Water Mold Achlya Research Articles

Potential of aquatic oomycete as a novel feedstock for microbial oil grown on waste sugarcane bagasse

Biodiesel production from vegetable oils is not sustainable and economical due to the food crisis worldwide. The development of a cost-effective non-edible feedstock is essential. In this study, we proposed to use aquatic oomycetes for microbial oils, which are cellulolytic fungus-like filamentous eukaryotic microorganisms, commonly known as water molds. They differ from true fungi as cellulose is present in their cell wall and chitin is absent. They show parasitic as well as saprophytic nature and have great potential to utilize decaying animal and plant debris in freshwater habitats. To study the triacylglycerol (TAG) accumulation in the aquatic oomycetes, the isolated water mold Achlya diffusa was cultivated under semi-solid-state conditions on waste sugarcane bagasse, which was compared with the cultivation in Czapek (DOX) medium. A. diffusa grown on waste sugarcane bagasse showed large lipid droplets in its cellular compartment and synthesized 124.03 ± 1.93 mg/gds cell dry weight with 50.26 ± 1.76% w/w lipid content. The cell dry weight and lipid content of this water mold decreased to 89.54 ± 1.21 mg/gds and 38.82% w/w, respectively, when cultivated on standard medium Czapek-Dox agar (CDA). For the fatty acid profile of A. diffusa grown in sugarcane bagasse and CDA, in situ transesterification (IST) and indirect transesterification (IDT) approaches were evaluated. The lipid profile of this mold revealed the presence of C12:0, C14:0, C16:0, C18:0, C18:1, C18:2, C20:0, and C21:0 fatty acids, which is similar to vegetable oils. The biodiesel properties of the lipids obtained from A. diffusa satisfied the limits as determined by international standards ASTM-D6751 and EN-14214 demonstrating its suitability as a fuel for diesel engines.

A steroid isolated from the water mold Achlya heterosexualis induces neurogenesis in vitro and in vivo

Using 22 R-hydroxycholesterol as a sub-structure to screen natural compound databases, we identified a naturally occurring steroid (sc-7) with a 16-acetoxy-22 R-hydroxycholesterol moiety, in which the hydroxyl groups in positions 3 and 22 are esterified by an acetoxy group and in which the carbon in position 26 carries a functional diacetylamino. sc-7 is an analog of the sex steroids dehydro-oogoniol and antheridiol, can be isolated from the water mold Achlya heterosexualis, and promoted neurogenesis in vitro and in vivo. Mouse embryonic teratocarcinoma P19 cells exposed to sc-7 for 2 days followed by a 5-day wash-out differentiated into cholinergic neurons that expressed specific neuronal markers and displayed axonal formation. Axons continued growing up to 28 days after treatment. In vivo, infusion of sc-7 for 2 weeks into the left ventricle of the rat brain followed by a 3-week wash-out induced bromodeoxyuridine uptake by cells of the ependymal layer and subventricular zone that co-localized with doublecortin and glial fibrillary acidic protein immunostaining, demonstrating induction of proliferation and differentiation of neuronal progenitors. Migrating neuroblasts were also observed in the corpus callosum. Thus, under these experimental conditions, adult ependymal cells resumed proliferation and differentiation. Taken together, these results suggest that sc-7 is an interesting molecule for stimulating in situ neurogenesis from resident neuronal progenitors as part of neuron replacement therapy. sc-7 did not bind to nuclear steroid receptors and was not metabolized as a steroid, supporting our hypothesis that the neurogenic effect of sc-7 is not likely due to a steroid-like effect.

Individual and combined effects of multiple pathogens on Pacific treefrogs

In nature, individual hosts often encounter multiple pathogens simultaneously, which can lead to additive, antagonistic, or synergistic effects on hosts. Synergistic effects on infection prevalence or severity could greatly affect host populations. However, ecologists and managers often overlook the influence of pathogen combinations on hosts. This is especially true in amphibian conservation, even though multiple pathogens coexist within amphibian populations, and several pathogens have been implicated in amphibian population declines and extinctions. Using an amphibian host, Pseudacris regilla (Pacific treefrog), we experimentally investigated interactive effects among three pathogens: the trematode Ribeiroia sp. (hereafter, Ribeiroia), the fungus Batrachochytrium dendrobatidis (hereafter, BD), and the water mold Achlya flagellata. We detected no effects of A. flagellata, but did find effects of Ribeiroia and BD that varied depending on context. Low doses of Ribeiroia caused relatively few malformations, while higher Ribeiroia doses caused numerous deformities dominated by missing and reduced limbs and limb elements. Exposure to low doses of BD accelerated larval host development, despite there being no detectable BD infections, while exposure to higher BD doses caused infection but did not alter developmental rate. Hosts exposed to both Ribeiroia and BD exhibited the highest mortality, although overall evidence of interactive effects of multiple pathogens was limited. We suggest further research on the influence of multi-pathogen assemblages on amphibians, particularly under a variety of ecological conditions and with a wider diversity of hosts and pathogens.

