in length and consists of 11,620 (34.38%) adenine, 5,047 (14.93%) cytosine, 6,025 (17.83%) guanosine and 11,108 (32.86%) thymine. The genome contains 19 protein-coding genes, 24 tRNA genes and 2 rRNA genes. Phylogenetic analysis based on the combined mitochondrial gene set showed that Blastocladiella sp. has a close relationship with Allomyces macrogynus and Blastocladiella emersonii.

Ultrastructure of early stages of Rozella allomycis (Cryptomycota) infection of its host, Allomyces macrogynus (Blastocladiomycota)

This study reconstructs early stages of Rozella allomycis endoparasitic infection of its host, Allomyces macrogynus. Young thalli of A. macrogynus were inoculated with suspensions of R. allomycis zoospores and allowed to develop for 120 h. Infected thalli at intervals were fixed for electron microscopy and observed. Zoospores were attracted to host thalli, encysted on their surfaces, and penetrated their walls with an infection tube. The parasite cyst discharged its protoplast through an infection tube, which invaginated the host plasma membrane. The host plasma membrane then surrounded the parasite protoplast and formed a compartment confining it inside host cytoplasm. The earliest host-parasite interface within host cytoplasm consisted of two membranes, the outer layer the host plasma membrane and the inner layer the parasite plasma membrane. At first a wide space separated the two membranes and no material was observed within this space. Later, as the endoparasite thallus expanded within the compartment, the two membranes became closely appressed. As the endoparasite thallus continued to enlarge, the interface developed into three membrane layers. Thus, host plasma membrane surrounded the parasite protoplast initially without the parasite having to pierce the host plasma membrane for entry. Significantly, host-derived membrane was at the interface throughout development.

The Fungal Sexual Pheromone Sirenin Activates the Human CatSper Channel Complex.

The basal fungus Allomyces macrogynus (A. macrogynus) produces motile male gametes displaying well-studied chemotaxis toward their female counterparts. This chemotaxis is driven by sirenin, a sexual pheromone released by the female gametes. The pheromone evokes a large calcium influx in the motile gametes, which could proceed through the cation channel of sperm (CatSper) complex. Herein, we report the total synthesis of sirenin in 10 steps and 8% overall yield and show that the synthetic pheromone activates the CatSper channel complex, indicated by a concentration-dependent increase in intracellular calcium in human sperm. Sirenin activation of the CatSper channel was confirmed using whole-cell patch clamp electrophysiology with human sperm. Based on this proficient synthetic route and confirmed activation of CatSper, analogues of sirenin can be designed as blockers of the CatSper channel that could provide male contraceptive agents.

Cleavage of cytoplasm within the oligonucleate zoosporangia of Allomyces macrogynus

Allomyces macrogynus produces zoosporangia that discharge uninucleate zoospores after cleavage of multinucleate cytoplasm. Cleavage of cytoplasm within the oligonucleate zoosporangia of A. macrogynus was visualized by constructing three-dimensional models based on electron micrographs and confocal images. In oligonucleate zoosporangia, three adjacent nuclei can form three cleavage planes with a line of intersection of the planes. The position and boundary of the cleavage planes are thought to be determined by the relative positions of the nuclei. The establishment of three cleavage planes by cleavage membranes occurred sequentially, and the nuclear axis connecting the centers of two nuclei affected the development of cleavage membranes on each cleavage plane. In multinucleate zoosporangia, groups of three neighboring nuclei near the cell cortex may initiate the sequential establishment of cleavage planes and then may interact with the nuclei further from the cortex until the interactions of nuclei are propagated to the central region of the cytoplasm.

