Ambient pH Controls Glycogen Levels by Regulating Glycogen Synthase Gene Expression in Neurospora crassa. New Insights into the pH Signaling Pathway

Abstract

Glycogen is a polysaccharide widely distributed in microorganisms and animal cells and its metabolism is under intricate regulation. Its accumulation in a specific situation results from the balance between glycogen synthase and glycogen phosphorylase activities that control synthesis and degradation, respectively. These enzymes are highly regulated at transcriptional and post-translational levels. The existence of a DNA motif for the Aspergillus nidulans pH responsive transcription factor PacC in the promoter of the gene encoding glycogen synthase (gsn) in Neurospora crassa prompted us to investigate whether this transcription factor regulates glycogen accumulation. Transcription factors such as PacC in A. nidulans and Rim101p in Saccharomyces cerevisiae play a role in the signaling pathway that mediates adaptation to ambient pH by inducing the expression of alkaline genes and repressing acidic genes. We showed here that at pH 7.8 pacC was over-expressed and gsn was down-regulated in wild-type N. crassa coinciding with low glycogen accumulation. In the pacCKO strain the glycogen levels and gsn expression at alkaline pH were, respectively, similar to and higher than the wild-type strain at normal pH (5.8). These results characterize gsn as an acidic gene and suggest a regulatory role for PACC in gsn expression. The truncated recombinant protein, containing the DNA-binding domain specifically bound to a gsn DNA fragment containing the PacC motif. DNA-protein complexes were observed with extracts from cells grown at normal and alkaline pH and confirmed by ChIP-PCR analysis. The PACC present in these extracts showed equal molecular mass, indicating that the protein is already processed at normal pH, in contrast to A. nidulans. Together, these results show that the pH signaling pathway controls glycogen accumulation by regulating gsn expression and suggest the existence of a different mechanism for PACC activation in N. crassa.

Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor

“…The tub-2 gene was used as the reference gene and the wild-type sample at pH 5.8 as the reference sample. Expression of the pac-3 gene in the wild-type strain was significantly higher at alkaline pH ( P < 0.01), as expected (Fig 4) and consistent with previous results obtained by Cupertino et al [23]. Additionally, the pal-1 (the palA homolog), pal-2 (the palB homolog), and pal-9 (the palI homolog) genes were over-expressed at alkaline pH in the wild-type strain, and their expression was dependent on the PAC-3 transcription factor since they were down-regulated in Δ pac-3 strain under the same pH (Fig 4).…”

“…The Δ pac-3 strain was generated as described in Cupertino et al [23]. Details for all mutant strains can be found in S1 Table.…”

Deletion of pH Regulator pac-3 Affects Cellulase and Xylanase Activity during Sugarcane Bagasse Degradation by Neurospora crassa

“…Since the growth rate of the Δ pac-3 mutant on solid medium is affected by pH [31], we investigated the final growth (total protein content) of 74A, Δ pac-3 , and Δ mus-52 in the cultured medium at pH 3.0, 5.0, 7.0, 8.0, and 10.0. The Δ pac-3 mutant grew better at acidic (3.0 and 5.0) rather than neutral (7) or alkaline (8.0 and 10.0) pH when compared with 74A and Δ mus-52 strains (Fig 1A).…”

“…A N . crassa Δ pac-3 knockout strain ( pac-3 KO ) was generated following the knockout procedures described previously [22], based on the strain FGSC N° 9568 (mat a, mus-52::hyg) [23]. Strains were maintained on agar-based Vogel’s minimal (VM) medium at pH 5.8 [24], containing 2% sucrose at 30°C.…”

Expression and promoter characterization of BbPacC, a pH response transcription factor gene of the entomopathogenic fungus Beauveria bassiana

“…Under alkaline conditions, PacC upregulates some alkaline pH inducible genes but prevents expression of acid-expressed genes (Then Bergh et al, 1998;Cupertino et al, 2012;Espeso & Arst, 2000;Kanda et al, 2008;Merhej et al, 2011;Tilburn et al, 1995). The transcription of the PacC gene is self-regulated in a feedback manner.…”