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They were classified using pairwise blastp searches with a threshold E-value of CYTH4 of the three strains using the blastclust programme under strict conditions (i.e. a threshold of 60% identity and 70% length coverage). The 2554 (KT2440), 2533 (PAO1) and 2572 (Pf0-1) proteins were classified into 2454 conserved clusters in the three host strains. In contrast, 2016 (KT2440), 2468 (PAO1) and 2122 (Pf0-1) proteins were classified into a strain-specific cluster (Table?2). The relative quantification of pyoverdine levels was performed as follows: 24?h cultures grown in King's B medium (King et?al., 1954) were adjusted to OD600?=?1.0 before isolation of the cell culture supernatant. Absorbance spectra from 350 to 450?nm were measured using a DU800 spectrophotometer (Beckman Coulter, Fullerton, CA); only the maximal absorbance of pyoverdine at a wavelength of 403?nm is shown. The culture densities of pCAR1-free, pCAR1-containing and pCAR1��1- or pCAR1��2-containing strains in iron-deficient media were measured in 15?ml tubes (Corning, Corning, NY) cultivated at 30��C and 300 strokes min?1. Cell Cycle inhibitor The growth of each culture was assessed based on its turbidity at 600?nm (OD600); the initial cultures were adjusted to OD600?=?0.02. The culture densities were measured using a DU800 spectrophotometer (Beckman Coulter) at the early log phase (7.5?h for KT2440, 6.5?h for PAO1, and 4.5?h for the Pf0-1 after the inoculation; OD600?=?0.15�C0.25). pBBRCAR for constitutive expression of the CARDO system was prepared from the 6.8?kb EcoRI-SacI fragment containing the carAaAaBaBbCAcAd genes on pCAR1 inserted into pBBR1MCS-5 (Kovach et?al., find more 1995). The growth of each culture was also assessed based on its OD600; the initial cultures were adjusted to OD600?=?0.02. The culture densities were measured using a DU800 spectrophotometer (Beckman Coulter) at the early log phase (8?h for KT2440, 6?h for PAO1, and 7.5?h for Pf0-1 after the inoculation respectively; OD600?=?0.15�C0.25). Mixtures of 300?��l of each host culture with l-media and 3?ml of top agar (LB containing 0.7% agar) were poured onto separate LB plates. Next, 2?��l of filtered culture supernatant was spotted from each of the plasmid-free and plasmid-containing host strains onto the plates. The plates were then incubated at 30��C and examined for plaque formation. We thank Prof Dr M. Tsuda and Dr S. Yuhara of Tohoku University for their advice regarding our experiments. This work was supported by the Programme for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) of Japan. Fig. S1.