The discovery of fungal sex hormones: II. Antheridiol

Antheridiol, originally termed hormone A, was discovered by John Raper during a study on mating in the oomycete water mould Achlya that extended from the mid-1930s to 1950. The hormone, which is produced by the female and induces the development of antheridia in the male, was postulated by Raper in 1939, its existence proved in 1940, and obtained by Raper and the chemist Haagen-Smit in a highly concentrated state by 1942. The study of mating in Achlya was resumed, by Alma Barksdale, in the mid-1950s. She and the chemist Trevor McMorris reported the isolation of antheridiol in 1967. A steroid, its structure was determined in 1968. Barksdale's career came to an end in 1975 through ill-health, but other workers continue to study antheridiol and to exploit its properties in their research.

Biological response of the female strain Achlya ambisexualis 734 to dehydro-oogoniol and analogues

Oogonium development in female strains of the water mould Achlya is initiated in nature by a steroid mixture of oogoniol, dehydro-oogoniol and thei

Extension growth of the water mold Achlya: interplay of turgor and wall strength.

When hyphae of the water mold Achlya were subjected to osmotic stress, imposed with polyethylene glycol (PEG)-300 or sucrose, turgor pressure fell in proportion to the increase in external osmotic pressure. There was no evidence of turgor regulation, even over a period of days, yet the extension rate was unaffected until turgor was reduced to less than a third of the normal level of 0.6-0.8 MPa (6-8 bars). Measurements of the pressure at which the hyphae burst indicate that they respond to osmotic stress by softening their apical cell walls, sustaining extension growth despite reduced turgor pressure. The effect of osmolytes excluded by the wall was very different; superfusion of growing hyphae with PEG-6000 or dextran-6000 reduced turgor and stopped extension but did not induce wall softening. Furthermore, the hyphae did not resume growth during an hour or more of continuous exposure to these substances. Although the two classes of osmolytes have the same effect on turgor, they may induce different strains within the cell wall; this might then affect the capacity of the organism to detect the drop in turgor or to soften its cell wall. The interplay between turgor and wall strength supports the proposition that turgor supplies the driving force for extension and that production of the standard hyphal form requires a balance between hydrostatic pressure and a resistive cell wall.

Heat shock protein 88 and Aspergillus infection

Immunoblotting was used to dissect the antibody responses in the sera of 50 patients with proven invasive aspergillosis, 28 patients with suspected invasive aspergillosis, 35 patients with allergic bronchopulmonary aspergillosis, and 10 patients with an aspergilloma. This demonstrated the immunodominance of antigenic bands at 88, 84, 51, and 40 kDa. Monoclonal antibodies against the heat shock protein 90 complexes of Candida albicans and the water mold Achlya ambisexualis identified these four antigenic bands as homologous proteins. Similar antigens have been described in humans, mice, Saccharomyces cerevisiae, chickens, and Drosophila species. The antibody against A. ambisexualis has previously been shown to cross-react with antigens belonging to the human heat shock protein 90 complex. Aspergillus heat shock protein 90 was extracted from the sera of patients with invasive aspergillosis by affinity chromatography. This was done with both a rabbit hyperimmune antiserum raised against an extract of Aspergillus fumigatus NCPF 2109 and a monoclonal antibody against the heat shock protein 90 of C. albicans. In vivo expression of the antigen was demonstrated in an aspergilloma surgically removed from a patient. The role of the antigen as an allergen in allergic bronchopulmonary aspergillosis is also discussed.

