Figure 5.

Effect of aromatic amino acids on the protein levels of MepS and MepM. (A) Effect of the addition of aromatic amino acids on the growth of the mepS mepM double mutant in the M9 minimal medium. Indicated strains were serially diluted from 10⁸ to 10⁴ cells/ml in 10-fold steps and spotted onto a glucose M9 minimal medium plate (Glc MM) and a glucose M9 minimal medium plate containing tryptophan (W), phenylalanine (F), and tyrosine (Y) (10 mM each). (B) Effect of aromatic amino acids on the protein level of MepS. MG1655 MepS-Flag cells were grown to the early exponential phase (OD_600nm = 0.4) in the glucose M9 minimal medium containing 1% casamino acids or/and the indicated amino acids (10 mM each). Harvested cells (5 x 10⁷ cells) were used to determine the intracellular levels of MepS using an anti-Flag antibody. DnaK was used as the loading control. W, tryptophan; Y, tyrosine; F, phenylalanine; Q, glutamine. (C) Effect of aromatic amino acids on the protein level of MepM. MG1655 MepM-Flag cells were grown to the early exponential phase (OD_600nm = 0.4) in the glucose M9 minimal medium containing 1% casamino acids or/and the indicated amino acids (10 mM each). Harvested cells (5 x 10⁸ cells) were used to determine the intracellular levels of MepM using an anti-Flag antibody. (D) The mepS mutant phenocopies the mepS mepM double mutant only in the minimal medium containing aromatic amino acids. Indicated strains were serially diluted from 10⁸ to 10⁴ cells/ml in 10-fold steps and spotted onto a glucose M9 minimal medium plate, a glucose M9 minimal medium plate containing phenylalanine-tryptophan dipeptide (F-W) and phenylalanine-tyrosine dipeptide (F-Y) (each 3 mM), and a glucose M9 minimal medium plate containing 1% casamino acids and tryptophan (10 mM).