Arthrospore Formation Research Articles

The arthrospore-related gene Acaxl2 is involved in cephalosporin C production in industrial Acremonium chrysogenum by the regulatory factors AcFKH1 and CPCR1

Cephalosporin C (CPC) production is often accompanied by a typical morphological differentiation of Acremonium chrysogenum, involving the fragmentation of its hyphae into arthrospores. The type I integral plasma membrane protein Axl2 is a central component of the bud site selection system (BSSS), which was identified as the regulatory factor involved in the hyphal septation process and arthrospore formation. Using CRISPR/Cas9 technology and homologous recombination (HR), we inserted an egfp donor DNA sequence into the Acaxl2 locus, causing the generation of the deletion strain Ac-ΔAcaxl2:eGFP from Acremonium chrysogenum FC3-5-23, the industrial producer of CPC. The mycelial morphology of the deletion strain Ac-ΔAcaxl2:eGFP was mainly composed of arthrospores with a characteristic diameter of 2–8 µm, which increased from 75% at 48 h to 90% at 72 h post culture and were maintained until the end of the fermentation process. However, the deletion strain showed accelerated production of CPC, and the final titer was 5573 μg/ml, which was nearly three times higher than that of the control strain FC3-5-23. The up-regulation of genes related to the biosynthesis gene cluster in Ac-ΔAcaxl2:eGFP, especially the “late” genes, was one reason why its CPC production was higher than that of the original strain. Furthermore, compared with FC3-5-23, the more significant increase of genes involved in the BSSS (Acbud3 and Acbud4) in Ac-ΔAcaxl2:eGFP in the late stage of fermentation, may be responsible for this increase in arthrospore formation. Similarily, the transcription of the regulatory factors AcFKH1 and CPCR1 were also markedly increased at this time and may be the factors responsible for the regulation of CPC synthesis. These results indicated that Acaxl2 plays an important role in both arthrospore formation and CPC production, strongly implicating these regulatory factors as having pivotal links between mycelial morphology and secondary metabolite production in high-yielding A. chrysogenum. To the opposite, the axl2 gene knockout of wild strain CGMCC 3.3795 did not significantly influence the CPC production, which reflected the complexity of the secondary metabolic process and the differences in the function of axl2 gene in high- and low-yielding strains.

AcAxl2 and AcMst1 regulate arthrospore development and stress resistance in the cephalosporin C producer Acremonium chrysogenum.

The filamentous fungus Acremonium chrysogenum is the primordial producer of the β-lactam antibiotic cephalosporin C. This antibiotic is of major biotechnological and medical relevance because of its antibacterial activity against Gram-positive and Gram-negative bacteria. Antibiotic production during the lag phase of fermentation is often accompanied by a typical morphological feature of A. chrysogenum, the fragmentation of the mycelium into arthrospores. Here, we sought to identify factors that regulate the hyphal septation process and present the first comparative functional characterization of the type I integral plasma membrane protein Axl2 (axial budding pattern protein 2), a central component of the bud site selection system (BSSS) and Mst1 (mammalian Sterile20-like kinase), a septation initiation network (SIN)-associated germinal center kinase (GCK). Although an Acaxl2 deletion strain showed accelerated arthrospore formation after 96h in liquid culture, deletion of Acmst1 led to a 24h delay in arthrospore development. The overexpression of Acaxl2 resulted in an arthrospore formation similar to the A3/2 strain. In contrast to this, A3/2::Acmst1 OE strain displayed an enhanced arthrospore titer. Large-scale stress tests revealed an involvement of AcAxl2 in controlling osmotic, endoplasmic reticulum, and cell wall stress response. In a similar approach, we found that AcMst1 plays an essential role in regulating growth under osmotic, cell wall, and oxidative stress conditions. Microscopic analyses and plating assays on media containing Calcofluor White and NaCl showed that arthrospore development is a stress-dependent process. Our results suggest the potential for identifying candidate genes for strain improvement programs to optimize industrial fermentation processes.

Deactivation of the autotrophic sulfate assimilation pathway substantially reduces high-level β-lactam antibiotic biosynthesis and arthrospore formation in a production strain from Acremonium chrysogenum.

