Project description:The multiple modes of response to ambient pH were explored and new regulatory structures determined. Biological triplicates from the mid-exponential growth phase of controlled bioreactor batch-cultivations of A. niger.
Project description:BackgroundThe filamentous fungus Aspergillus niger is an exceptionally efficient producer of organic acids, which is one of the reasons for its relevance to industrial processes and commercial importance. While it is known that the mechanisms regulating this production are tied to the levels of ambient pH, the reasons and mechanisms for this are poorly understood.MethodsTo cast light on the connection between extracellular pH and acid production, we integrate results from two genome-based strategies: A novel method of genome-scale modeling of the response, and transcriptome analysis across three levels of pH.ResultsWith genome scale modeling with an optimization for extracellular proton-production, it was possible to reproduce the preferred pH levels for citrate and oxalate. Transcriptome analysis and clustering expanded upon these results and allowed the identification of 162 clusters with distinct transcription patterns across the different pH-levels examined. New and previously described pH-dependent cis-acting promoter elements were identified. Combining transcriptome data with genomic coordinates identified four pH-regulated secondary metabolite gene clusters. Integration of regulatory profiles with functional genomics led to the identification of candidate genes for all steps of the pal/pacC pH signalling pathway.ConclusionsThe combination of genome-scale modeling with comparative genomics and transcriptome analysis has provided systems-wide insights into the evolution of highly efficient acidification as well as production process applicable knowledge on the transcriptional regulation of pH response in the industrially important A. niger. It has also made clear that filamentous fungi have evolved to employ several offensive strategies for out-competing rival organisms.
Project description:Microarray analysis of Aspergillus niger under conditions with differing combinations of carbon source, nitrogen source, nitrogen concentration, and culture pH Fermentor cultures were grown in minimal medium (MM) at a constant temperature of 30 ± 0.5 ºC and with differing combinations of carbon source (either 277.5 mM glucose or 333.0 mM xylose), nitrogen source (NH4Cl or NaNO3) and nitrogen concentration (4x: 282.4 mM; 8x: 564.8 mM), and pH (pH4 or pH5) of the medium (M. Braaksma, A.K. Smilde, M.J. van der Werf, P.J. Punt, submitted for publication). At different time points samples were collected, quenched immediately in methanol at -45 ºC and centrifuged at -20 ºC to remove supernatant. Part of the biomass was frozen into liquid nitrogen and stored at -80 ºC for microarray analysis. For each of the 16 culture conditions one sample was selected for microarray analysis; samples were collected either around the time point carbon source depleted or a considerable time (~24 h) after carbon souce depletion. In addition some technical duplicates were included.
Project description:There is a growing interest for the use of post-fermentation mycelial waste to obtain cell wall chitin as an added-value product. In the pursuit to identify suitable production strains that can be used for post-fermentation cell wall harvesting, we turned to an Aspergillus niger strain in which the kexB (also named pclA in literature) gene was deleted. Previous work has shown that deletion of kexB causes hyper-branching and thicker cell walls, which is beneficial as these properties reduce fermentation viscosity and lysis. The deletion of kexB has been shown to exhibit a pH-dependent hyper-branching on solid agar plates at pH 6.0, but not at pH 5.0, whereas this phenotype was reported to be less pronounced during submerged growth. Here, we show a series of controlled batch cultivations performed at a pH range of 5, 5.5, and 6 to examine the pellet phenotype in liquid medium of the ΔkexB strain. Morphological analysis showed that the ΔkexB formed wild type-like pellets at pH 5.0 whereas the characteristic hyper-branching ΔkexB phenotype was found at pH 6.0. Cultivations at pH 5.5 showed that the ΔkexB strain grows as an intermediate phenotype of pH 5.0 and pH 6.0. Analyzing the cell walls of the ΔkexB strain from these controlled pH-conditions showed an increase in chitin content compared to wild type at all three pH values tested. Surprisingly, the increase in chitin content was found to be irrespective of the hyper-branching morphology. Evidence for alterations in cell wall make-up are corroborated by transcriptional analysis that showed a significant cell wall stress response in addition to upregulation of genes encoding other unrelated cell well biosynthetic genes.
