Unai Ugalde

Aspergillus nidulans asexual development: making the most of cellular modules

Asexual development in Aspergillus nidulans begins in superficial hyphae as the programmed emergence of successive pseudohyphal modules, collectively known as the conidiophore, and is completed by a layer of specialized cells (phialides) giving rise to chains of aerial spores. A discrete number of regulatory factors present in hyphae play different stage-specific roles in pseudohyphal modules, depending on their cellular localization and protein-protein interactions. Their multiple roles include the timely activation of a sporulation-specific pathway that governs phialide and spore formation. Such functional versatility provides for a new outlook on morphogenetic change and the ways we should study it.

FlbC is a putative nuclear C2H2 transcription factor regulating development in Aspergillus nidulans

Asexual development (conidiation) in Aspergillus is governed by multiple regulators. Here, we characterize the upstream developmental activator FlbC in Aspergillus nidulans. flbC mRNA is detectable throughout the life cycle, at relatively high levels during vegetative growth, early asexual and late sexual developmental phases. The deletion of flbC causes a delay/reduction in conidiation, brlA and vosA expression, and conidial germination. While overexpression of flbC (OEflbC) does not elaborate conidiophores, it inhibits hyphal growth and activates expression of brlA, abaA and vosA, but not wetA. FlbC is conserved in filamentous Ascomycetes containing two C2H2 zinc fingers at the C‐terminus and a putative activation domain at the N‐terminus. FlbC localizes in the nuclei of both hyphae and developmental cells. Localization and expression of FlbC are not affected by the absence of FlbB or FlbE, and vice versa. Importantly, overexpression of flbC causes growth inhibition and activation of abaA and vosA in the absence of brlA and abaA respectively. In vitro DNA‐binding assay reveals that FlbC binds to the brlA, abaA and vosA, but not the wetA, promoters. In summary, FlbC is a putative nuclear transcription factor necessary for proper activation of conidiation, and its balanced activity is crucial for governing growth and development in A. nidulans.

Basic-Zipper-Type Transcription Factor FlbB Controls Asexual Development in Aspergillus nidulans†

ABSTRACT The fungal colony is a complex multicellular unit consisting of various cell types and functions. Asexual spore formation (conidiation) is integrated through sensory and regulatory elements into the general morphogenetic plan, in which the activation of the transcription factor BrlA is the first determining step. A number of early regulatory elements acting upstream of BrlA (fluG and flbA-E) have been identified, but their functional relations remain to be further investigated. In this report we describe FlbB as a putative basic-zipper-type transcription factor restricted to filamentous fungi. FlbB accumulates at the hyphal apex during early vegetative growth but is later found in apical nuclei, suggesting that an activating modification triggers nuclear import. Moreover, proper temporal and quantitative expression of FlbB is a prerequisite for brlA transcription, and misscheduled overexpression inhibits conidiation. We also present evidence that FlbB activation results in the production of a second diffusible signal, acting downstream from the FluG factor, to induce conidiation.

Conidiation induction in Penicillium

Asexual spores or conidia are dispersive propagules produced as an alternative to vegetative growth by a diverse group of filamentous fungi. The cellular development programmes which govern conidiation have been intensely studied in the last few decades, although important gaps stand in the way of our understanding of this phenomenon, namely in the areas of the environmental sensing mechanisms and signal transduction pathways. The aim of this review is to summarize the current advances in conidiation induction in the genus Penicillium, and to put them into context with the state of our knowledge stemming from work in widely studied fungal model systems.

The bZIP‐type transcription factor FlbB regulates distinct morphogenetic stages of colony formation in Aspergillus nidulans

Conidiophore formation in Aspergillus nidulans involves a developmental programme in which vegetative hyphae give rise to an ordered succession of differentiated cells: foot cell, stalk, vesicle, metulae, phialides and conidia. The developmental transition requires factors that are expressed in vegetative hyphae that activate the expression of the main regulator of conidiation, BrlA. One such element is the bZIP‐type transcription factor FlbB. We found that flbB‐ mutants show defective branching patterns and are susceptible to autolysis under high sorbitol or sucrose concentrations, revealing a role in vegetative growth. In addition, FlbB plays a role in conidiophore initiation, as its upregulation reduces conidiophore vesicle swelling and generates a reduced number of metulae. FlbB was located at the tip of growing metulae, following a similar pattern as described in vegetative hyphae. In wild‐type strains, the transition from metulae to phialides could be reversed to generate vegetative hyphae, indicating the existence of a specific control point at this stage of conidiophore formation. The combined evidence points to FlbB as a key factor in the transition to asexual development, playing a role at various control points in which the process could be reversed.

Conidiation in Penicillium cyclopium is induced by conidiogenone, an endogenous diterpene.

