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Ruyters S.,Campus Management | Mukherjee V.,Campus Management | Thevelein J.M.,Institute of Botany and Microbiology | Willems K.A.,Campus Management | Lievens B.,Campus Management
Journal of Industrial Microbiology and Biotechnology | Year: 2015

Bioethanol fermentations expose yeasts to a new, complex and challenging fermentation medium with specific inhibitors and sugar mixtures depending on the type of carbon source. It is, therefore, suggested that the natural diversity of yeasts should be further exploited in order to find yeasts with good ethanol yield in stressed fermentation media. In this study, we screened more than 50 yeast isolates of which we selected five isolates with promising features. The species Candida bombi, Wickerhamomyces anomalus and Torulaspora delbrueckii showed better osmo- and hydroxymethylfurfural tolerance than Saccharomyces cerevisiae. However, S. cerevisiae isolates had the highest ethanol yield in fermentation experiments mimicking high gravity fermentations (25 % glucose) and artificial lignocellulose hydrolysates (with a myriad of inhibitors). Interestingly, among two tested S. cerevisiae strains, a wild strain isolated from an oak tree performed better than Ethanol Red, a S. cerevisiae strain which is currently commonly used in industrial bioethanol fermentations. Additionally, a W. anomalus strain isolated from sugar beet thick juice was found to have a comparable ethanol yield, but needed longer fermentation time. Other non-Saccharomyces yeasts yielded lower ethanol amounts. © 2014, Society for Industrial Microbiology and Biotechnology.


Duitama J.,Agrobiodiversity research area | Quintero J.C.,Agrobiodiversity research area | Cruz D.F.,Agrobiodiversity research area | Quintero C.,Agrobiodiversity research area | And 5 more authors.
Nucleic Acids Research | Year: 2014

Recent advances in high-throughput sequencing (HTS) technologies and computing capacity have produced unprecedented amounts of genomic data that have unraveled the genetics of phenotypic variability in several species. However, operating and integrating current software tools for data analysis still require important investments in highly skilled personnel. Developing accurate, efficient and user-friendly software packages for HTS data analysis will lead to a more rapid discovery of genomic elements relevant to medical, agricultural and industrial applications. We therefore developed Next-Generation Sequencing Eclipse Plug-in (NGSEP), a new software tool for integrated, efficient and user-friendly detection of single nucleotide variants (SNVs), indels and copy number variants (CNVs). NGSEP includes modules for read alignment, sorting, merging, functional annotation of variants, filtering and quality statistics. Analysis of sequencing experiments in yeast, rice and human samples shows that NGSEP has superior accuracy and efficiency, compared with currently available packages for variants detection. We also show that only a comprehensive and accurate identification of repeat regions and CNVs allows researchers to properly separate SNVs from differences between copies of repeat elements. We expect that NGSEP will become a strong support tool to empower the analysis of sequencing data in a wide range of research projects on different species. © The Author(s) 2013. Published by Oxford University Press.


Castermans D.,Institute of Botany and Microbiology | Somers I.,Institute of Botany and Microbiology | Kriel J.,Institute of Botany and Microbiology | Louwet W.,Institute of Botany and Microbiology | And 5 more authors.
Cell Research | Year: 2012

The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucose-deprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation. © 2012 IBCB, SIBS, CAS All rights reserved.


Vandamme J.,Institute of Botany and Microbiology | Castermans D.,Institute of Botany and Microbiology | Thevelein J.M.,Institute of Botany and Microbiology
Cellular Signalling | Year: 2012

The cAMP-protein kinase A (PKA) pathway is a major signalling pathway in the yeast Saccharomyces cerevisiae, but also in many other eukaryotic cell types, including mammalian cells. Since cAMP plays a crucial role as second messenger in the regulation of this pathway, its levels are strictly controlled, both in the basal condition and after induction by agonists. A major factor in the down-regulation of the cAMP level after stimulation is PKA itself. Activation of PKA triggers feedback down-regulation of the increased cAMP level, stimulating its return to the basal concentration. This is accomplished at different levels. The best documented mechanisms are: inhibition of cAMP synthesis by down-regulation of adenylate cyclase and/or its regulatory proteins, stimulation of cAMP breakdown by phosphodiesterases and spatial regulation of cAMP levels in the cell by A-Kinase Anchoring Proteins (AKAPs). In this review we describe these processes in detail for S. cerevisiae, for cells of mammals and selected other organisms, and we hint at other possible targets for feedback regulation of intracellular cAMP levels. © 2012 Elsevier Inc.


