Research Paper Volume 6, Issue 7 pp 587—601

Figure 2. Stationary phase-specific pyruvate decarboxylases are regulated by Phx1. (A) The phylogenetic relatedness of various fungal PDC proteins. Amino acid sequences were aligned with ClustalW program, and a phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 5 program. A Bootstrap test was performed for 1000 replicates and the values were indicated at each node. (B) Expression levels of pdc101⁺, pdc102⁺, pdc201⁺, and pdc202⁺ genes in the wild type (JH43) and Δphx1 mutant (ESX5) at two growth phases. RNA samples were obtained from cells grown in EMM for 18 and 50 h for exponential and stationary phase cultures, respectively. The amounts of gene-specific mRNAs were estimated by qRT-PCR, along with that of act1⁺ mRNA as an internal control. Relative expression values to act1⁺ mRNA were obtained from three independent experiments, and were presented as an average with standard deviations. (C) Phx1-dependent PDC enzyme activity. Cell extracts were obtained from cells as described in (B). Pyruvate decarboxylase activity was measured as described in the text. Average values from three independent experiments were presented with standard deviations. (D) Effect of TPP addition on PDC activity. Experiments were done as in (C), except that TPP was added at 100 μM (final) to cell extracts.