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

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.

Figure 1

Growth and phenotypic profiles of HacA^WT and HacA^CA strains. (A) Differences on colony size (diameter) of HacA^WT and HacA^CA 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. HacA^CA phenotype is characterized by a slower growth/colony size as well as a delay in sporulation compared to the HacA^WT. Bars indicate standard deviations from three individual measurements.

Figure 2

Growth profiles of one of the triplicate A. niger HacA^WT (A) and HacA ^CA (B) batch cultures. Dry weight biomass concentration (g_DWkg^-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 (C_biomass) 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.

Figure 3

Analysis of differentially expressed genes in HacA^CA at all three time points in comparison to HacA^WT . Venn diagrams of the number of overlapping and non-overlapping induced (A) or repressed (B) genes on A. niger HacA^CA mutant strain at different time points in comparison to HacA^WT strain

Figure 4

Functional classification of differentially expressed genes in HacA^CA. Representation of the main significant induced and repressed biological processes in the HacA^CA mutant strain in comparison to HacA^WT strain.

Figure 5

Analysis of differentially expressed genes in HacA^CA (this study) and Guillemette’ study[[37]]. Venn diagrams of the number of overlapping and non-overlapping induced or repressed genes of A. niger HacA^CA-1/HacA^WT(A) and genes from Guillemette et al. (2007) induced (or repressed) at least in two conditions (B) or induced in all conditions (C).

Figure 6

Effects of the constitutive activation of the UPR on the utilization of starch and starch related carbon sources. The wild-type strain (HacA^WT), the strain containing a constitutive active form of hacA (HacA^CA) and the AmyR disruptant (ΔamyR) strain were grown on MM containing 1% of the different carbon sources indicated at 30 °C for 3 days.

Figure 7

Effects of the constitutive activation of the UPR on the utilization of different polimeric and monomeric carbon sources. The wild-type strain (HacA^WT), the strain containing a constitutive active form of hacA (HacA^CA) the amyR disruptant (ΔamyR) and inuR disruptant (ΔinuR) strains were grown on MM containing 1% of the different carbon sources or 1% dried skim milk at 30 °C for 4 days.