Project description:Microarray analysis of Aspergillus niger under conditions with differing combinations of carbon source, nitrogen source, nitrogen concentration, and culture pH Fermentor cultures were grown in minimal medium (MM) at a constant temperature of 30 ± 0.5 ºC and with differing combinations of carbon source (either 277.5 mM glucose or 333.0 mM xylose), nitrogen source (NH4Cl or NaNO3) and nitrogen concentration (4x: 282.4 mM; 8x: 564.8 mM), and pH (pH4 or pH5) of the medium (M. Braaksma, A.K. Smilde, M.J. van der Werf, P.J. Punt, submitted for publication). At different time points samples were collected, quenched immediately in methanol at -45 ºC and centrifuged at -20 ºC to remove supernatant. Part of the biomass was frozen into liquid nitrogen and stored at -80 ºC for microarray analysis. For each of the 16 culture conditions one sample was selected for microarray analysis; samples were collected either around the time point carbon source depleted or a considerable time (~24 h) after carbon souce depletion. In addition some technical duplicates were included. 20 samples were analyzed from 16 different fermentation conditions. The fermentation conditions were varied according to a full factorial design of four factors tested at two levels. From one fermentation two different time samples were analyzed, from another fermentation four samples, two technical replicates of two different time samples, were analyzed. Samples were collected either around the time point carbon source depleted or a considerable time (~24 h) after carbon souce depletion.
Project description:The grey mould fungus Botrytis cinerea is capable of developing on a wide variety of host plants and different organs that differ greatly in their pH values. The strategy developed by this necrotrophic fungus is partly based on the acidification of the plant tissues via the secretion of several organic acids including oxalic acid. That’s why B.cinerea is considered as an “acidic” fungus. However, we have previously shown that it is also capable of alkalinizing its ambient environment during sunflower cotyledons or bean leaves infection and that the alkalinization is achieved by amino acids catabolism and ammonia secretion ( Billon-grand et al, 2012). This capacity to modulate the surrounding pH reveals also an ability to regulate the expression and the secretion of an arsenal of enzymes in order to produce virulence factors in accordance with the ambient pH and enzymes requirement for optimal activity. Expression of pH-regulated genes is controlled by the highly conserved signaling pathway Pal/Pac that leads to activation of the zinc finger transcription factor PACC under neutral or alkaline conditions. Investigations of the role of this pH regulator in the infectious process of B.cinerea are presented.
Project description:Cells in ectothermic organisms often maintain homeostatic function over a considerable range of ambient temperatures. However, as temperature has pronounced effects on all biological processes, but not necessarily in a uniform manner on each of the myriad of distinct processes, cellular acclimation to ambient temperature change is predicted to involve complex regulation. To assess the effects of temperature change within the readily tolerated range on the transcriptome, we have performed analyses with the ectothermic organism Drosophila melanogaster. Both adult male flies and S2R+ cells were analyzed. 3' RNA-Seq was applied to study effects of ambient temperature change on transcript levels and on polyadenylation site selection.
Project description:As a successful commensal and pathogen of humans, Candida albicans encounters a wide range of environmental changes. Among them, ambient pH is an important factor, which changes frequently and affects many biological processes in this species. The ability to adapt to pH changes is tightly linked with pathogenesis and morphogenesis. In this study, we report that pH has a profound effect on white-opaque switching and sexual mating in C. albicans. Acidic pHs promote white-to-opaque switching but repress sexual mating of opaque cells. The cAMP signaling and Rim101-mediated pH sensing pathways are involved in the regulation of pH-regulated white-opaque switching. Interestingly, white and opaque cells of the cyr1/cyr1 mutant, which is defective in producing cAMP, show distinct growth defects under acidic and alkaline conditions. Phr2 could play a major role in acidic pHs-induced opaque cell formation. We further discover that acidic pH conditions repress sexual mating due to the failure of activation of the Ste2-mediated a-pheromone response pathway. The effects of pH changes on phenotypic switching and sexual mating could be a balance behavior between host adaptation and sexual reproduction.