The filamentous ascomycete Acremonium chrysogenum is the only industrial producer of the β-lactam antibiotic cephalosporin C. Synthesis of all β-lactam antibiotics starts with the three amino acids l-α-aminoadipic acid, l-cysteine and l-valine condensing to form the δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine tripeptide. The availability of building blocks is essential in every biosynthetic process and is therefore one of the most important parameters required for optimal biosynthetic production. Synthesis of l-cysteine is feasible by various biosynthetic pathways in all euascomycetes, and sequencing of the Acr. chrysogenum genome has shown that a full set of sulfur-metabolizing genes is present. In principle, two pathways are effective: an autotrophic one, where the sulfur atom is taken from assimilated sulfide to synthesize either l-cysteine or l-homocysteine, and a reverse transsulfuration pathway, where l-methionine is the sulfur donor. Previous research with production strains has focused on reverse transsulfuration, and concluded that both l-methionine and reverse transsulfuration are essential for high-level cephalosporin C synthesis. Here, we conducted molecular genetic analysis with A3/2, another production strain, to investigate the autotrophic pathway. Strains lacking either cysteine synthase or homocysteine synthase, enzymes of the autotrophic pathway, are still autotrophic for sulfur. However, deletion of both genes results in sulfur amino acid auxotrophic mutants exhibiting delayed biomass production and drastically reduced cephalosporin C synthesis. Furthermore, both single- and double-deletion strains are more sensitive to oxidative stress and form fewer arthrospores. Our findings provide evidence that autotrophic sulfur assimilation is essential for growth and cephalosporin C biosynthesis in production strain A3/2 from Acr. chrysogenum.

Identification of degenerate nuclei and development of a SCAR marker for Flammulina velutipes.

Flammulina velutipes is one of the major edible mushrooms in the world. Recently, abnormalities that have a negative impact on crop production have been reported in this mushroom. These symptoms include slow vegetative growth, a compact mycelial mat, and few or even no fruiting bodies. The morphologies and fruiting capabilities of monokaryons of wild-type and degenerate strains that arose through arthrospore formation were investigated through test crossing. Only one monokaryotic group of the degenerate strains and its hybrid strains showed abnormal phenotypes. Because the monokaryotic arthrospore has the same nucleus as the parent strain, these results indicated that only one aberrant nucleus of the two nuclei in the degenerate strain was responsible for the degeneracy. A sequence-characterized amplified region marker that is linked to the degenerate monokaryon was identified based on a polymorphic sequence that was generated using random primers. Comparative analyses revealed the presence of a degenerate-specific genomic region in a telomere, which arose via the transfer of a genomic fragment harboring a putative helicase gene. Our findings have narrowed down the potential molecular targets responsible for this phenotype for future studies and have provided a marker for the detection of degenerate strains.

A correlative evaluation of morphology and rheology ofAspergillus terreus during lovastatin fermentation

Lovastatin, a secondary metabolite, was produced by fermentation process usingAspergillus terreus in an internal loop airlift reactor. It is a highly aerobic fermentation process. Biomass concentration and cell morphology were evaluated and observed to contribute significantly to the high viscosity and pseudoplastic non-Newtonian behavior of the broth. Typical morphological changes over 10 days in the fermentation broth were studied. The viscosity increased from the start of the fermentation with an increasing cell mass content, reached to a maximum of 60 N/m2·s at 160 h and then declined after the branching of the hyphae with the formation of arthrospores. Rheological parameters like consistency index and fluidity index were evaluated. The consistency index was observed to increase from 9.8 to 66.85 N/m2, while fluidity index decreased from 0.69 to 0.48 s−1 during 10 days of lovastatin production. A correlation between growth and consistency index of the broth has been evaluated.

