The minimal bactericidal concentrations
(MBC) were expressed as the range a–b, in which a corresponds to the highest concentration in which bacterial growth was observed and b corresponds to the lowest concentration that kills GPCR Compound Library supplier 100% of the cells. Three biological replicates were used for the determination of MBC. Total RNA was isolated from mid-log suspensions of the strain 9a5c incubated or not with 50 μM of gomesin at 28 °C for 15, 30 and 60 min using Trizol reagent (Invitrogen, Carlsbad, CA). The RNA concentration was determined using an ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE) after treatment with RQ1 RNAse-free DNAse (Promega, Madison, WI), and its reliability was evaluated by electrophoresis on formaldehyde-agarose gels. cDNA labeling and hybridization of microarray slides were performed as described previously (Zaini et al., 2008). A detailed description of the microarray can be found in Koide et al. (2004) and Zaini et al. (2008) and at the NCBI’s Gene www.selleckchem.com/products/poziotinib-hm781-36b.html Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo) under accession number GPL2708. Data represent six biological replicates with at least two technical replicates each. Microarray data acquisition, normalization and analysis were performed as detailed by Koide et al. (2004). A gene was considered differentially expressed when >66.5% of the biological replicates were
outside the intensity-dependent cutoff curves obtained by self–self hybridization experiments (Koide et al., 2004; Zaini et al., 2008) and exhibited a fold-change >2.0. The complete data set has been submitted to the GEO database according to MIAME guidelines and is accessible through GEO series accession number GSE17605 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17605).
Forskolin cell line RT-qPCR was performed in the GeneAmp 5700 thermocycler (Applied Biosystems, Carlsbad, CA) as described previously (Zaini et al., 2008). Data from two biological replicates with three technical replicates were used to calculate the relative changes in coding sequence (CDS) expression levels as described (Zaini et al., 2008). Mid-log suspensions of the strain 9a5c of X. fastidiosa were transferred to glass flasks and incubated with 50 μM gomesin, streptomycin 1 μg mL−1 or water as control. After 4 days at 28 °C, the walls of the glass flasks were examined to evaluate biofilm production as described (Kjaergaard et al., 2000). Two independent biological experiments were assayed in triplicate. Mid-log suspensions of strain 9a5c or strain J1a12 of X. fastidiosa were incubated with 25 or 50 μM gomesin or water as a control. After 18 h at 28 °C, cells were harvested by centrifugation at 3000 g for 10 min at 25 °C, washed twice in sterile phosphate-buffered saline (PBS) and suspended in PBS. The resulting bacterial suspensions were used to mechanically inoculate Nicotiana clevelandii plants as detailed (Lopes et al., 2000).