1. DAPA, EMSA, and ChIP-PCR analyses demonstrating in vitro and in vivo interaction of GlnR with the GlnR boxes in pureI.
DAPA, EMSA, and ChIP-PCR analyses demonstrating in vitro and in vivo interaction of GlnR with the GlnR boxes in pureI. (A) DAPA demonstrating specific interaction between GlnR and the putative GlnR boxes. A 1-µg amount of the total cell lysate of S. salivarius 57.I was used in the input control (I). Amounts of 500 µg of the total cell lysate were incubated with biotin-labeled probes specific for GlnR box 1 (an annealing product of GlnR_box-1_S and GlnR_box-1_AS) and box 2 (an annealing product of GlnR_box-2_S and GlnR_box-2_AS), respectively (II), and with probes with mutated bases in the putative GlnR boxes (annealing products of mGlnR_box-1_S plus mGlnR_box-1_AS and mGlnR_box-2_S plus mGlnR_box-2_AS) (III). The GlnR protein in the reaction mixtures was detected by immunoblotting with anti-GlnR antiserum. (B) EMSA demonstrating interaction between GlnR and the putative GlnR box 1 (lanes 1 to 5) and GlnR box 2 (lanes 6 to 10) of pureI. Lanes 1 and 6, probe only; lanes 2 to 4 and 7 to 9, 0.2 to 0.8 µM MalE-GlnR in 2-fold increments; lanes 5 and 10, 0.8 µM MalE-GlnR with a specific competitor. All reactions were carried out with 0.01 pmol biotin-labeled probe. (C) ChIP-PCR assay demonstrating in vivo interaction between GlnR and pureI. (I) Input control; (II) PCR product obtained from a reaction mixture containing anti-GlnR antiserum; (III) final result from a reaction with preimmune rabbit serum.
Szu-Chuan Huang, and Yi-Ywan M. Chen mSphere 2016; doi: /mSphere