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: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.
Project description:We aim to compare the genomic discrepancies across de novo Ph+ ALL, Ph+ MPAL and Ph+ AML, three diseases characterized by the occurrence of BCR-ABL1 transcripts but showing varied immunophenotypes. The data we are now submitting is the genomic copy number variants of these three groups. The following is the abstract with associated manuscript. The chromosome abnormality of Philadelphia (Ph) is typically seen in de novo acute lymphoblastic leukemia (ALL). It has also been identified in mixed phenotype acute leukemia (MPAL) and acute myeloid leukemia (AML) in the revisions to World Health Organization classification of myeloid neoplasms and actue leukemia. The discrepancies between these patients and potential mechanisms underlying differentiation fate of the leukemia cells remain poorly defined. We evaluated the clinical, genomic and transcriptomic features of Ph+ ALL, Ph+ MPAL and Ph+ AML by taking advantage of high-density genomic analysis, including next-generation sequencing array comparative genomic hybridization and gene expression profiling for transcriptomic analysis. Our results showed that the three cohorts demonstrated diversified clinical features. Ph+ ALL had the best response to induction therapy, with a complete remission (CR) rate of 93.5 and molecular response of 43.5%. Ph+ MPAL had a 90.0% CR rate but only 5.9% of molecular response. The CR rate of Ph+ AML was only 68.8%. Ph+ ALL was characterized by loss and mutations of B-cell development gene IKZF1 and PAX5, and frequent histone H3K36 trimethyltransferase SETD2 mutations. SETD2 mutations were detected in 11.3% of Ph+ ALL patients and predicted higher relapse rate. Ph+ MPAL and Ph+ AML featured high frequency of RUNX1 mutations. Further studies showed RUNX1-R177X mutation inhibited 32D cell differentiation induced by G-Csf, and cooperated with BCR-ABL1 to lead to myeloid differentiation arrest of human cord blood CD34+ cells. It is therefore presumed that these additional mutations work in synergy with BCR-ABL1 fusion gene to facilitate the development of Ph-positive acute leukemia in different immunophenotypic classifications.