Carbon starvation-induced synthesis of GDH2 and PEPCK is essential for the survival of Pichia pastoris.

The industrial yeast Pichia pastoris can utilize amino acids as the sole source of carbon. It possesses a post-transcriptional regulatory circuit that governs the synthesis of cytosolic glutamate dehydrogenase 2 (GDH2) and phosphoenolpyruvate carboxykinase (PEPCK), key enzymes of amino acid catabolism. Here, we demonstrate that the post-transcriptional regulatory circuit is activated during carbon starvation resulting in the translation of GDH2 and PEPCK mRNAs. GDH2 and PEPCK synthesis is abrogated in Δatg1 indicating a key role for autophagy or an autophagy-related process. Finally, carbon-starved Δgdh2 and Δpepck exhibit poor survival. This study demonstrates a key role for amino acid catabolism during carbon starvation, a phenomenon hitherto unreported in other yeast species.

Antimicrobial peptide CGA-N12 decreases the Candida tropicalis mitochondrial membrane potential via mitochondrial permeability transition pore.

Amino acid sequence from 65th to 76th residue of the N-terminus of Chromogranin A (CGA-N12) is an antimicrobial peptide (AMP). Our previous studies showed that CGA-N12 reduces Candida tropicalis mitochondrial membrane potential. Here, we explored the mechanism that CGA-N12 collapsed the mitochondrial membrane potential by investigations of its action on the mitochondrial permeability transition pore (mPTP) complex of C. tropicalis. The results showed that CGA-N12 induced cytochrome c (Cyt c) leakage, mitochondria swelling and led to polyethylene glycol (PEG) of molecular weight 1000 Da penetrate mitochondria. mPTP opening inhibitors bongkrekic acid (BA) could contract the mitochondrial swelling induced by CGA-N12, but cyclosporin A (CsA) could not. Therefore, we speculated that CGA-N12 could induce C. tropicolis mPTP opening by preventing the matrix-facing (m) conformation of adenine nucleotide transporter (ANT), thereby increasing the permeability of the mitochondrial membrane and resulted in the mitochondrial potential dissipation.

Regulation of Acetate Metabolism and Acetyl Co-a Synthetase 1 (ACS1) Expression by Methanol Expression Regulator 1 (Mxr1p) in the Methylotrophic Yeast Pichia pastoris.

Methanol expression regulator 1 (Mxr1p) is a zinc finger protein that regulates the expression of genes encoding enzymes of the methanol utilization pathway in the methylotrophic yeast Pichia pastoris by binding to Mxr1p response elements (MXREs) present in their promoters. Here we demonstrate that Mxr1p is a key regulator of acetate metabolism as well. Mxr1p is cytosolic in cells cultured in minimal medium containing a yeast nitrogen base, ammonium sulfate, and acetate (YNBA) but localizes to the nucleus of cells cultured in YNBA supplemented with glutamate or casamino acids as well as nutrient-rich medium containing yeast extract, peptone, and acetate (YPA). Deletion of Mxr1 retards the growth of P. pastoris cultured in YNBA supplemented with casamino acids as well as YPA. Mxr1p is a key regulator of ACS1 encoding acetyl-CoA synthetase in cells cultured in YPA. A truncated Mxr1p comprising 400 N-terminal amino acids activates ACS1 expression and enhances growth, indicating a crucial role for the N-terminal activation domain during acetate metabolism. The serine 215 residue, which is known to regulate the expression of Mxr1p-activated genes in a carbon source-dependent manner, has no role in the Mxr1p-mediated activation of ACS1 expression. The ACS1 promoter contains an Mxr1p response unit (MxRU) comprising two MXREs separated by a 30-bp spacer. Mutations that abrogate MxRU function in vivo abolish Mxr1p binding to MxRU in vitro. Mxr1p-dependent activation of ACS1 expression is most efficient in cells cultured in YPA. The fact that MXREs are conserved in genes outside of the methanol utilization pathway suggests that Mxr1p may be a key regulator of multiple metabolic pathways in P. pastoris.

Real-time imaging of hydrogen peroxide dynamics in vegetative and pathogenic hyphae of Fusarium graminearum.