Van Zeebroeck G.,Institute of Botany and Microbiology | Rubio-Texeira M.,Institute of Botany and Microbiology | Schothorst J.,Institute of Botany and Microbiology | Thevelein J.M.,Institute of Botany and Microbiology
Molecular Microbiology | Year: 2014

The Saccharomyces cerevisiae amino acid transceptor Gap1 functions as receptor for signalling to the PKA pathway and concomitantly undergoes substrate-induced oligo-ubiquitination and endocytosis. We have identified specific amino acids and analogues that uncouple to certain extent signalling, transport, oligo-ubiquitination and endocytosis. l-lysine, l-histidine and l-tryptophan are transported by Gap1 but do not trigger signalling. Unlike l-histidine, l-lysine triggers Gap1 oligo-ubiquitination without substantial induction of endocytosis. Two transported, non-metabolizable signalling agonists, β-alanine and d-histidine, are strong and weak inducers of Gap1 endocytosis, respectively, but both causing Gap1 oligo-ubiquitination. The non-signalling agonist, non-transported competitive inhibitor of Gap1 transport, l-Asp-γ-l-Phe, induces oligo-ubiquitination but no discernible endocytosis. The Km of l-citrulline transport is much lower than the threshold concentration for signalling and endocytosis. These results show that molecules can be transported without triggering signalling or substantial endocytosis, and that oligo-ubiquitination and endocytosis do not require signalling nor metabolism. Oligo-ubiquitination is required, but apparently not sufficient to trigger endocytosis. In addition, we demonstrate intracellular cross-induction of endocytosis of transport-defective Gap1Y395C by ubiquitination- and endocytosis-deficient Gap1K9R,K16R. Our results support the concept that different substrates bind to partially overlapping binding sites in the same general substrate-binding pocket of Gap1, triggering divergent conformations, resulting in different conformation-induced downstream processes. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.


van den Ende W.,Institute of Botany and Microbiology | Coopman M.,Institute of Botany and Microbiology | Clerens S.,Agresearch Ltd. | Vergauwen R.,Institute of Botany and Microbiology | And 3 more authors.
Plant Physiology | Year: 2011

About 15% of flowering plants accumulate fructans. Inulin-type fructans with β(2,1) fructosyl linkages typically accumulate in the core eudicot families (e.g. Asteraceae), while levan-type fructans with β(2,6) linkages and branched, graminan-type fructans with mixed linkages predominate in monocot families. Here, we describe the unexpected finding that graminan- and levan-type fructans, as typically occurring in wheat (Triticum aestivum) and barley (Hordeum vulgare), also accumulate in Pachysandra terminalis, an evergreen, frost-hardy basal eudicot species. Part of the complex graminan- and levan-type fructans as accumulating in vivo can be produced in vitro by a sucrose:fructan 6-fructosyltransferase (6-SFT) enzyme with inherent sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan 6-exohydrolase side activities. This enzyme produces a series of cereal-like graminan- and levan-type fructans from sucrose as a single substrate. The 6-SST/6-SFT enzyme was fully purified by classic column chromatography. In-gel trypsin digestion led to reverse transcription-polymerase chain reaction-based cDNA cloning. The functionality of the 6-SST/6-SFT cDNA was demonstrated after heterologous expression in Pichia pastoris. Both the recombinant and native enzymes showed rather similar substrate specificity characteristics, including peculiar temperature-dependent inherent 1-SST and fructan 6-exohydrolase side activities. The finding that cereal-type fructans accumulate in a basal eudicot species further confirms the polyphyletic origin of fructan biosynthesis in nature. Our data suggest that the fructan syndrome in P. terminalis can be considered as a recent evolutionary event. Putative connections between abiotic stress and fructans are discussed. © 2010 American Society of Plant Biologists.


Huysmans S.,Institute of Botany and Microbiology | Verstraete B.,Institute of Botany and Microbiology | Smets E.,Institute of Botany and Microbiology | Smets E.,Leiden University | Chatrou L.W.,Wageningen University
Plant Ecology and Evolution | Year: 2010

Background and aims - Orbicules or Ubisch bodies have been recorded in many angiosperm families and although the first observations date back to 1865, their function in the anther remains enigmatic. In flowering plants a general evolutionary trend is observed from common occurrence of orbicules in early diverging lineages towards a more patchy distribution in derived clades of eudicots. Annonaceae was our family of choice for an in depth study of orbicule distribution in early diverging angiosperms since it met the following three criteria: (1) high tapetum diversity, (2) orbicule presence and absence recorded, and (3) recent phylogeny at genus level available. Key results - Our SEM data of eighteen species show that orbicules are more common in Annonaceae than previously perceived. The resulting orbicule distribution pattern on the family topology indicates a consistent absence of orbicules in the 'long branch clade' while orbicules are present in Anaxagorea, the ambavioids, and the 'short branch clade'. Presence of orbicules is the ancestral condition in the family. Morphologically, Annonaceae orbicules are small (< 1 μm), spherical and smooth. Conclusions - The orbicule distribution pattern in Annonaceae reflects the general evolutionary trend in flowering plants. We hypothesize that orbicule presence can be considered as a powerful proxy for nonamoeboid tapetum characterization in Annonaceae. © 2010 National Botanic Garden of Belgium and Royal Botanical Society of Belgium.


