In the presence of PCA, PcaU acts as an activator for the transcription of the pca operon (Gerischer et al., 1998; Trautwein & Gerischer, 2001). In contrast, the reports on IclR-type repressors involved in the regulation of catabolic genes for aromatic compounds are limited to HmgR of P. putida U (Arias-Barrau et al., 2004), CatR of Rhodococcus erythropolis CCM2595 (Veselý et al., 2007), and PraR of Paenibacillus sp. strain JJ-1b (Kasai et al., 2009), which negatively regulate the homogentisate pathway genes, the catechol ortho-cleavage pathway genes, and the PCA 2,3-cleavage pathway
genes, respectively. Among these IclR-type repressors, only the research of the HmgR showed the binding of this repressor to the operator. Here, we focused on the regulation of iphACBDR operon controlled
by an IclR-type repressor, IphR. This Apoptosis Compound Library clinical trial is the first report to determine the transcription start site of iph operon, binding region of IphR, and effector molecule of IphR. Comamonas sp. strain E6 and its SCH772984 manufacturer mutants, DEIR and DEIA (Fukuhara et al., 2010) were grown in Luria–Bertani (LB) medium or in 0.2× LB medium at 30 °C. When required, 50 mg of kanamycin/liter or 30 mg of chloramphenicol/liter were added to the media. Escherichia coli strains JM109 and BL21(DE3) were grown in LB medium at 37 °C. For cultures of E. coli cells carrying antibiotic resistance markers, the media were supplemented with 100 mg of ampicillin/liter or 25 mg of kanamycin/liter. A set of deletion plasmids of pZSH2 (Fukuhara et al., 2010), pZSM1, pZSP08, pZSN06, pZSNE530, pZSNE347, and pZSNE198, was constructed by deletion using restriction enzymes or a Kilosequence kit (Takara Bio Inc.). To construct pZ347, pZ284, pZ274, and pZ255, the DNA fragments amplified by PCR using specific primer pairs (Supporting Information, Table Quisqualic acid S1) and pKS24 (Fukuhara et al., 2010) as a template were cloned into a promoter probe vector pPR9TZ (Kamimura et al.,
2010). Nucleotide sequences of the insert fragments were determined by the dideoxy termination method using a CEQ2000XL genetic analysis system (Beckman Coulter Inc.) The lacZ reporter plasmids were introduced into cells of E6 and DEIA by the triparental mating procedure. Cells of E6 and DEIA harboring each reporter plasmid pre-grown in 0.2× LB medium containing chloramphenicol were inoculated into the same fresh medium to an absorbance at 600 nm of 0.2. After 90 min of incubation at 30 °C, 5 mM IPA was added, and the cultures were incubated for another 120 min. The cells were washed twice with 20 mM Tris-HCl (pH 8.0) and resuspended in the same buffer, and broken by ultrasonication. The supernatant was collected by centrifugation (19 000 g, 15 min, 4 °C) and used as a crude enzyme. The β-galactosidase activities were measured using 4-methylumbelliferyl-β-d-galactopyranoside (Kamimura et al., 2010). The protein concentration was determined by the Bradford method (Bradford, 1976).