Journal: Journal of Bacteriology
Article Title: Protein Modulator of Multidrug Efflux Gene Expression in Pseudomonas aeruginosa ▿
Figure Lengend Snippet: PA3719 and MexR control of in vitro mexA transcription. (A) Impact of PA3719 on MexR repression of mexA transcription. Phage T7 RNA polymerase-based in vitro transcription reactions were initiated from a 836-bp PCR-generated template (250 ng) carrying a T7 promoter and mexA sequences extending from the MexR operator to 585 bp into the mexA gene (lanes 1 to 4). The results for control reactions with a T7 promoter-containing DNA template, pTRI-Xef, provided with the T7 in vitro transcription kit, are shown in lanes 5 to 7. Reactions were carried out at 37°C for 3 h, and RNA was purified and subjected to electrophoresis prior to visualization with ethidium bromide. MexR (1 μM) and PA3719 (150 nM [+ 1 ] or 500 nM [+ 2] ) were included in the reaction mixtures as indicated. Equal volumes of RNA were loaded in each instance. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 1.1 μg; lane 3, 2.0 μg; lane 4, 4.2 μg; lane 5, 1.7 μg; lane 6, 1.6 μg; and lane 7, 1.6 μg. (B) Impact of PA3719 on mexA transcription. In vitro transcription of the mex (lanes 1 to 3) and pTRI-Xef (lanes 4 to 6) templates was carried out as described above with or without PA3719 (500 nM [+ 2 ] or 2 μM [+ 3 ]), as indicated. The following amounts of RNA were produced: lane 1, 4.4 μg; lane 2, 4.5 μg; lane 3, 4.6 μg; lane 4, 4.6 μg; lane 5, 4.6 μg; and lane 6, 4.4 μg. (C) Differential impact of PA3719 on the repressor activities of MexR and MexR I104F . In vitro transcription of the mex (lanes 1 to 5) and pTRI-Xef (lanes 6 to 8) templates was carried out as described above with or without PA3719 (500 nM) and MexR/MexR I104F (1 μM), as indicated. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 0.8 μg; lane 3, 3.8 μg; lane 4, 0.9 μg; lane 5, 1.9 μg; lane 6, 6.6 μg; lane 7, 6.5 μg; and lane 8, 6.4 μg. RNA was quantitated prior to loading, and the values reported reflect the amount loaded and not the total amount produced. Because reaction, recovery, and loading volumes were kept constant throughout, these values accurately reflect the relative transcript levels produced in each instance.
Article Snippet: Annealing was achieved by incubating 800 pmol of each oligonucleotide in T4 ligation buffer at 95°C for 3 min and allowing the reaction mixture to cool at room temperature for 5 h, after which the 3′ PA3719 DNA was purified using a gel and PCR cleanup kit (Promega).
Techniques: In Vitro, Polymerase Chain Reaction, Generated, Purification, Electrophoresis, Produced