Fernandez O.,CNRS Research Unit on Grapevine and Wines in Champagne | Vandesteene L.,Institute of Botany and Microbiology | Feil R.,Max Planck Institute of Molecular Plant Physiology | Baillieul F.,CNRS Research Unit on Grapevine and Wines in Champagne | And 2 more authors.
Planta | Year: 2012

During the last decade, there has been growing interest in the role of trehalose metabolism in tolerance to abiotic stress in higher plants, especially cold stress. So far, this metabolism has not yet been studied in Vitis vinifera L., despite the economic importance of this crop. The goal of this paper was to investigate the involvement of trehalose metabolism in the response of grapevine to chilling stress, and to compare the response in plants bacterised with Burkholderia phytofirmans strain PsJN, a plant growth-promoting rhizobacterium that confers grapevine chilling tolerance, with mock-inoculated plants. In silico analysis revealed that the V. vinifera L. genome contains genes encoding the enzymes responsible for trehalose synthesis and degradation. Transcript analysis showed that these genes were differentially expressed in various plant organs, and we also characterised their response to chilling. Both trehalose and trehalose 6-phosphate (T6P) were present in grapevine tissues and showed a distinct pattern of accumulation upon chilling. Our results suggest a role for T6P as the main active molecule in the metabolism upon chilling, with a possible link with sucrose metabolism. Furthermore, plants colonised by B. phytofirmans and cultivated at 26°C accumulated T6P and trehalose in stems and leaves at concentrations similar to non-bacterised plants exposed to chilling temperatures for 1 day. Overall, our data suggest that T6P and trehalose accumulate upon chilling stress in grapevine and might participate in the resistance to chilling stress conferred by B. phytofirmans. © 2012 Springer-Verlag.


Vandesteene L.,Institute of Botany and Microbiology | Ramon M.,Harvard University | Le Roy K.,Institute of Botany and Microbiology | Van Dijck P.,Vlaams Institute for Biotechnology | And 2 more authors.
Molecular Plant | Year: 2010

Higher plants typically do not produce trehalose in large amounts, but their genome sequences reveal large families of putative trehalose metabolism enzymes. An important regulatory role in plant growth and development is also emerging for the metabolic intermediate trehalose-6-P (T6P). Here, we present an update on Arabidopsis trehalose metabolism and a resource for further detailed analyses. In addition, we provide evidence that Arabidopsis encodes a single trehalose-6-P synthase (TPS) next to a family of catalytically inactive TPS-like proteins that might fulfill specific regulatory functions in actively growing tissues.


Kong E.F.,University of Maryland, Baltimore | Kucharikova S.,Vlaams Institute for Biotechnology | Kucharikova S.,Institute of Botany and Microbiology | Van Dijck P.,Vlaams Institute for Biotechnology | And 5 more authors.
Infection and Immunity | Year: 2015

The clinical significance of polymicrobial interactions, particularly those between commensal species with high pathogenic potential, remains largely understudied. Although the dimorphic fungal species Candida albicans and the bacterium Staphylococcus aureus are common cocolonizers of humans, they are considered leading opportunistic pathogens. Oral candidiasis specifically, characterized by hyphal invasion of oral mucosal tissue, is the most common opportunistic infection in HIV+ and immunocompromised individuals. In this study, building on our previous findings, a mouse model was developed to investigate whether the onset of oral candidiasis predisposes the host to secondary staphylococcal infection. The findings demonstrated that in mice with oral candidiasis, subsequent exposure to S. aureus resulted in systemic bacterial infection with high morbidity and mortality. Histopathology and scanning electron microscopy of tongue tissue from moribund animals revealed massive C. albicans hyphal invasion coupled with S. aureus deep tissue infiltration. The crucial role of hyphae in the process was demonstrated using a non-hypha-producing and a noninvasive hypha-producing mutant strains of C. albicans. Further, in contrast to previous findings, S. aureus dissemination was aided but not contingent upon the presence of the Als3p hypha-specific adhesion. Importantly, impeding development of mucosal C. albicans infection by administering antifungal fluconazole therapy protected the animals from systemic bacterial disease. The combined findings from this study demonstrate that oral candidiasis may constitute a risk factor for disseminated bacterial disease warranting awareness in terms of therapeutic management of immunocompromised individuals. © 2015, American Society for Microbiology.

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