Balanced dynamics of reactive oxygen species in the phytopathogenic fungus Fusarium graminearum play key roles for development and infection. To monitor those dynamics, ratiometric analysis using the novel hydrogen peroxide (H2O2) sensitive fluorescent indicator protein HyPer-2 was established for the first time in phytopathogenic fungi. H2O2 changes the excitation spectrum of HyPer-2 with an excitation maximum at 405 nm for the reduced and 488 nm for the oxidized state, facilitating ratiometric readouts with maximum emission at 516 nm. HyPer-2 analyses were performed using a microtiter fluorometer and confocal laser scanning microscopy (CLSM). Addition of external H2O2 to mycelia caused a steep and transient increase in fluorescence excited at 488 nm. This can be reversed by the addition of the reducing agent dithiothreitol. HyPer-2 in F. graminearum is highly sensitive and specific to H2O2 even in tiny amounts. Hyperosmotic treatment elicited a transient internal H2O2 burst. Hence, HyPer-2 is suitable to monitor the intracellular redox balance. Using CLSM, developmental processes like nuclear division, tip growth, septation, and infection structure development were analyzed. The latter two processes imply marked accumulations of intracellular H2O2. Taken together, HyPer-2 is a valuable and reliable tool for the analysis of environmental conditions, cellular development, and pathogenicity.

GATA-Dependent Glutaminolysis Drives Appressorium Formation in Magnaporthe oryzae by Suppressing TOR Inhibition of cAMP/PKA Signaling.

Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR) signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development.

Effect of an organophosphate pesticide, monocrotophos, on phosphate-solubilizing efficiency of soil fungal isolates.

Soil is a sink of pesticide residues as well as microorganisms. Fungi are well known for solubilization of inorganic phosphates, and this activity of fungal isolates may be affected by the presence of pesticide residues in the soil. In the present study, five generically different fungal isolates, viz. Aspergillus niger JQ660373, Aspergillus flavus, Penicillium aculeatum JQ660374, Fusarium pallidoroseum and Macrophomina sp., were tested and compared for their phosphate-solubilizing ability in the absence and presence of monocrotophos (500 mg L(-1)). After 168 h of incubation, four times high amount of tricalcium phosphate was solubilized by isolates in the growth medium containing monocrotophos in comparison to control (without monocrotophos). Concurrently, 78 % of the applied monocrotophos was degraded by these fungal isolates. Kinetics of phosphate solubilization shifted from logarithmic to power model in the presence of monocrotophos. Similarly, the phosphatase activity was also found significantly high in the presence of monocrotophos. The combined order of phosphate solubilization as well as monocrotophos degradation was found to be A. niger JQ660373 > P. aculeatum JQ660374 > A. flavus > F. pallidoroseum > Macrophomina sp. On the contrary, phosphate solubilization negatively correlated with the pH of the growth medium. Hence, it could be concluded that these fungal species efficiently solubilize inorganic phosphates and monocrotophos poses a positive effect on their ability and in turn degraded by them. To the best of our knowledge, this is the first report on P solubilization by Macrophomina sp. and F. pallidoroseum.

Mineral supplementation increases erythrose reductase activity in erythritol biosynthesis from glycerol by Yarrowia lipolytica.

The aim of this study was to examine the impact of divalent copper, iron, manganese, and zinc ions on the production of erythritol from glycerol by Yarrowia lipolytica and their effect on the activity of erythrose reductase. No inhibitory effect of the examined minerals on yeast growth was observed in the study. Supplementation with MnSO4 · 7H2O (25 mg l(-1)) increased erythritol production by Y. lipolytica by 14.5%. In the bioreactor culture with manganese ion addition, 47.1 g l(-1) of erythritol was produced from 100.0 g l(-1) of glycerol, which corresponded to volumetric productivity of 0.87 g l(-1) h(-1). The addition of Mn(2+) enhanced the intracellular activity of erythrose reductase up to 24.9 U g(-1) of dry weight of biomass (DW), hence, about 1.3 times more than in the control.

Promotion of activity and thermal stability of chloroperoxidase by trace amount of metal ions (M2+/M3+).

The effect of M(2+) (Zn(2+), Cu(2+), Cd(2+), Mn(2+), Pb(2+)) and M(3+) (Cr(3+), La(3+), Fe(3+), Ce(3+), Y(3+), Al(3+)) metal ions on the activity and thermal stability of chloroperoxidase (CPO) was investigated in this work. It was found that the lower concentration of metal ions was favorable to CPO activity whereas the higher concentration reversed the results. CPO activity could be increased to 116.4-127.1% in the presence of a trace amount of these M(2+)/M(3+) metal ions at a concentration range of 0-25 µmol L(-1) after 2 h of incubation at 25 °C. The activating effect of M(3+) is better than that of M(2+), and Cr(3+) was mostly efficient. The thermal stability of the enzyme was also improved significantly. Only 30.3% of CPO activity was retained at 50 °C whereas 82.6% of CPO activity was maintained in the presence of Cr(3+) after 2 h of incubation at the same temperature. The activation of CPO by metal ions at their low concentration was studied through intrinsic fluorescence, circular dichroism (CD), and UV-Vis spectra assay. A favorable environment around the active site was achieved in the presence of metal ions. Intrinsic fluorescence and CD spectra indicated that the α-helix structure of CPO was strengthened in metal ion-contained media. More exposure of the heme ring was achieved for easy access of the substrate, which was suggested by UV-Vis spectrum analysis. This strategy for enhancing CPO activity is very simple and useful. It will be favorable to the practical application of this enzyme.

Differential expression of ATP-binding cassette and/or major facilitator superfamily class efflux pumps contributes to voriconazole resistance in Aspergillus flavus.

Invasive aspergillosis remains a life-threatening infection in immunocompromised patients. Although clinical failures are attributed to poor host immunity, antifungal drug resistance may be a contributing factor. Reports of voriconazole (VRC) resistance (VRC-R) in clinical isolates of Aspergillus spp. continue to emerge from various centers around the world, and mechanisms contributing to drug resistance are poorly understood. The aim of this study is to study the role of multidrug resistance efflux pumps (MDR-EPs) in VRC-R in Aspergillus flavus using efflux pump inhibitors and quantitative reverse transcriptase polymerase chain reaction. Relative quantification of various MDR-EPs was performed pre-exposure and postexposure to VRC, which demonstrated an increase in 1 or more efflux pump gene transcripts to varying degrees in VRC-susceptible and VRC-R isolates of A. flavus. Exposure to sub-MIC of VRC causes up-regulation of genes encoding MDR-EPs, contributing to triazole resistance in A. flavus and may not be detected during routine antifungal susceptibility testing in vitro.

Modelling the activation of alkaline pH response transcription factor PacC in Aspergillus nidulans: involvement of a negative feedback loop.

Alkaline pH adaptation represents an important environmental stress response in Aspergillus nidulans. It is mediated by the pal signalling pathway and the PacC transcription factor. Although studied extensively experimentally, the activation mechanism of PacC has not been quantified, and it is not clear how this activation is regulated. Here, by constructing mathematical models, we first show that the pattern of PacC activation observed in previously published experiments cannot be explained based on existing knowledge about PacC activation. Extending the model with a negative feedback loop is necessary to produce simulation results that are consistent with the data, suggesting the existence of a negative feedback loop in the PacC activation process. This extended model is then validated against published measurements for cells with drug treatment and mutant cells. Furthermore, we investigate the role of an intermediate form of PacC in the PacC activation process, and propose experiments that can be used to test our predictions. Our work illustrates how mathematical models can be used to uncover regulatory mechanisms in the transcription regulation, and generate hypotheses that guide further laboratory investigations.

Agonist-specific conformational changes in the yeast alpha-factor pheromone receptor.

The yeast alpha-factor pheromone receptor is a member of the G-protein-coupled receptor family. Limited trypsin digestion of yeast membranes was used to investigate ligand-induced conformational changes in this receptor. The agonist, alpha-factor, accelerated cleavage in the third intracellular loop, whereas the antagonist, desTrp1,Ala3-alpha-factor, reduced the cleavage rate. Thus, the enhanced accessibility of the third intracellular loop is specific to the agonist. alpha-Factor inhibited cleavage weakly at a second site near the cytoplasmic terminus of the seventh transmembrane helix, whereas the antagonist showed a stronger inhibition of cleavage at this site and at another site in the C-terminal domain of the receptor. The alpha-factor-induced conformational changes appeared to be inherent properties of the receptor, as they were retained in G-protein-deficient mutants. Moreover, a mutant receptor (ste2-L236H) that affects the third loop and is defective for G-protein coupling retained the ability to undergo the agonist-induced conformational changes. These results are consistent with a model in which G-protein activation is limited by the availability of specific contacts between the G protein and the third intracellular loop of the receptor. The antagonist appears to promote a distinct conformational state that differs from either the unoccupied or the agonist-occupied state.