The protein concentration was 4C5 M
The protein concentration was 4C5 M. growth, as well as of new drug targets, is usually a prominent question. J2315 possesses four QS systems composed by a synthase (I) and a receptor (R): CepIR, CciIR, CT5.1 the Diffusible Signal Factor (BDSF)-based system RpfFBC, and the recently discovered non-ribosomal peptide synthetase-like cluster (Coenye, 2010; Spadaro et al., 2016; Jenul et al., 2018). The characterization of mutants lacking the synthases CepI and/or CciI and RpfFBC exhibited an involvement of CepI in biofilm formation, protease production, and virulence, as well as an interplay among the Acyl Homoserine Lactone (AHL) systems CepIR and CciIR and the BDSF-based system (Udine et al., 2013). We recently identified new diketopiperazine molecules, able to inhibit CepI to produce proteases, siderophores, and to form biofilm (Scoffone et al., 2016). These molecules did not possess any antimicrobial activity, nevertheless their administration significantly increased the survival of nematodes infected with (Scoffone et al., 2016). The current lack of molecular structure data on CepI prevents the possibility of 3D structure-assisted optimization studies of these new inhibitors. In our previous work, we generated a CepI homology model, and used it to perform docking analyses of the diketopiperazine inhibitor 8b (Physique ?Physique11) (Supplementary Materials and Methods). Using this approach, we identified multiple candidate binding sites, localized far from the enzyme catalytic site, but in regions possibly still implicated in substrate recognition and catalysis (Scoffone et al., 2016). Open in a separate window Physique 1 Chemical structure of the (3S)-3-Benzyl-6-(3,6-dioxocyclohexa-1,4-dien-1-yl)piperazine-2,5-dione (8b) CepI inhibitor. Here, we confirmed that this cellular effects of 8b are indeed related to the inhibition of CepI, by analyzing the proteome of cells treated with the compound, and compared with that of the knock-out strain. Moreover, we exploited a site-directed mutagenesis strategy to better define the crucial amino acid residues responsible for catalysis and recognition of the 8b inhibitor. Taken together, our results suggest a possible mechanism of CepI inhibition by the 8b compound through perturbations of a flexible loop involved in recognition and stabilization of the BL21(DE3) cells and purified as previously described (Scoffone et al., 2016). Far-UV circular dichroism (CD) measurements were performed with a JascoJ-700 spectropolarimeter (Jasco-Europe, Cremella, Italy) using a 1 mm path cell. Scans were conducted between 190 and 250 nm at a velocity of 50 nm/min with a spectral band width of 2 nm and a sensitivity of 20 mdeg. CD spectrum measurements were performed at 25C in 50 mM sodium phosphate pH 8.0, 50 mM KCl, and represent the average of 10 scans. The protein concentration was 4C5 M. Spectra were analyzed using the DichroWeb online platform (Whitmore and Wallace, 2008). CepI activity was decided according to Christensen et al. (2013). Reaction mixtures contained 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5, 0.005% Nonidet P-40, 0.13 mM 2,6-dichlorophenylindophenol Tubacin (DCPIP), 70 M Octanoyl-ACP (C8-ACP, prepared as reported previously) (Quadri et al., 1998; Cronan and Thomas, 2009), 40 M (Udine et al., 2013) and J2315 (with or without 25 M of 8b compound) were produced in 10 ml of LB medium until Tubacin OD600 nm > 2. Cells were then harvested, resuspended in 0.2 ml of Tris-HCl 50 mM, pH 7.5, disrupted by sonication, and centrifuged at 12,000 rpm for 1 h at 4C. The amount of proteins present in the supernatant was quantified by bicinchoninic acid method (Smith et al., 1985), then 300 g were precipitated with 10% (v/v) trichloroacetic acid. The obtained protein pellet was dissolved in 125 L of rehydration buffer (8 M Tubacin urea, 4% CHAPS (w/v), 65 mM.