Sediment Enzyme Activities and Microbial Community Diversity in an Oligotrophic Drinking Water Reservoir, Eastern China

Drinking water reservoir plays a vital role in the security of urban water supply, yet little is known about microbial community diversity harbored in the sediment of this oligotrophic freshwater environmental ecosystem. In the present study, integrating community level physiological profiles (CLPPs), nested polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and clone sequence technologies, we examined the sediment urease and protease activities, bacterial community functional diversity, genetic diversity of bacterial and fungal communities in sediments from six sampling sites of Zhou cun drinking water reservoir, eastern China. The results showed that sediment urease activity was markedly distinct along the sites, ranged from 2.48 to 11.81 mg NH3-N/(g·24h). The highest average well color development (AWCD) was found in site C, indicating the highest metabolic activity of heterotrophic bacterial community. Principal component analysis (PCA) revealed tremendous differences in the functional (metabolic) diversity patterns of the sediment bacterial communities from different sites. Meanwhile, DGGE fingerprints also indicated spatial changes of genetic diversity of sediment bacterial and fungal communities. The sequence BLAST analysis of all the sediment samples found that Comamonas sp. was the dominant bacterial species harbored in site A. Alternaria alternate, Allomyces macrogynus and Rhizophydium sp. were most commonly detected fungal species in sediments of the Zhou cun drinking water reservoir. The results from this work provide new insights about the heterogeneity of sediment microbial community metabolic activity and genetic diversity in the oligotrophic drinking water reservoir.

Cytokinesis of the binucleate zoosporangia of Allomyces macrogynus

Allomyces macrogynus, a true fungus, produces zoosporangia which discharge uninucleate zoospores after cytoplasmic cleavage. Binucleate zoosporangia of A. macrogynus were induced and examined to understand the basic principles of cytokinesis associated with the multinucleate zoosporangia. Development of cleavage membranes was visualized by constructing three dimensional models based on electron micrographs and confocal images. Cleavage membranes on the cleavage plane showed asymmetric ingression from the cortex, but cleavage of cytoplasm was completed by the fusion of cleavage membranes with plasma membrane. Also, the position of the cleavage plane was continuously rotated until settled at the last stage. These studies suggest that the positions of the numerous cleavage planes within a multinucleate zoosporangium are continuously adjusted during development of cleavage membranes. The final settlement of cleavage planes would define the exact boundary of cleavage planes and the expansion of cleavage membranes toward the boundary could complete the cleavage of cytoplasm.

Convergent evolution of RFX transcription factors and ciliary genes predated the origin of metazoans

BackgroundIntraflagellar transport (IFT) genes, which are critical for the development and function of cilia and flagella in metazoans, are tightly regulated by the Regulatory Factor X (RFX) transcription factors (TFs). However, how and when their evolutionary relationship was established remains unknown.ResultsWe have identified evidence suggesting that RFX TFs and IFT genes evolved independently and their evolution converged before the first appearance of metazoans. Both ciliary genes and RFX TFs exist in all metazoans as well as some unicellular eukaryotes. However, while RFX TFs and IFT genes are found simultaneously in all sequenced metazoan genomes, RFX TFs do not co-exist with IFT genes in most pre-metazoans and thus do not regulate them in these organisms. For example, neither the budding yeast nor the fission yeast possesses cilia although both have well-defined RFX TFs. Conversely, most unicellular eukaryotes, including the green alga Chlamydomonas reinhardtii, have typical cilia and well conserved IFT genes but lack RFX TFs. Outside of metazoans, RFX TFs and IFT genes co-exist only in choanoflagellates including M. brevicollis, and only one fungus Allomyces macrogynus of the 51 sequenced fungus genomes. M. brevicollis has two putative RFX genes and a full complement of ciliary genes.ConclusionsThe evolution of RFX TFs and IFT genes were independent in pre-metazoans. We propose that their convergence in evolution, or the acquired transcriptional regulation of IFT genes by RFX TFs, played a pivotal role in the establishment of metazoan.

