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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.

Carvalho ND, Jørgensen TR, Arentshorst M, Nitsche BM, van den Hondel CA, Archer DB, Ram AF - BMC Genomics (2012)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

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

ABSTRACT

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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Growth and phenotypic profiles of HacAWT and HacACA strains. (A) Differences on colony size (diameter) of HacAWT and HacACA strains growing at different temperatures. 104 spores were spotted on solid CM plates and growth was monitored for 6 days. (B) Strains phenotype on CM after 3 and 6 days of growth at 30 °C. HacACA phenotype is characterized by a slower growth/colony size as well as a delay in sporulation compared to the HacAWT. Bars indicate standard deviations from three individual measurements.
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Figure 1: Growth and phenotypic profiles of HacAWT and HacACA strains. (A) Differences on colony size (diameter) of HacAWT and HacACA strains growing at different temperatures. 104 spores were spotted on solid CM plates and growth was monitored for 6 days. (B) Strains phenotype on CM after 3 and 6 days of growth at 30 °C. HacACA phenotype is characterized by a slower growth/colony size as well as a delay in sporulation compared to the HacAWT. Bars indicate standard deviations from three individual measurements.

Mentions: To obtain an A. niger strain with a constitutively activated HacA (HacACA) transcription factor, the wild-type hacA gene was replaced by the spliced form of hacA that lacks the 20 nucleotide intron. For the construction of a reference strain and a strain only expressing the hacA induced form, plasmids pHacWT and pHacCA were used [Additional file 1 (A and B)]. Transformants with the correct integration pattern for each plasmid were selected after Southern blot analysis (data not shown) and the absence of the intron was confirmed in the HacACA strain [Additional file 1 (C and D)]. Growth assays were performed with both strains at different temperatures (Figure 1A and B). At each temperature tested, radial growth rate (colony size) of the HacACA strain was reduced compared to the HacAWT strain, and this growth impairment was more pronounced at 37 and 42 °C (Figure 1A). Differences in phenotype between both strains were also apparent as HacACA showed a delay in growth and conidiation in comparison to HacAWT (Figure 1B). As no phenotypic differences were found between our reference strain HacAWT and N402 (data not shown), we conclude that the phenotypic effects observed in HacACA are due to the presence of only the UPR-induced form of hacA. The effects of having a constitutive activation of the UPR are different from the absence of a functional UPR. The deletion of the HacA transcription factor in A. niger has a profound effect on growth and morphology of the fungus, resulting in smaller and more compact colonies that hardly form conidia [30,47].


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.

Carvalho ND, Jørgensen TR, Arentshorst M, Nitsche BM, van den Hondel CA, Archer DB, Ram AF - BMC Genomics (2012)

Growth and phenotypic profiles of HacAWT and HacACA strains. (A) Differences on colony size (diameter) of HacAWT and HacACA strains growing at different temperatures. 104 spores were spotted on solid CM plates and growth was monitored for 6 days. (B) Strains phenotype on CM after 3 and 6 days of growth at 30 °C. HacACA phenotype is characterized by a slower growth/colony size as well as a delay in sporulation compared to the HacAWT. Bars indicate standard deviations from three individual measurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472299&req=5

Figure 1: Growth and phenotypic profiles of HacAWT and HacACA strains. (A) Differences on colony size (diameter) of HacAWT and HacACA strains growing at different temperatures. 104 spores were spotted on solid CM plates and growth was monitored for 6 days. (B) Strains phenotype on CM after 3 and 6 days of growth at 30 °C. HacACA phenotype is characterized by a slower growth/colony size as well as a delay in sporulation compared to the HacAWT. Bars indicate standard deviations from three individual measurements.
Mentions: To obtain an A. niger strain with a constitutively activated HacA (HacACA) transcription factor, the wild-type hacA gene was replaced by the spliced form of hacA that lacks the 20 nucleotide intron. For the construction of a reference strain and a strain only expressing the hacA induced form, plasmids pHacWT and pHacCA were used [Additional file 1 (A and B)]. Transformants with the correct integration pattern for each plasmid were selected after Southern blot analysis (data not shown) and the absence of the intron was confirmed in the HacACA strain [Additional file 1 (C and D)]. Growth assays were performed with both strains at different temperatures (Figure 1A and B). At each temperature tested, radial growth rate (colony size) of the HacACA strain was reduced compared to the HacAWT strain, and this growth impairment was more pronounced at 37 and 42 °C (Figure 1A). Differences in phenotype between both strains were also apparent as HacACA showed a delay in growth and conidiation in comparison to HacAWT (Figure 1B). As no phenotypic differences were found between our reference strain HacAWT and N402 (data not shown), we conclude that the phenotypic effects observed in HacACA are due to the presence of only the UPR-induced form of hacA. The effects of having a constitutive activation of the UPR are different from the absence of a functional UPR. The deletion of the HacA transcription factor in A. niger has a profound effect on growth and morphology of the fungus, resulting in smaller and more compact colonies that hardly form conidia [30,47].

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

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

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus