Table 1.
Growth of ΔMoglk1 and ΔMohxk1 mutants on different carbon sources.
Figure 1.
Ammonium utilization of ΔMoglk1 and ΔMohxk1 mutants.
Guy11, ΔMoglk1 and ΔMohxk1 mutants were grown on MM containing 1% glucose and 100 mM ammonium. Photographs (A) were taken from one transformant of each mutant at indicated days after incubation at 30°C. Colony diameter (B) and medium pH (C) were measured at the same days. The experiment was performed with three strains for the ΔMoglk1 and ΔMohxk1 mutants, respectively; and three independent replicates provided the same results. Different letter represent difference (P<0.05) among Guy11 and ΔMoglk1, ΔMohxk1.
Figure 2.
The ΔMoglk1 mutants show reduced acidification on ammonium and glucose containing medium.
(A) The effects of different sugars (1% glucose, or 1% fructose) and medium pH (4.5, 6.5, or 8.0) on extracellular acidification. The extracellular pH was measured in liquid cultures of wild type Guy11 and ΔMoglk1 strains grown in the presence of 100 mM ammonium for various times, as indicated (days). The experiments were performed in six independent replicates. Error bars show the standard deviation of the mean for six replicates. (B) Guy11 and ΔMoglk1 mutants were grown on MM containing 1% glucose and 100 mM ammonium agar plates with initial pH 4.5 or pH 8.0. Photographs were taken at 9 days after incubation at 30°C. The experiments were performed in triplicate.
Figure 3.
The ΔMoglk1 mutants show decreased ammonium utilization in the presence of glucose as sole carbon source.
(A) The dry weight of mycelia from Guy11 and ΔMoglk1 strains grown in 100 mM ammonium-containing MM liquid culture with 1% glucose or 1% fructose as sole carbon source in indicated pH. Error bars indicate standard deviations from the mean. P values were calculated comparing Guy11 with ΔMoglk1 mutant mycelia dry weight. * indicates P<0.05. (B) The relationship between the drop in extracellular pH and the increase of fungus biomass. X-axis represents the time interval. 1, between 2nd and 1st day; 2, between 3rd and 2nd day; 3, between 5th and 3rd day; 4, between 7th and 5th day. The Y-axis represents the ratio of pH and biomass. pH indicates the drop of pH between the interval days (data from fig. 2A); biomass indicates the increase of biomass between the interval day (data from fig. 3A). (C) The medium (MM+1% glucose) ammonium concentration after incubation of Guy11 and ΔMoglk1 mutant for indicated days. The initial concentration of ammonium in the medium was 100 mM. Error bars indicate standard deviations from the mean. The experiment was repeated three times with similar results.
Figure 4.
Catalytically inactive Moglk1 alleles restore ammonium utilization in the ΔMoglk1 mutant.
(A) Complementation of yeast Δhxk1, Δhxk2, and ΔMoglk1 triple mutant. Yeast cells transformed with MoGLK1, MoGLK1G69D and MoGLK1S156A were cultured in SD medium with glucose (Glc) as the sole carbon source. Photographs were taken after culturing at 30°C for 4 days. 1 and 2 represent two clones of each strain. (B) RT-PCR used to monitor the expression of MoGLK1, MoGLK1G69D and MoGLK1S156A in the ΔMoglk1 mutant, using ACTIN as control. Total RNA was isolated from mycelium cultured in CM liquid medium for 2 days. The experiments were performed in triplicate. (C) The ΔMoglk1 mutants transformed with wild-type MoGLK1, MoGLK1G69D and MoGLK1S156A were grown on MM agar plates containing 1% glucose and 100 mM ammonium. Photographs were taken at 9 days after incubation at 30°C. Three transformants for each strain were tested, and three experiments were performed and provided the same results. (D) G-6-P levels in mycelium of Guy11 and ΔMoglk1 mutant and ΔMoglk1 mutants transformed with wild-type MoGLK1, MoGLK1G69D and MoGLK1S156A in indicated medium conditions. Error bars indicate standard deviations from the mean. The experiments were performed in triplicate.
Figure 5.
Gene expression analysis from Guy11 and ΔMoglk1 mutants.
(A) Expression levels of the MoPMA1 gene in indicated conditions by semi-quantitative RT-PCR, using ACTIN as control. 1, MM+1% glucose+100 mM nitrate; 2, MM+1% glucose+100 mM ammonium; 3, MM+5% glucose+100 mM ammonium. (B) Relative transcript abundance of MoPMA1 of Guy11 and ΔMoglk1 mutant and ΔMoglk1 mutants transformed with wild-type MoGLK1, MoGLK1G69D and MoGLK1S156A grown in MM with 1% glucose and 100 mM ammonium for 3 days. Each profile was normalized against ACTIN and compared relative to the expression of MoPMA1 in Guy11. (C) qRT-PCR analysis of gene expression from Guy11 and ΔMoglk1 mutant grown for 36 h in CM liquid medium and MM liquid medium containing either 100 mM ammonium or 100 mM nitrate as the sole nitrogen source. Gene transcript levels were normalized to the level of ACTIN and compared relative to the expression of each gene in Guy11 grown in CM. Error bars indicate standard deviations from the mean. Two independent biological repeats were performed and three technical replicates of each repeat were analyzed.