Isolation of male strains of the aquatic Phycomycete Allomyces macrogynus

Treatment of gametophytic or sporophytic thalli of the aquatic Phycomycete Allomyces macrogynus with acridine compounds (acriflavin, acriflavin hydrochloride, ethidium bromide, acridine orange) results in gametophytic thalli which are predominantly male in phenotype (95–99% male). When the male gametes of these acridine-treated strains are crossed to wild-type female gametes or the reciprocal cross is made, using female gametes from the predominantly male strains, there is no segregation of the predominantly male phenotype among the F1 gametophytes. The acridine-induced male strains are able to grow and differentiate under conditions of oxygen starvation. It is suggested that the extreme male phenotype of acridine-treated strains mimic the wild-type male by an induced mitochondrial disfunction through the specific action of the dyes used.

The mitochondrial view of Blastocladiella emersonii

The mitochondrial genome of the chytrid Blastocladiella emersonii was sequenced and annotated, revealing the complete set of oxidative phosphorylation genes and tRNAs/rRNAs necessary for the translation process. Phylogenetic reconstructions reinforce the proposal of the new phylum Blastocladiomycota. Evidences of gene duplication due to inserted elements suggest shared susceptibility to gene invasion/exchange between chytrids and zygomycetes. The gene content of B. emersonii is very similar to Allomyces macrogynus but the content of intronic and changeable elements is much lower suggesting a stronger resistance to this kind of exchange. In addition, a total of 401 potential nuclear transcripts encoding mitochondrial proteins were obtained after B. emersonii EST database scanning using Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana data as probes and TargetP tool to find N-terminal mitochondrial signal in translated sequences.

Origin, evolution, and mechanism of 5' tRNA editing in chytridiomycete fungi.

5' tRNA editing has been demonstrated to occur in the mitochondria of the distantly related rhizopod amoeba Acanthamoeba castellanii and the chytridiomycete fungus Spizellomyces punctatus. In these organisms, canonical tRNA structures are restored by removing mismatched nucleotides at the first three 5' positions and replacing them with nucleotides capable of forming Watson-Crick base pairs with their 3' counterparts. This form of editing seems likely to occur in members of Amoebozoa other than A. castellanii, as well as in members of Heterolobosea. Evidence for 5' tRNA editing has not been found to date, however, in any other fungus including the deeply branching chytridiomycete Allomyces macrogynus. We predicted that a similar form of tRNA editing would occur in members of the chytridiomycete order Monoblepharidales based on the analysis of complete mitochondrial tRNA complements. This prediction was confirmed by analysis of tRNA sequences using a tRNA circularization/RT-PCR-based approach. The presence of partially and completely unedited tRNAs in members of the Monoblepharidales suggests the involvement of a 5'-to-3' exonuclease rather than an endonuclease in removing the three 5' nucleotides from a tRNA substrate. Surprisingly, analysis of the mtDNA of the chytridiomycete Rhizophydium brooksianum, which branches as a sister group to S. punctatus in molecular phylogenies, did not suggest the presence of editing. This prediction was also confirmed experimentally. The absence of tRNA editing in R. brooksianum raises the possibility that 5' tRNA editing may have evolved twice independently within Chytridiomycota, once in the lineage leading to S. punctatus and once in the lineage leading to the Monoblepharidales.

Functional Necessity of the Cytoskeleton during Cleavage Membrane Development and Zoosporogenesis in Allomyces macrogynus

Cleavage membrane development and cytokinesis were examined in zoosporangia of Allomyces macrogynus treated with cytoskeletal inhibitors and compared to zoosporogenesis under control conditions. Developing membranes were visualized in living zoosporangia with laser-scanning confocal microscopy using the lipophilic membrane dye FM4-64. Under control conditions, cleavage membranes developed in four discrete stages, ultimately interconnecting to delimit the cytoplasm into polygonal uninucleate domains of near uniform size. Disruption of microtubules did not impede the normal four-stage development of cleavage membranes, and cytokinesis occurred with only minor detectable anomalies, although zoospores lacked flagella. Disruption of actin microfilaments did not inhibit membrane formation but blocked nuclear migration and significantly disrupted membrane alignment and cytoplasmic delimitation. This resulted in masses of membrane that remained primarily in cortical regions of the zoosporangia, as did nuclei, throughout zoosporogenesis. Zoospores formed in the absence of microtubules had only a slightly larger mean diameter than control zoospores, although nearly 50% of spores contained two or more nuclei. Microfilament inhibitor treatments produced spores with substantially larger mean diameters and correspondingly larger numbers of nuclei per spore, with greater than 85% containing three or more nuclei. These results showed that a functional actin microfilament cytoskeleton was required for proper alignment of cleavage elements and cytokinesis in Allomyces zoosporangia while microtubules played a less significant role.