Chemotactic auto-aggregation in the water mould Achlya

SUMMARY: Free-swimming zoospores of several isolates of the oomycetous water mould Achlya demonstrate mutual attraction (adelphotaxis) which may result in auto-aggregation. In the apparent absence of exogenous chemotactic signals macroscopic globular aggregates or microscopic plaques of cysts are formed. Auto-aggregation was favoured by high concentrations of zoospores in suspension; the growth rate of spontaneous aggregates was estimated at 4--10 x 103spores min−1; growth of an aggregate was limited to about 7 min. By means of chemotactic assays, attractant activity for zoospores was detected in the vicinity of aggregates, and in zoospore or cyst supernatants. The attractant activity extracted from spore aggregates was heat-stable. In the absence of exogenous nutrients, the macroscopic aggregates of an homothallic isolate produced sporangia and oogonia. Aggregation was readily initiated as a result of zoospore chemotaxis towards capillary tubes containing exogenous attractant in an agar medium. Aggregating zoospores frequently assumed a slender swarm configuration moving directly towards the aggregate at the capillary-tube tip. Analysis of video recordings showed that many zoospores joining the swarm were attracted directly by the swarm rather than by the exogenous attractant. Such aggregation was a time-limited process, the duration of which was extended by the presence of amino acids in the zoospore suspension. Under natural conditions zoospore aggregation may be a dual-component chemotactic process triggered by exogenous attractants and amplified by chemical signals from the aggregating spores. The possible consequences of adelphotaxis and auto-aggregate formation, namely enhanced inoculum potential, syngamy and spore production, are discussed.

A method of sequencing without subcloning and its application to the identification of a novel ORF with a sequence suggestive of a transcriptional regulator in the water mold Achlya ambisexualis

Genomic amplification with transcript sequencing (GAWTS) is a method of direct sequencing that involves amplification with PCR using primers containing phage promoters, transcription of the amplified product, and sequencing with reverse transcriptase. GAWTS requires the generation of PCR primers that are specific for the sequences on both sides of a region. Here we describe promoter ligation and transcript sequencing (PLATS), a direct method for rapidly obtaining novel sequences that utilizes generic primers and only requires knowledge of the sequence on one side of a region. PLATS involves restriction digestion of the amplified vector insert, ligation with a phage promoter, and then GAWTS using phage promoter sequences as the PCR primers. The method is rapid and economical because it uses a limited set of oligonucleotides, and it potentially amenable to automation because it does not require in vivo manipulations. PLATS facilitates the determination of a genomic sequence responsible for cross-hybridization in a Southern blot. Using PLATS, sequence has been obtained from a 1.1-kb segment in Achlya ambisexualis, which cross-hybridizes to the DNA-binding region of the chicken and Xenopus estrogen receptors. To our knowledge, this represents the first sequence reported from the Oomycetes, a large and widely distributed group of fungi. The sequence reveals a large, transcribed open reading frame that is markedly deficient in the dinucleotide TpA. A putative zinc finger containing three cysteines and one histidine (C-X 2-C-X 12-H-X 3-C) and an acidic segment hint that this clone may be a member of a novel class of transcriptional regulators.

Synthesis of dehydro-oogoniol and oogoniol: the adrenosterone route

Dehydro-oogoniol(3β,11α,15β,29-tetrahydroxystigmasta-5,24(28)(E)-dien-7-one), a female-activating hormone of the water mold Achlya, has been synthesized from 4-androstene-3,11,17-trione by a series of highly stereoselective reactions. One of these involved 1,4-addition of the magnesium cyanocuprate derivative of 3-(1,3-dioxolan-2-yl)-4-methylpentyl bromide to 3β-hydroxy-15β,16β-epoxy-11-oxo-(17E)-pregna -5, 17(20)-diene 3-tert -butyidimethylsilyl ether. The structure of an intermediate, 3β,11β,15β-triacetoxy-5-cholesten-24-one, was confirmed by X-ray crystallographic analysis. Oogoniol [(24R)-3β,11α,15β,29-tetrahydroxy-stigmast-5-en-7-one] was synthesized in a similar manner by reaction of the magnesium cyanocuprate of (S)-3-(1-methylethyl)-5-[(tert-butyldimethylsilyl)oxy]pentyl bromide with the above epoxy pregnadiene

Cationic Interactions Regulate the Initiation and Termination of Zoospore Activity in the Water Mould Achlya heterosexualis

The zoosporic phase of the water mould Achlya heterosexualis, initiated when zoospores emerge from cysts, is strongly influenced by cations. The concentrations of Ca2+ and K+ present in the medium can determine whether or not zoospores will be released from cysts, whether zoospore motion will be approximately linear or circular, rapid or slow, and when zoospore activity will be terminated by encystment. Ionic effects on zoospore motility are rapid and reversible over short time spans; within a 2 min period motility can be strongly suppressed by increasing the [K+] and then restored by increasing the [Ca2+]. Trifluoperazine, considered to be an anti-calmodulin drug, both inhibits zoospore release, and arrests zoospore motility.

