The Secret Dominate The Tryptophan synthase-Scene Is Rather Clear-Cut!

Their phenotypes are shown in Table?4. The cbrB mutant strain contains a mini Tn5-luxAB-Km insertion at a position 669?bp downstream of the translational start of CbrB. Pseudomonas putida strains were grown at 30��C in LB medium or in M9 minimal medium selleck screening library (Mandelbaum et?al., 1993), the latter containing sodium succinate (20?mM) as the carbon source and ammonium chloride (1?g?l?1) as the nitrogen source. For growth tests, 25?ml cultures in 100?ml flasks were inoculated at an initial A600 of 0.05, incubated in M9 minimal medium containing proline, arginine, ornithine, glutamate, histidine (20?mM) or tyrosine (5?mM) as carbon, nitrogen or carbon and nitrogen sources and were monitored. Growth was followed by measuring turbidity at 600?nm (A600) every 2?h for up to 21?h in some cases. For expression analysis, inocula were incubated with shaking (180?r.p.m.) at 30��C until they reached the exponential phase (A600?=?0.6). The cells were collected and resuspended in 100?ml of fresh M9 minimal medium containing a mixture of the 20 amino acids as carbon and nitrogen sources at A600?=?0.15. This composition allowed specific induction of the pathways for the uptake or assimilation of any of the amino acids assayed. The cultures were then incubated for 2?h for specific induction, and cells were then collected by centrifugation, frozen in liquid nitrogen and stored at ?80��C for RNA purification. Antibiotics were added, when necessary, at the following concentrations: 500??g?ml?1 carbenicilin, 25??g?ml?1 kanamycin, 5??g?ml?1 tetracycline and 20??g?ml?1 rifampicin. For solid media, Bacto-Agar (Difco, Detroit, MI) was added to a final concentration Tryptophan synthase of find more 15?g?l?1. The phenotype microarray analysis was carried out by the Biolog commercial service (Biolog, Hayward, CA) using the PM Kit (Biolog Catalog # 12191), which contains 20 microplates containing the same medium but different carbon or nitrogen sources (Bochner et?al., 2001). Phenotype microarray analysis use Biolog's redox chemistry, employing cell respiration as a universal reporter and the tetrazolium dye as redox indicator. The OmniLog instrument captures a digital image into kinetic graphs for 24?h. Differences in the plotted areas of redox activity monitored for 24?h between the wild type and cbrB mutant strains are quantified as scores. An aliquot (600??l) of overnight culture grown on LB medium was diluted and placed in a 1?ml spectrophotometric cuvette, and the absorbance was monitored over time. Sedimentation of the wild type and the cbrB mutant strains, estimated as the absorbance decay over time, is plotted in Fig.?3D. Cell pellets were resuspended in 1?ml of TriPure Isolation Reagent (Tri Reagent LS, Molecular Research Center, Cincinnati, OH) and incubated at 60��C for 10?min for complete cell lysis. The solution was centrifuged at 13?000 r.p.m. for 10?min at 4��C, and the pellet was discarded. The samples were transferred to a 2?ml tube of Phase Lock Gel (Eppendorf) and 0.