Functional necessity of the cytoskeleton during cleavage membrane development and zoosporogenesis in Allomyces macrogynus

Cleavage membrane development and cytokinesis were examined in zoosporangia of Allomyces macrogynus treated with cytoskeletal inhibitors and compared to zoosporogenesis under control conditions. Developing membranes were visualized in living zoosporangia with laser-scanning confocal microscopy using the lipophilic membrane dye FM4-64. Under control conditions, cleavage membranes developed in four discrete stages, ultimately interconnecting to delimit the cytoplasm into polygonal uninucleate domains of near uniform size. Disruption of microtubules did not impede the normal four-stage development of cleavage membranes, and cytokinesis occurred with only minor detectable anomalies, although zoospores lacked flagella. Disruption of actin microfilaments did not inhibit membrane formation but blocked nuclear migration and significantly disrupted membrane alignment and cytoplasmic delimitation. This resulted in masses of membrane that remained primarily in cortical regions of the zoosporangia, as did nuclei, throughout zoosporogenesis. Zoospores formed in the absence of microtubules had only a slightly larger mean diameter than control zoospores, although nearly 50% of spores contained two or more nuclei. Microfilament inhibitor treatments produced spores with substantially larger mean diameters and correspondingly larger numbers of nuclei per spore, with greater than 85% containing three or more nuclei. These results showed that a functional actin microfilament cytoskeleton was required for proper alignment of cleavage elements and cytokinesis in Allomyces zoosporangia while microtubules played a less significant role.

Microtubules Are Required for Motility and Positioning of Vesicles and Mitochondria in Hyphal Tip Cells of Allomyces macrogynus

We have used video-enhanced light microscopy and digital image processing to characterize the intracellular motility and positioning of vesicles (∼1-μm diameter) and mitochondria in growing hyphal tip cells of Allomyces macrogynus. These observations were coupled with cytoskeletal inhibitory experiments to define the roles of the microtubule and actin cytoskeletons in organelle translocation and positioning. Vesicles and mitochondria were abundant in apical and subapical hypha regions. Vesicles traveled along paths that were parallel to the longitudinal axis of the cell. Anterograde (i.e., toward the hyphal apex) and retrograde (i.e., away from the hyphal apex) movements of vesicles occurred at average rates of 4.0 and 2.2 μm/s, respectively. Bidirectional travel of vesicles along common paths was noted in the cortical cytoplasm. Mitochondria were aligned mostly parallel to the long axis of the hypha, except those extending into the hyphal apex, which were oriented toward the Spitzenkörper. In regions of the subapical hypha mitochondria were often restricted to the cortical cytoplasm and nuclei occupied the central cytoplasmic region. Mitochondria displayed rapid anterograde movements reaching speeds of 3.0 μm/s, but primarily maintained a constant position relative to either the advancing cytoplasm or the lateral cell wall. Cytoskeletal disruption experiments showed that the positioning of mitochondria and motility of vesicles and mitochondria were microtubule-based and suggested that the actin cytoskeleton played uncertain roles.