Synthesis of dehydro-oogoniol, a female-activating hormone of achlya: The progesterone route

The structure of dehydro-oogoniol (3β, 11α, 15β, 29-tetrahydroxystigmasta-5,24(28) (E)-dien-7-one), a female-activating hormone of the water mold Achlya, has been confirmed by synthesis. The starting material was progesterone, which was converted to the 11α, 15β-dihydroxy derivative by microbiological hydroxylation with Aspergillus giganteus (ATCC 10059). The side chain was constructed in a stepwise manner by means of Wittig and Horner-Emmons reactions, and the C-7 ketone was then introduced by allylic oxidation. The biological activity of the synthetic compound was the same as that of the natural hormone.

Presence of DNA sequence homologous toRAS gene in the aquatic fungusAchlya ambisexualis: Possible role in sporangiogenesis

The water moldAchlya ambisexualis reproduces asexually by the formation of zoospores produced in sporangia. The differentiation of vegetative hyphae to sporangia involves a series of morphologic and biochemical changes. Thus far, very little is known about the regulation of sporogenesis in this organism. Using DNA-DNA and DNA-RNA hybridizations, we investigated the presence of DNA sequences homologous to the yeastRAS gene inA. ambisexualis. A 1.8-kilobase (kb)HindIII fragment containing the completeRAS sc1 gene specifically hybridized toBamHI,PstI, andSalI digestedA. ambisexualis DNA. RNA blot analysis revealed the presence of a detectable level of RNA transcript (1.3 kb) that hybridizes toRAS sc1 , only in differentiating mycelia. In addition, the35S-labeled polypeptide profile of sporulating hyphae differs from that of vegetative hyphae. The selective transcription of 1.3-kb RNA during sporangiogenesis indicates that it may play some significant role in the asexual sporulation inA. ambisexualis.

Transcellular Ion Currents during Sporangium Development in the Water Mould Achlya bisexualis

Changes in the pattern of electric currents that accompany the transformation of growing hyphae of Achlya bisexualis into sporangia have been examined. When hyphae were transferred to a non-nutrient buffer, they continued to extend for several hours and then gave rise to sporangia. Throughout this process, current (positive charge) flowed into the apical region that corresponds approximately to the future sporangium. The current ceased after the crosswall appeared. The sporangium then remained electrically quiescent, except for a brief intense burst of outward current at the 'homogeneous' stage of spore cleavage. The inward current during sporangium formation largely represents an influx of protons. Addition of nitrate abolished the flow of electric current with little effect on sporulation. The late burst of outward current is most probably an artefact, generated by the discharge of salts from the sporangial vacuole. The transcellular electric current apparently plays no role in sporangium formation or in spore cleavage. Calcium ions, however, are required and may traverse the plasma membrane.

Effects of Antheridiol on Growth, Branching and Electrical Currents of Hyphae of Achlya ambisexualis

SUMMARY: Treatment of male hyphae of the water mould Achlya ambisexualis with the female sex pheromone, antheridiol, caused them to stop extending. This response was accompanied by a marked decline in the characteristic inward current just behind the hyphal apex. After a hypha had stopped, a site of inward current developed some way behind the apex. Then an antheridial branch developed at this site. These responses only occurred in the absence of nutrients; in the presence of nutrients the hyphal extension rate and current pattern were unaffected by the addition of antheridiol.

Allomonal functions of steroid hormone, antheridiol, in water moldAchlya

The steroid hormone antheridiol has previously been shown to play a number of key roles in regulating development of the male sex organs, antheridia, of the water moldAchlya ambisexualis. We now demonstrate that synthetic antheridiol can inhibit both sexual (determined by oogonial counts) and asexual reproduction (determined by counts of asexual spores) in an isolate of the homothallic speciesA. heterosexualis. The implications of this dual role of antheridiol as hormone and allomone are discussed.

Electrophysiological properties of Achlya hyphae: ionic currents studied by intracellular potential recording.

