Fig. 4.

Characterization and functional analysis of the promoter region and the 5′UTR of the met operon. (a) Characterization of the promoter. The small green arrow represents the promoter (P₁₂₅₁) of the operon. TSS represents the identified transcriptional start site. The promoter-containing DNA sequences used for the construction of reporter plasmids are shown in green, while the 5’UTR sequence is in magenta. The −10/–35 region and the Shine-Dalgarno sequence (SD) are indicated. (b) Functional analysis of the promoter. The upper part shows the genetic organization of the reporter plasmids pP1251L and pP1251L. The lower part shows the GUS activities produced by the reporter strains 8004/pP1251L (pP1251L) and 8004/pP1251S (pP1251S), as well as the negative control strain 8004/pNPgusA (pNPgusA) grown for 72 h in MMX alone (Met−) and MMX with 1 mM methionine (Met+). Data are from a representative experiment, and similar results were obtained in two other independent experiments. The letters above the columns represent no significant difference at P=0.05 by t-test. (c) and (d) Functional analysis of the 5′UTR. The upper part shows the genetic organization of the 5′UTR-gusA transcriptional fusion reporter plasmid (p5UTR-SD⁺) and the translational fusion reporter plasmid (p5UTR-SD⁻). The lower part shows the GUS activities produced by the reporter strains 8004/p5′UTR-SD⁺ (p5UTR-SD⁺) and 8004/p5′UTR-SD⁻ (p5UTR-SD⁻) grown in MMX alone (Met−) and MMX with the addition of 1 mM methionine (Met+) or 1 mM glycine (Gly+). The asterisks above the columns represent significant difference at P<0.05 by t-test. The data in blue and black above the columns are the absolute and relative values, respectively. One unit of GUS activity was defined as milligrams (mg) of ρ-nitrophenol released from ρ-nitrophenyl β-d-glucuronide per minute per ml of bacterial culture (cell density: OD₆₀₀=1.0). Data presented are from a representative experiment, and similar results were obtained in two other independent experiments.