Double-Hairpin Elements in the Mitochondrial DNA of Allomyces: Evidence for Mobility

The mitochondrial DNA (mtDNA) of the chytridiomycete fungus Allomyces macrogynus contains 81 G+C-rich sequence elements that are 26-79 bases long and can be folded into a unique secondary structure consisting of two stem-loops. At the primary sequence level, the conservation of these double-hairpin elements (DHEs) is variable, ranging from marginal to complete identity. Forty of these DHEs are inserted in intergenic regions, 35 in introns, and 6 in variable regions of rRNA genes. Ten DHEs are inserted into other DHE elements (twins); two even form triplets. A comparison of DHE sequences shows that loop regions contain more sequence variation than helical regions and that the latter often contain compensatory base changes. This suggests a functional importance of the DHE secondary structure. We further identified nine DHEs in a 4-kb region of Allomyces arbusculus, a close relative of A. macrogynus. Eight of these DHEs are highly similar in sequence (90%-100%) to those in A. macrogynus, but only five are inserted at the same positions as in A. macrogynus. Interestingly, DHEs are also found in the mtDNAs of other chytridiomycetes, as well as certain zygomycete and ascomycete fungi. The overall distribution pattern of DHEs in fungal mtDNAs suggests that they are mobile elements.

Use of the Vital Stain FM4-64 for Visualizing Membrane Dynamics in Living Fungal Cells

Abstract Allomyces macrogynus is a zoosporic fungus (Chytridiomycota) that exhibits a true alternation of generations between the diploid and haploid thalli. During the diploid stage in the life cycle, sporothalli are produced which are capable of forming asexual zoosporangia containing a multinucleate protoplast bound by a single plasma membrane and cell wall. Though able to remain in a quiescent, coenocytic state for an extended period of time, under certain environmental conditions the cytoplasm of mature zoosporangia is cleaved and gives rise to numerous uninucleate, uniflagellate zoospores within 40 to 50 min. Previous studies of cleavage membrane development m Allomyces zoosporangia have been conducted using light microscopy and transmission electron microscopy (TEM). Despite these investigations, our understanding of cytokinesis in zoosporangia of Allomyces and other zoosporic organisms remains incomplete. This is due, in part, to the difficulty of detecting cleavage elements using standard light microscope optics (i.e., phase contrast, differential interference contrast) and because ultrastructural analysis often provides limited temporal and spatial detail and may suffer from artifacts of sample preparation.

Cytoplasmic cleavage in living zoosporangia of Allomyces macrogynus.

We have used the vital fluorescent dye, FM4-64, as a marker of membrane development during zoospore formation in living zoosporangia of Allomyces macrogynus. Membrane development was visualized and documented using standard epifluorescence and laser scanning confocal microscopy. Video-enhanced light microscopy and transmission electron microscopy, using cryopreparation methods, were also employed in this study. In the first 10-12 min after the induction of zoospore formation, only the plasma membrane labelled with FM4-64. During this time, nuclei were strictly located in the cortical cytoplasm with their associated centrosomes positioned immediately adjacent to the plasma membrane (Lowry & Roberson, 1997). Between 12 and 20 min post-induction, increased fluorescence appeared along regions of the plasma membrane adjacent to the nuclei. From these sites, membranes (i.e. cleavage elements) extended laterally within the cortex and then, in conjunction with nuclear migration, rapidly elongated into the sporangial cytoplasm. By 25-35 min post-induction, cleavage elements had ramified throughout the cytoplasm forming a complex, interconnected membranous network. Transmission electron microscopy revealed that cleavage elements were paired membrane sheets with a lumen consisting of an electron opaque, granular matrix. Cleavage elements developed into a highly ordered network by 35-40 min post-induction, which fully delimited zoospore initials into polyhedral-shaped cells. Zoospore discharge occurred between 40 and 50 min post-induction. Our results have shown that cleavage elements undergo four stages of development during zoospore formation in A. macrogynus: (i) development of membrane initials, (ii) cortical extension, (iii) cytoplasmic elongation and ramification and (iv) zoospore initial delimitation.

