Publications by authors named "Arthur Fj Ram"

4 Publications

  • Page 1 of 1

A new vector for efficient gene targeting to the locus in .

Fungal Biol Biotechnol 2015 14;2. Epub 2015 Mar 14.

Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands.

Background: The possibility for efficient gene targeting for the controlled integration of DNA constructs is an important tool in fungal genetics.

Findings: In this study, we report a new targeting vector based on the marker in . The DNA sequence to be targeted is surrounded by two fragments of the gene to allow homologous recombination of the recombinant DNA at the locus. The 5' end of the targeting cassette contains a non-functional truncated open reading frame (first 112 bases deleted) and the 3' untranslated region (3' UTR). At the 3' end, the targeting cassette consists of the 3' flanking region of the gene. A unique I site between the flanks allows the insertion of a gene of interest. The linearized targeting cassette is transformed to the mutant strain AB4.1 or a derivative thereof. By using a constitutively expressed luciferase reporter gene () as an example, it is shown that the targeting system is efficient as 4 out of 6 (67%) AB4.1 transformants and 51 out of 66 (77%) MA169.4 ( ) transformants contained the reporter gene at the locus. A luciferase (lux) activity assay, performed with independently obtained transformants in which the reporter was integrated at the locus, showed comparable and reproducible lux activities.

Conclusion: The new targeting vector is an important improvement to the existing method for gene targeting in Although the vector is specific for the presented design and approach is easily applicable for constructing integration vectors for other fungi.
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March 2015

The capacity of to sense and respond to cell wall stress requires at least three transcription factors: RlmA, MsnA and CrzA.

Fungal Biol Biotechnol 2014 1;1. Epub 2014 Dec 1.

Institute of Biotechnology, Department Applied and Molecular Microbiology Berlin University of Technology, Gustav-Meyer-Allee 25, Berlin, D-13355 Germany.

Background: Cell wall integrity, vesicle transport and protein secretion are key factors contributing to the vitality and productivity of filamentous fungal cell factories such as . In order to pioneer rational strain improvement programs, fundamental knowledge on the genetic basis of these processes is required. The aim of the present study was thus to unravel survival strategies of when challenged with compounds interfering directly or indirectly with its cell wall integrity: calcofluor white, caspofungin, aureobasidin A, FK506 and fenpropimorph.

Results: Transcriptomics signatures of and phenotypic analyses of selected null mutant strains were used to predict regulator proteins mediating the survival responses against these stressors. This integrated approach allowed us to reconstruct a model for the cell wall salvage gene network of that ensures survival of the fungus upon cell surface stress. The model predicts that (i) caspofungin and aureobasidin A induce the cell wall integrity pathway as a main compensatory response via induction of RhoB and RhoD, respectively, eventually activating the mitogen-activated protein kinase kinase MkkA and the transcription factor RlmA. (ii) RlmA is the main transcription factor required for the protection against calcofluor white but it cooperates with MsnA and CrzA to ensure survival of when challenged with caspofungin and aureobasidin A. (iii) Membrane stress provoked by aureobasidin A via disturbance of sphingolipid synthesis induces cell wall stress, whereas fenpropimorph-induced disturbance of ergosterol synthesis does not.

Conclusion: The present work uncovered a sophisticated defence system of which employs at least three transcription factors - RlmA, MsnA and CrzA - to protect itself against cell wall stress. The transcriptomic data furthermore predicts a fourth transfactor, SrbA, which seems to be specifically important to survive fenpropimorph-induced cell membrane stress. Future studies will disclose how these regulators are interlocked in different signaling pathways to secure survival of under different cell wall stress conditions.
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December 2014

Identification of the UDP-glucose-4-epimerase required for galactofuranose biosynthesis and galactose metabolism in .

Fungal Biol Biotechnol 2014 14;1. Epub 2014 Oct 14.

Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Leiden University, Sylviusweg 72, Leiden, 2333 BE The Netherlands.

Background: Galactofuranose (Gal)-containing glycoconjugates are important to secure the integrity of the cell wall of filamentous fungi. Mutations that prevent the biosynthesis of Gal-containing molecules compromise cell wall integrity. In response to cell wall weakening, the cell wall integrity (CWI)-pathway is activated to reinforce the strength of the cell wall. Activation of CWI-pathway in is characterized by the specific induction of the gene, which encodes a cell wall α-glucan synthase.

Results: In this study, we screened a collection of cell wall mutants with an induced expression of for defects in Gal biosynthesis using a with anti-Gal antibody (L10). From this collection of mutants, we previously identified mutants in the UDP-galactopyranose mutase encoding gene (). Here, we have identified six additional UDP-galactopyranose mutase () mutants and one mutant (named mutant #41) in an additional complementation group that displayed strongly reduced Gal-levels in the cell wall. By using a whole genome sequencing approach, 21 SNPs in coding regions were identified between mutant #41 and its parental strain which changed the amino acid sequence of the encoded proteins. One of these mutations was in gene An14g03820, which codes for a putative UDP-glucose-4-epimerase (UgeA). The A to G mutation in this gene causes an amino acid change of Asn to Asp at position 191 in the UgeA protein. Targeted deletion of resulted in an even more severe reduction of Gal in N-linked glucans, indicating that the UgeA protein in mutant #41 is partially active. The gene is also required for growth on galactose despite the presence of two UgeA homologs in the genome.

Conclusion: By using a classical mutant screen and whole genome sequencing of a new Gal-deficient mutant, the UDP-glucose-4-epimerase gene () has been identified. UgeA is required for the biosynthesis of Gal as well as for galactose metabolism in .
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October 2014

Genome-wide expression analysis upon constitutive activation of the HacA bZIP transcription factor in Aspergillus niger reveals a coordinated cellular response to counteract ER stress.

BMC Genomics 2012 Jul 30;13:350. Epub 2012 Jul 30.

Institute of Biology Leiden, Leiden University, Molecular Microbiology and Biotechnology, BE Leiden, The Netherlands.

Background: HacA/Xbp1 is a conserved bZIP transcription factor in eukaryotic cells which regulates gene expression in response to various forms of secretion stress and as part of secretory cell differentiation. In the present study, we replaced the endogenous hacA gene of an Aspergillus niger strain with a gene encoding a constitutively active form of the HacA transcription factor (HacACA). The impact of constitutive HacA activity during exponential growth was explored in bioreactor controlled cultures using transcriptomic analysis to identify affected genes and processes.

Results: Transcription profiles for the wild-type strain (HacAWT) and the HacACA strain were obtained using Affymetrix GeneChip analysis of three replicate batch cultures of each strain. In addition to the well known HacA targets such as the ER resident foldases and chaperones, GO enrichment analysis revealed up-regulation of genes involved in protein glycosylation, phospholipid biosynthesis, intracellular protein transport, exocytosis and protein complex assembly in the HacACA mutant. Biological processes over-represented in the down-regulated genes include those belonging to central metabolic pathways, translation and transcription. A remarkable transcriptional response in the HacACA strain was the down-regulation of the AmyR transcription factor and its target genes.

Conclusions: The results indicate that the constitutive activation of the HacA leads to a coordinated regulation of the folding and secretion capacity of the cell, but with consequences on growth and fungal physiology to reduce secretion stress.
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July 2012