Figure 2.

Activity and processing of constitutively active truncated IutY variants. (A) Schematic representation of the P. putida sigma/anti-sigma hybrid protein IutY drawn to scale. The cytosolic sigma factor domain, transmembrane (TM) domain, and periplasmic anti-sigma factor domain are shown. The number of amino acids of the various truncated variants tested are indicated. (B) β-Galactosidase activity of the P. putida KT2440 wild-type strain (black bars) and its isogenic Δprc (black striped bars) or ΔrseP (white striped bars) mutants bearing the transcriptional fusion iutA::lacZ (pMPK4 plasmid) and a pMMB67EH-derivate expressing one of the indicated truncated IutY variants grown in LB supplemented with 1 mM IPTG. (C) β-Galactosidase activity of the P. putida ΔiutY mutant bearing the transcriptional fusion iutA::lacZ (pMPK4 plasmid) and the pMMB67EH (empty), pMMBK1-HA (WT), pMMBK1-HA-168, -201, -236, or -365 construct expressing one of the IutY variants grown in low iron medium supplemented with 1 mM IPTG in the absence (light gray bars) or presence (dark gray bars) of aerobactin. (D) The P. putida KT2440 wild-type strain (WT) and its isogenic Δprc or ΔrseP mutant expressing one of the indicated truncated IutY variants were grown in LB supplemented with 1 mM IPTG. Proteins were detected by Western-blot using an anti-HA antibody. Position of the full-length truncate protein is indicated by an arrow. The protein band corresponding to the σ^IutY domain and the molecular size marker (in kDa) are also indicated. Vertical dotted line indicates combination of two separate images to show prolonged exposure of the HA-IutY wild-type protein blot, which was not visible in this condition.