ABSTRACT The filamentous fungus Penicillium cyclopium conidiates in the presence of calcium ions in submerged culture without nutrient limitation according to a precisely timed program. Conidiation could be prematurely induced in a nutritionally sufficient medium which had previously supported growth, suggesting that a metabolite which influenced the process was produced. A diterpenoid with conidiation-inducing activity, conidiogenone, was purified from the culture medium, along with conidiogenol, a putative derivative with delayed activity. Contrary to previous thought, the presence of calcium was demonstrated to only decrease the threshold concentration of conidiogenone required for the induction to proceed. In light of these results, a mechanism of conidiation induction is presented according to which the mycelium produces a conidiation inducer (conidiogenone) that accumulates extracellularly. When a threshold concentration is reached, induction likely takes place by interaction with a specific cellular receptor. The results indicate that conidiogenone is both sufficient and necessary to induce conidiation.

Continuous hydrolysis of whey proteins in a membrane recycle reactor

Whey proteins were hydrolyzed in a membrane recycle reactor (MRR) in the presence of Alcalase 0.6L under varying enzyme to substrate ratios and residence times. Substrate conversion was directly dependent on these two parameters, and at a fixed enzyme-substrate ratio of 10%, conversion levels could be controlled through residence time, resulting in peptide permeates with homogeneous molecular and functional properties. In addition, recycling of the enzyme and elimination of peptide products resulted in much improved enzyme yields and process productivity levels. However, the process was only operational for 7 h, given membrane fouling and enzyme inactivation phenomena whose contribution occurred in a time-dependent manner. Changes in process design leading to increased reactor stability were examined, and the system is considered to provide a suitable basis for the production of peptides with specific functional and nutritional properties at the industrial level in the future.

Aspergillus nidulans FlbE is an upstream developmental activator of conidiation functionally associated with the putative transcription factor FlbB

Aspergillus nidulans switches from vegetative growth to conidiation when aerial hyphae make contact with the atmosphere, or are subjected to specific environmental stress. The activation of the central conidiation pathway led by the transcription factor brlA is a critical milestone in this morphogenetic transition. A number of upstream developmental activators (UDAs), expressed in vegetative cells, are required for this process to occur in conjunction with cessation of vegetative growth. Mutants affected in these factors remain aconidial (fluffy) with low brlA expression levels (flb). In this report, we describe FlbE as a UDA containing two conserved but hitherto uncharacterized domains, which functions in close association with putative transcription factor FlbB. Both UDAs are functionally interdependent, and colocalize at the hypha tip in an actin cytoskeleton‐dependent manner. Moreover, bimolecular fluorescence studies show that they physically interact in vivo. These findings add evidence in favour of the existence of a signalling complex at or near the Spitzenkörper as an important part of the machinery controlling the morphogenetic transition between vegetative growth and conidiation.

Highly efficient assimilation of lactose by a metabolically engineered strain of Saccharomyces cerevisiae.

A diploid strain of Saccharomyces cerevisiae able to metabolize lactose with high efficiency has been obtained. Haploid strains of Saccharomyces able to grow on lactose were constructed by cotransformation with two genes of Kluyveromyces lactis required for the utilization of the sugar, LAC4 and LAC12, encoding β‐galactosidase and lactose permease respectively. Both genes were placed under the control of a galactose‐inducible promoter and targeted to the rDNA encoding region (RDN1 locus) of the Saccharomyces genome. Lac+ transformants were selected on medium with lactose as the only carbon source. These transformants were mitotically stable, they maintained the Lac+ phenotype after growing in non‐selective medium for more than 60 generations, but their growth was slow. We found that this lack of vigour was caused by their genetic background and not by a deficient expression of the heterologous genes. Therefore, their performance could be improved by crossing with a wild‐type strain. Among the offspring of the crosses, two strains of opposite mating type were selected and mated to obtain a fast‐growing Lac+ diploid. This diploid strain showed the typical fermentative behaviour of S. cerevisiae when it was grown in aerated liquid medium with glucose. In lactose medium, it exhibited a respiro‐fermentative metabolism similar to that of K. lactis, with low ethanol production and high biomass yield.

Signaling the Induction of Sporulation Involves the Interaction of Two Secondary Metabolites in Aspergillus nidulans

When growing Aspergillus nidulans hyphae encounter the atmosphere, they initiate a morphogenetic program leading to the production of spores. Mutants that are defective in the fluG gene fail to undergo sporulation because they lack an endogenous diffusible factor that purportedly accumulates on aerial hyphae, thus signaling the initiation of development. In this study, the defect could be reversed by adding culture extracts from a wild-type strain onto a mutant colony. Moreover, a bioassay-guided purification of the active culture extract resulted in the identification of the active agent as dehydroaustinol. However, this meroterpenoid was active only when administered in conjunction with the orsellinic acid derivative diorcinol. These two compounds formed an adduct that was detected by HRMS in an LC-MS experiment. The diorcinol-dehydroaustinol adduct prevented crystal formation of the signal on the surface of aerial hyphae and on an artificially prepared aqueous film and also increased the signal lipophilicity.