CPCR1, but not its interacting transcription factor AcFKH1, controls fungal arthrospore formation in Acremonium chrysogenum

Fungal morphogenesis and secondary metabolism are frequently associated; however, the molecular determinants connecting both processes remain largely undefined. Here we demonstrate that CPCR1 (cephalosporin C regulator 1 from Acremonium chrysogenum), a member of the winged helix/regulator factor X (RFX) transcription factor family that regulates cephalosporin C biosynthesis, also controls morphological development in the beta-lactam producer A. chrysogenum. The use of a disruption strain, multicopy strains as well as several recombinant control strains revealed that CPCR1 is required for hyphal fragmentation, and thus the formation of arthrospores. In a DeltacpcR1 disruption strain that exhibits only hyphal growth, the wild-type cpcR1 gene was able to restore arthrospore formation; a phenomenon not observed for DeltacpcR1 derivatives or non-related genes. The intracellular expression of cpcR1, and control genes (pcbC, egfp) was determined by in vivo monitoring of fluorescent protein fusions. Further, the role of the forkhead transcription factor AcFKH1, which directly interacts with CPCR1, was studied by generating an Acfkh1 knockout strain. In contrast to CPCR1, AcFKH1 is not directly involved in the fragmentation of hyphae. Instead, the presence of AcFKH1 seems to be necessary for CPCR1 function in A. chrysogenum morphogenesis, as overexpression of a functional cpcR1 gene in a DeltaAcfkh1 background has no effect on arthrospore formation. Moreover, strains lacking Acfkh1 exhibit defects in cell separation, indicating an involvement of the forkhead transcription factor in mycelial growth of A. chrysogenum. Our data offer the potential to control fungal growth in biotechnical processes that require defined morphological stages for optimal production yields.

Stress-induced morphological and physiological changes in γ-linolenic acid production by Mucor fragilis in batch and continuous cultures

Batch and continuous cultures conditions were studied in order to increase γ-linolenic acid production by Mucor fragilis CCMI 142, in response to the presence of ethanol. Continuous cultures were used to add ethanol pulses to steady state pellet cultures. It was demonstrated that pellet size, which allowed homogeneous fungal cultures, can be obtained by means of pH adjustment, thus enabling steady state continuous growth at 2.90±0.05. The 5 and 2% (v/v) ethanol pulses induced hyphal morphological changes with arthrospore formation. A 1% (v/v) pulse of ethanol did not immediately affect growth, but induced morphological changes, which led to autolysis at the pellet core. A 0.5% (v/v) pulse of ethanol did not affect neither the morphology nor the physiology of the microorganism to any significant extent. The 0.5 and 1% (v/v) ethanol pulses resulted in an increase in the proportion of γ-linolenic acid production up to 11%. Data from batch cultures showed a higher enhancement of ethanol, attaining 30% of γ-linolenic acid. The increase of γ-linolenic acid content observed in batch and continuous conditions appears to be a response associated with stress induced by the ethanol which seems to be of value as an industrial medium component.

Acremonium chrysogenum differentiation and biosynthesis of cephalosporin

On the basis of structure-functional analysis of the development of Acremonium chrysogenum, e.g. under conditions either stimulating antibiotic synthesis or not conductive to production, a scheme was proposed representing the various ways in which morphological differentiation occurs in the culture in dependence on the directions of its metabolism. Three types of culture differentiation were determined. Type 1 differentiation is characterized by the transition of the vegetative stage into the reproductive one with the formation of conidia. Type 2 differentiation is characterized by the formation of typical arthrospores also being the reproductive form. Type 3 differentiation is characterized by the multistage transformation of the mycelium organization into the yeast-like one which is metabolically more active and is a producer of antibiotics and enzymes. In addition to the defined regularities in the development and differentiation of Acremonium chrysogenum structural peculiarities were observed which could be helpful to the search for regulators or specific enzymes taking part in the culture development.

Autoinhibition of germination and growth in Geotrichum candidum

Dense, self-inhibitory arthrospore suspensions of Geotrichum candidum release trimethylamine which arrests germination and inhibits extension growth of germlings and mature hyphae. When assayed at low concentrations trimethylamine induced arthrospore formation in the terminal compartments of leader hyphae of G. candidum .

Formation and ultrastructure of Mucor rouxii arthrospores.