The electrical properties of the water mold Achlya bisexualis were investigated using intracellular microelectrodes. Hyphae growing in a defined medium maintained a membrane potential (Vm) of -150 to -170 mV, interior negative. Under the conditions used here, this potential was insensitive to changes in the inorganic ion composition of the medium. Changes in external pH did affect Vm, but only outside the physiological pH range. By contrast, the addition of respiratory inhibitors caused a rapid depolarization without affecting the conductance of the plasma membrane. Taken together these findings strongly suggest that the membrane potential is governed by an electrogenic ion pump rather than by an ionic diffusion potential. Previous work from this laboratory showed that Achlya hyphae generate a transcellular proton current that enters the growing tip, flows along the hyphal length, and exits distally from the trunk. These initial experiments used an extracellular vibrating electrode, and I now report intracellular electrical recordings which support the hypothesis that protons enter the tip by symport with amino acids and are expelled distally by a proton-translocating ATPase. Most significantly, current flowing intracellularly along the hyphal length is associated with a cytoplasmic electric field of 0.2 V/cm or greater. Conditions that inhibit the current also abolish the internal field, suggesting that these two phenomena are closely linked.

The molybdate-stabilized glucocorticoid binding complex of L-cells contains a 98–100 kdalton steroid binding phosphoprotein and a 90 kdalton nonsteroid-binding phosphoprotein that is part of the murine heat-shock complex

This paper summarizes our work performed with glucocorticoid-binding complexes in molybdate-stabilized cytosol prepared from 32P-labeled L-cells. In our early work, we showed that cytosol prepared from 32P-labeled L-cells contains two phosphoproteins (a 90 and a 98–100 kdalton protein) that elute from an affinity resin of deoxycorticosterone agarose in a manner consistent with the predicted behavior of the glucocorticoid receptor. Both phosphoproteins are immunoadsorbed onto protein-A-Sepharose from molybdate-stabilized cytosol incubated with a monoclonal antibody against the receptor. The 98–100 kdalton phosphoprotein binds steroid and the 90 kdalton phosphoprotein is a structurally different, nonsteroid-binding protein that is bound to the untransformed, molybdate-stabilized glucocorticoid receptor. The 90 kdalton protein reacts on Western blots with a monoclonal antibody raised against a 90 kdalton protein from the water mold Achlya ambisexualis. This antibody recognizes an epitope that is conserved in 90 kdalton phosphoproteins from rodent and human cells, and it reacts with the 90 kdalton phosphoprotein that copurifies with the molybdate-stabilized, untransformed chick oviduct progesterone receptor. The 90 kdalton nonsteroid-binding phosphoprotein is an abundant cytosolic protein that dissociates from the glucocorticoid receptor when it is transformed, and unlike the steroid-binding protein, it does not bind to DNA. The 90 kdalton phosphoprotein determines the acidic behavior of the untransformed glucocorticoid receptor on DEAE-cellulose. This abundant cytosolic 90 kdalton phosphoprotein reacts with rabbit antiserum raised against the gel purified 89 kdalton chicken heat-shock protein (hsp89). This antiserum recognizes 90 kdalton heat-shock proteins in human, rodent, frog and Drosophila cells. Immunoadsorption of molybdate-stabilized cytosol with antibody directed against the 98–100 kdalton steroid receptor results in the immune-specific adsorption of a 90 kdalton phosphoprotein that reacts with anti-hsp89 antibody on Western blots. These observations suggest that, like the transforming proteins from several avian sarcoma viruses, the untransformed glucocorticoid receptor exists in a complex with the 90 kdalton heat-shock protein.

Calmodulin from the water mold [formula omitted]: Isolation and characterization

A protein-activator of bovine cyclic nucleotide phosphodiesterase from the water mold Achlya ambisexualis has been affinity-purified to apparent electrophoretic homogeneity. The heat-stable protein is similar in amino acid content and electrophoretic mobility on SDS acrylamide gels, to bovine brain calmodulin. It also crossreacts with antibodies raised to the bovine protein. Achlya calmodulin activates PDE increasing its activity up to 9-fold in a Ca 2+-dependent manner. The mold protein appears unusual in that its tyrosine fluorescence is unaltered by Ca 2+ or by EGTA.

Chemotropism of Achlya ambisexualis to Methionine and Methionyl

SUMMARY: The chemotropic response of the water mould Achlya ambisexualis to nutrients was investigated. Among individual amino acids only methionine was active. Other amino acids were active only in combinations containing cysteine. Methionine was unique among the amino acids tested in its ability, when incorporated uniformly into the water agar substratum, to disrupt chemotropism towards an attractant mixture of amino acids. Among non-amino acid compounds tested for chemotropic activity only S-adenosylmethionine was active. Carboxy1 methylation of protein was promoted by amino acid mixtures.