Hyperpolarisation-activated calcium currents found only in cells from the elongation zone of Arabidopsis thaliana roots: Kiegle E, Gilliham M, Haselhoff J, Tester M: Plant J 2000, 21:225–229

Electrical currents are generated by tip-growing cells and have been proposed to be important in the establishment and maintenance of cell polarity. Unlike the situation in all other fungi thus far examined, the electrical current of Allomyces macrogynus normally flows out of the hyphal tip, not into it. In this organism, as with another chytrid Blastocladiella emersonii, the electrical current enters the rhizoids. Extracellular ion-selective microelectrodes detected local acidification of the medium around hyphal apices, but no local accumulation of K+ or Ca2+ at this site. These results suggest that the outward current is generated by H+ efflux, not K+ or Ca2+ efflux. The hyphae of this fungus were found to be weakly chemotropic, in contrast to the rhizoids which were strongly chemotropic. Rhizoid growth was also directed strongly towards the anode of an applied electrical field; the hyphae were directed to the cathode. Chemotropism and galvanotropism are therefore unrelated. We suggest that, as with other chytridomycetes, the rhizoid of A. macrogynus is an organ specializing in nutrient transport. Since transport is often coupled to proton symport the inward electrical current at rhizoids may be the consequence of localized proton-driven solute transport. In hyphae of the oomycete Achlya bisexualis inward current, localized nutrient transport and chemotropic behaviour again are correlated. We conclude that for water moulds, inward currents indicate zones of localized nutrient transport. The current is therefore incidental to the process of tip growth.

Changes in the number of nuclei during minicycle sporogenesis of Allomyces macrogynus

Young thalli of Allomyces macrogynus were induced to develop small zoosporangia that released one to three zoospores. In the majority of thalli examined in this study, the number of nuclei per thallus increased after induction of sporogenesis, but, decreased before discharge of zoospores. The significance of the decrease in nuclear number during sporogenesis is discussed in relation to the formation of uniformly sized zoospores.

Changes in the Number of Nuclei during Minicycle Sporogenesis of Allomyces macrogynus

Changes in the Number of Nuclei during Minicycle Sporogenesis of Allomyces macrogynus

The Mitochondrial DNA of Allomyces macrogynus: The Complete Genomic Sequence from an Ancestral Fungus

We have determined the complete nucleotide sequence of the circular mitochondrial DNA (mtDNA) of the chytridiomycete fungus, Allomyces macrogynus(57,473 bp; A + T content 60.5%). The identified genes that are typical for most fungal mitochondria include those for the large ( rnl) and small subunit ( rns) ribosomal RNAs, a complete set of 25 tRNAs, three ATPase subunits ( atp6, atp8and atp9), apocytochrome b(cob), three subunits of the cytochrome oxidase complex ( cox1, cox2and cox3), and seven subunits of the NADH dehydrogenase complex ( nad1, nad2, nad3, nad4, nad4L, nad5and nad6). A total of 28 introns of both groups are found, some of which contain open reading frames (ORFs) coding for potential endonucleases (group I) or reverse-transcriptases (group II). All mitochondrial genes are transcribed from the same DNA strand, as is the case in many other eufungi. Particular features of the A. macrogynus mtDNA include: (1) the first documented case of a fungal mitochondrial ribosomal protein gene ( rps3) that is clearly identified by similarity with bacterial homologues; (2) four unique ORFs; (3) the presence of an insert in the atp6gene that may have been acquired by interspecific transfer; (4) more than 67 short, highly structured and conserved DNA elements inserted in intergenic spacers, introns, and variable regions of the rnland rnsgenes: these elements are unusually G + C rich; (5) rRNA structures that resemble more closely those of eubacteria than their counterparts in other fungal mitochondria. The high degree of conservation of the A. macrogynusmitochondrial rRNA secondary structures, the existence of a mitochondrial rps3gene (common to protist but unique in fungal mtDNAs), and phylogenetic relationships inferred from highly conserved protein genes, demonstrate consistently the ancestral character of this fungal mitochondrial genome.