The formation of arthrospores in Mucor rouxii was studied by transmission and scanning electron microscopy and light microscopy. The arthrospores formed in a random manner in terminal and internal regions of the hyphae. The earliest appearance of the arthrospores was seen by scanning electron microscopy as compartments delineated by double ridges. These ridges probably corresponded to the site of septal wall formation. The elongated compartments varied considerably in size. As the arthrospores matured, they tended to separate as a result of a gradual change in the shape of the arthrospores to a nearly spherical form and also as the result of simultaneous degradation of the outermost cell wall layer. The mature arthrospores were surrounded by a complex cell wall consisting of at least three distinct layers in addition to the original hyphal cell wall. Crystal-like structures were seen in the cytoplasm of some of the arthrospores in addition to the usual organelles such as mitochondria, nuclei, and ribosomes. Septum formation by centripetal cell wall growth from the lateral hyphal wall was documented by transmission electron microscopy. However, evidence was also found which suggested that not all internal cell wall development in the fungal hyphae during arthrosporogenesis necessarily led to the formation of mature arthrospores.

Sporulation of Geotrichum candidum in submerged culture

Spore formation was studied in batch and continuous cultures of Geotrichum candidum growing on carbon-limiting mineral salts medium. With glucose, glycerol or ethanol as carbon source and asparagine as nitrogen source cylindrical arthrospores were formed at an exponential rate during both the log and stationary phases of growth. The decline in culture pH which resulted if an inorganic nitrogen source was used largely prevented normal arthrospore formation during the stationary phase, while if the initial pH were adjusted to pH 2.8 neither filaments nor arthrospores were formed and growth occurred by the multiplication of a thick-walled ellipsoidal spore type. In continuous culture spores were produced at all dilution rates studied up to 0.31 h −1 although at the higher growth rates they constituted less than 5% of biomass weight. Arthrospores predominated in all instances and ellipsoidal spores were not produced at dilution rates greater than 0.125 h −1 .

The effects of temperature, incubation atmosphere, and medium composition on arthrospore formation in the fungus Trichophyton mentagrophytes

Several growth conditions were found to allow abundant arthrospore formation in T. mentagrophytes. These included growth at 32--37 degrees C on Sabouraud's medium (1% neopeptone, 4% glucose) and growth at temperatures below 32 degrees C solely on neopeptone or other complex peptide sources without the addition of glucose, a supplementary carbon source. Sabouraud's medium did not allow arthropsore formation at 30 degrees C under normal atmospheric conditions. However, if oxygen tension were reduced by partial replacement of air with either N2 or CO2 arthrosporulation did occur on Sabouraud's medium at 30 degrees C. The rate of germ tube elongation was lower under those conditions which supported arthrospore formation, suggesting a correlation between decreased rate of hyphal extension and arthrospore formation. Stimulation of arthrospore formation by sublethal concentrations of several antifungal agents tends to support this hypothesis.

Identification of carbon dioxide as a dermatophyte inhibitory factor produced by Candida albicans

A factor produced by Candida albicans, which inhibits dermatophyte growth and induces arthrospore formation is characterized and identified. Candida dermatophyte inhibitory factor (CDIF) is volatile and fungistatic. Analysis of volatile materials produced by C. albicans was subsequently identified as carbon dioxide. The involvement of carbon dioxide in the inhibition of dermatophytes was demonstrated by: (1) utilization of commercial carbon dioxide to produce dermatophyte inhibition as well as arthrospore formation, and (2) prevention of dermatophyte inhibition by C. albicans through incoporation of soda lime into the incubation atmosphere. The ability of carbon dioxide to inhibit dermatophyte growth was shared with other gases (methane and helium), but arthrospore formation was observed only with carbon dioxide. The possible significance of carbon dioxide's induction of arthrospores, a form occasionally observed in active dermatophyte lesions, is discussed.

Nuclei, septation, branching and growth of Geotrichum candidum.

A study was made of growth, septation and branching in Geotrichum candidum, a mould which forms physiologically complete septa. A correlation was observed between septation and branch initiation; branches were almost invariably formed just behind septa. Primary branches and their parent intercalary compartments initially increased in length at an exponential rate before eventually attaining a constant rate of extension. The whole branching system (which eventually contained seven tips) produced by an intercalary compartment increased in length exponentially until it attained a total length of at least 1-5 mm. The total length and the number of nuclei of undifferentiated mycelia increased exponentially at the same specific growth rate. The results suggest that nuclei divide just before or just after arthrospore formation.

Effect of norleucine on mycelial fragmentation in Cephalosporium acremonium

DL-Norleucine, which is known to replace methionine for stimulation of cephalosporin C formation, also mimics methionine's effect on arthrospore formation. Thus, hyphal fragmentation, like antibiotic biosynthesis, is divorced from a sulfur donation role.

