These results clearly indicated that both IR2 and the downstream half-site of IR1 are necessary for the binding of IphR. The requirement of an additional half-site with a palindrome is uncommon in regulator binding sites, but the PcaU binding region is known to contain three perfectly conserved 10 bp repeats (R1, R2, and R3), which form a palindrome (R1 and R2) and a direct repeat (R3) located 11 bp downstream of the palindrome (Popp et al.,

Bortezomib solubility dmso 2002; Jerg & Gerischer, 2008). R3 is a repetition of the half-site of the palindrome (R2) proximal to R3. The binding region of an IclR-type repressor, HmgR, also contains a 17-bp perfect palindromic motif and a 6-bp direct repetition of the palindrome (Arias-Barrau et al., 2004). However, the direct repeat motif located 4 bp upstream of the palindrome is

a repetition of the half-site of the palindrome distal Gefitinib price to the direct repeat motif. Although there was no obvious sequence similarity between the binding regions of IphR and HmgR, and the downstream half-site of IR1 is not a perfect direct repeat of the downstream half-site of IR2, the arrangement of both binding regions appeared to be similar; positions of the palindrome and additional repeat each overlap the transcription start site and −10 region, respectively. IPA and/or its metabolite were suggested to be an inducer of the iph operon by the analyses of promoter and primer extension. We examined the ability of IPA and its analogous substrates: phthalate, TPA, PCA, and 3-hydroxybenzoate (100 µM) to inhibit the ht-IphR binding to the IPH-60 fragment by EMSA. Among these substrates, only IPA abolished the binding of ht-IphR (Fig. 4). In addition, the iphA promoter activity of iphA mutant (DEIA) cells harboring reporter plasmid pZSH2, which accumulates IPA during incubation with IPA, was increased ca. 90-fold (21 ± 2.0 mU mg−1) GPX6 in the presence

of IPA. These results indicated that IPA itself is the specific effector that modulates IphR binding to the operator, acting as an inducer of the iph operon. IphR negatively autoregulates the transcription of IPA catabolic operon, iphACBDR, in E6. In the absence of IPA, IphR binds to the operator region containing an inverted repeat (IR2) and a downstream half-site of another inverted repeat (IR1) to repress the transcription of iph operon. Although further analysis is necessary to clarify the manner of binding of IphR, this regulator protein might bind to the operator as a dimer of dimers, as ht-IphR was suggested to mainly form a dimer in solution. N.K. and K.I. contributed equally to this work. This study has no conflict of interest between authors. “
“The goal of this study was to develop and validate a novel fosmid-clone-based metagenome isotope array approach – termed the community isotope array (CIArray) – for sensitive detection and identification of microorganisms assimilating a radiolabeled substrate within complex microbial communities.