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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 profiles of one of the triplicate A. niger HacAWT (A) and HacA CA (B) batch cultures. Dry weight biomass concentration (gDWkg-1) as a function of time (h) illustrates the growth of the cultures. The maximum specific growth rate for each culture was determined from the slope (α) of the ln transformation of biomass (Cbiomass) in the exponential growth phase as a function of time (h), as well from log transformation of alkali addition as a function of time (h). Dash-line represents the end of the exponential growth phase (depletion of glucose). Arrows indicate time-points where mycelium was harvested for transcriptomic analysis.
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Figure 2: Growth profiles of one of the triplicate A. niger HacAWT (A) and HacA CA (B) batch cultures. Dry weight biomass concentration (gDWkg-1) as a function of time (h) illustrates the growth of the cultures. The maximum specific growth rate for each culture was determined from the slope (α) of the ln transformation of biomass (Cbiomass) in the exponential growth phase as a function of time (h), as well from log transformation of alkali addition as a function of time (h). Dash-line represents the end of the exponential growth phase (depletion of glucose). Arrows indicate time-points where mycelium was harvested for transcriptomic analysis.

Mentions: Growth of batch cultures of the A. niger HacAWT and HacACA strains was characterized as filamentous and highly reproducible. The growth kinetics of a representative culture of each strain is shown in Figure 2 and results from all cultures are given in the supplemental material [Additional file 2. Cultures of the HacAWT strain exhibited exponential growth with a specific growth rate (μ) of 0.22 ±0.01 h-1 (n = 4) from exit of lag phase to depletion of glucose (Figure 2A). Initial growth of HacACA was similar to that of the HacAWT; it was exponential with a μ of 0.21 ±0.01 h-1 (n = 3). However, after 21–22 h of batch cultivation, when half of the glucose was consumed, the growth kinetics shifted from exponential to apparently linear (Figure 2B). It was not clear from the relatively few determinations of biomass concentration whether growth was truly linear in the second phase but this was strongly supported by analysis of the growth-dependent alkali addition (inset Figure 2A, B). We established a concordance between growth and alkali added to maintain constant pH in the cultures (not shown), and used this as an indirect measure of growth as described previously by Iversen et al. [48]. Linearity was then confirmed by log-transformation of alkali addition rates using the computer recorded titrant addition data and the LOS program [49]. During exponential growth, growth yield on substrate (Yxs) was comparable in both strains: 0.53 ± 0.02 for HacAWT and 0.52 ± 0.04 for HacACA.


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 profiles of one of the triplicate A. niger HacAWT (A) and HacA CA (B) batch cultures. Dry weight biomass concentration (gDWkg-1) as a function of time (h) illustrates the growth of the cultures. The maximum specific growth rate for each culture was determined from the slope (α) of the ln transformation of biomass (Cbiomass) in the exponential growth phase as a function of time (h), as well from log transformation of alkali addition as a function of time (h). Dash-line represents the end of the exponential growth phase (depletion of glucose). Arrows indicate time-points where mycelium was harvested for transcriptomic analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Growth profiles of one of the triplicate A. niger HacAWT (A) and HacA CA (B) batch cultures. Dry weight biomass concentration (gDWkg-1) as a function of time (h) illustrates the growth of the cultures. The maximum specific growth rate for each culture was determined from the slope (α) of the ln transformation of biomass (Cbiomass) in the exponential growth phase as a function of time (h), as well from log transformation of alkali addition as a function of time (h). Dash-line represents the end of the exponential growth phase (depletion of glucose). Arrows indicate time-points where mycelium was harvested for transcriptomic analysis.
Mentions: Growth of batch cultures of the A. niger HacAWT and HacACA strains was characterized as filamentous and highly reproducible. The growth kinetics of a representative culture of each strain is shown in Figure 2 and results from all cultures are given in the supplemental material [Additional file 2. Cultures of the HacAWT strain exhibited exponential growth with a specific growth rate (μ) of 0.22 ±0.01 h-1 (n = 4) from exit of lag phase to depletion of glucose (Figure 2A). Initial growth of HacACA was similar to that of the HacAWT; it was exponential with a μ of 0.21 ±0.01 h-1 (n = 3). However, after 21–22 h of batch cultivation, when half of the glucose was consumed, the growth kinetics shifted from exponential to apparently linear (Figure 2B). It was not clear from the relatively few determinations of biomass concentration whether growth was truly linear in the second phase but this was strongly supported by analysis of the growth-dependent alkali addition (inset Figure 2A, B). We established a concordance between growth and alkali added to maintain constant pH in the cultures (not shown), and used this as an indirect measure of growth as described previously by Iversen et al. [48]. Linearity was then confirmed by log-transformation of alkali addition rates using the computer recorded titrant addition data and the LOS program [49]. During exponential growth, growth yield on substrate (Yxs) was comparable in both strains: 0.53 ± 0.02 for HacAWT and 0.52 ± 0.04 for HacACA.

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