Differentition of mutants of Cephalosporium acremonium in complex medium: the formation of unicellular arthrospores and their germination.

Differentiation of swollen hyphal fragments to unicellular arthrospores accompanied the synthesis of cephalosporin C by a series of Cephalosporium acremonium mutants during propagation in a complex medium. The complex medium supported significantly higher synthesis than the defined medium used in previous studies of differentiation in C. acremonium. The mutants differed in their ability to form unicellular arthrospores and to synthesize cephalosporin C, but a one-to-one correspondence between the two properties was not observed. An inverse relation was observed between the growth rates of the mutants and their ability to synthesize cephalosporin C: each mutant produced more antibiotic but grew more slowly than its parent strain. Germination of the unicellular arthrospores occurred in complex medium but differed significantly from the germination of conidia in seed medium. The unicellular arthrospores were examined by electron microscopy and compared with swollen hyphal fragments and slender hyphal filaments. The unicellular arthrospores had a thicker cell wall, rougher cell surface, and had one or more small identations in their surface. The internal structure of the unicellular arthrospore resembled those of the swollen hyphal fragment and slender hyphal filament. Filaments had lower concentrations of lipid-containing vacuoles which were prevalent in both the swollen hyphal fragments and the unicellular arthrospores.

Septa ultrastructure and hyphal branching in the pleomorphic imperfect fungus Trichosporonoides oedocephalis

Incomplete septa, complete simple septa, septa with simple pores, but mostly dolipore septa without pore caps or parenthosomes are present in this organism. Walls are distinctly laminar in aspect, especially with glutaraldehyde – osmic acid fixation. Cell division is by septum formation between previously formed septa. Conidia result from either blastospore or endospore (arthrospore) formation. Branching occurs irrespective of the type of adjacent septa, with each cell capable of bipolar germination. The organism shows features of both ascomycetous and basidiomycetous yeasts.

Ultrastructure of polymorphic Mucor as observed by means of freeze-etching. I. Vegetative growth of mycelium and arthrospore formation in submerged and aerated cultures.

Ultrastructure of polymorphic Mucor as observed by means of freeze-etching. I. Vegetative growth of mycelium and arthrospore formation in submerged and aerated cultures.

Studies on the microorganisnns of general grain A new radio-resistant yeast of Trichosporon oryzae nov. sp. isolated from rice.

A radio-resistant asporogenous yeast has been isolated from unpolished new crop rice. On the cultivation of this species, formation of true mycelium, arthrospores and blastospores were observed, and it could be classified as in one group of genus Trichosporon. Its taxonomic characteristics were found to be sufficiently different from all the described species in this genus to warrant its description as a new species and was named as Trichosporon oryzae nov. sp. This species exhibited high resistance to gamma-radiation as same as Pullularia pullulans, and the D10 value of radio-resistant cells was to be ca. 120 krad in 0.067M phosphate buffer which was about 4 to 5 times larger than that of Saccharomyces cerevisiae. In this study, high resistance to gamma-radiation was also observed on the species of Trichosporon capitatum and Tr. inkin.

Morphogenesis and ultrastructure of Geotrichum candidum septa.

The ultrastructure and mode of formation of septa of Geotrichum candidum were investigated by light and electron microscopy. The invaginations of the lateral membrane and wall appear to initiate at multiple points around the circumference of the cell; the immature septum subsequently assumes a cart-wheel shape, with branched spokes radiating from the center of the septum. Each face of the septum is covered with a membrane possessing hitherto undescribed structural differentiation; the membrane substructures are comprised of two central subunits encircled by 12 identical subunits. The diameter of the entire 12 plus 2 structure is 24 to 25 nm, and the diameter of each individual subunit is approximately 4 nm. The maturation of the septum appears to occur by further deposition of material along the branched skeletal regions. Numerous small openings (micropores), formed as a result of incomplete deposition, ultimately give rise to plasmodesmata. During arthrospore formation, the plasmodesmal canals and associated micropores are occluded by electron-dense materials, rendering each segment of the hyphae completely independent of the rest of the hyphae.