Rapidly Solutions For Tryptophan synthase Troubles

As shown in Fig.?1C, in the presence of iron, the cbrB mutant showed a longer lag phase in this minimal medium, but grew at the same rate as the wild type (doubling Selleck Alectinib time of 60 and 67?min for the wild type and cbrB mutant respectively). Pseudomonas putida is a very efficient iron scavenger and can grow efficiently in minimal medium with no iron added (growth rate 70% of that with iron added), and reached the same cell density as the culture with iron added. However, the cbrB mutant was clearly more sensitive to iron limitation as it grew very slowly and reached half of the cell density of the culture with iron added (after 25?h, not shown). This result suggests that it has diminished iron scavenging capability. We showed above that several genes from the flagellar motility/chemotaxis cluster are differentially expressed in the presence of the cbrB mutation. To test whether these changes in expression lead to alterations in motility behaviour, we performed swimming motility assays using minimal medium soft agar plates, as described previously (Robleto et?al., 2003). The results are shown in Fig.?2. The cbrB mutant strain showed about a 10-fold decrease as compared with wild type in swimming motility through M9 minimal medium soft agar, which was supplemented with 0.2% proline as the sole carbon and nitrogen source. Because the cbrB mutant exhibits slow growth in these conditions (Table?1, Fig.?S1), a limited Tryptophan synthase energy supply may be at least partly responsible for the observed defect. In order to avoid effects due to the growth rate, the same assay was also performed in soft agar medium http://www.selleckchem.com/PI3K.html supplemented with succinate and ammonium as a carbon and nitrogen source, respectively, which support growth of the cbrB strain at the same rate as the wild type (Fig.?S1). Again, the cbrB mutant strain showed a clear, although somewhat smaller (2.5-fold), decrease in motility as compared with the wild type. The fleQ mutant used as a control was non-motile in both media. Assayed this way, the altered motility phenotype of the mutant strain may be due to a defect in flagellar structure or function, impaired chemotaxis or both. In order to discriminate between these possibilities, we used microscopy to visualize the flagella of the cbrB mutant and wild type strains. Flagella staining and visualization revealed that P.?putida KT2442 has several polar flagella, and the frequency of flagellated cells in the cbrB mutant is similar to that in the wild type (see Fig.?S2). Confocal laser scanning microscopy and immunofluorescent staining of the flagellum with anti-flagellin antiserum failed to reveal significant differences in flagellar number or structure between the wild type and the cbrB mutant (Fig.?S2). In addition, phase contrast microscopy of fresh cells grown in LB revealed that both strains were similarly motile (not shown), thus indicating that flagella are functional in the cbrB strain.