DNA from the deletion strains did not hybridize with the gene probe, and showed the expected size decrease when probed with the gene’s upstream region. Since the deletions in both parent strains S9 and R1 exhibited the same phenotype, they will be discussed together in the following sections. As independent biological replicates, the use of two parent strains gives a high degree of certainty for the phenotypic findings. OE2401F and OE2402F are essential for chemotaxis and phototaxis To examine the effect of RG7112 solubility dmso the deletions
on chemotaxis and motility, the deletion strains were analyzed by swarm plate assays. A swarm plate is a semi-solid agar plate in which the cells are inoculated. The agar Y-27632 datasheet concentration is low enough to allow movement of the cells in the agar. After point inoculation the cells grow, metabolize various nutrients, and create a concentration gradient. Cells which are motile and capable of chemotaxis move along this gradient away from the inoculation site, forming extended rings, called swarm rings. Figure 3 shows representative swarm plates for each
deletion in S9, compared to wildtype (see Additional file 3 for all swarm plates). After three days of growth, the wild type strains formed large swarm rings. The deletion strains Δ1, Δ2, and Δ2–4 did not show any swarming. Δ4 cells produced swarm GSK3235025 rings, but of a reduced size. Figure 3 Swarming ability of the deletion strains. Representative swarm plate for each deletion in S9 after three days of growth at 37°C. Reduced
or impaired ring formation on swarm plates can be due to defects in signal transduction or flagellar motility. In order to determine the defects of the deletion strains, PtdIns(3,4)P2 their swimming ability was evaluated by microscopy, and the frequency of reversal of their swimming direction was measured with a computer-based cell-tracking system (Figure 4; see Additional file 4 for details). This system automatically determines the rate of reversing cells over a certain observation time . Figure 4 Reversals of the wild type and deletion strains as measured by computer-based cell-tracking. The percent reversal in a 4 second interval was determined either without stimulation (spontaneous, gray bar), after a blue light pulse (blue bar), or after a step down in orange light (orange bar). Error bars represent the 95% confidence interval. The dashed line indicates the estimated maximal tracking error of 5%. Two clones of each deletion strain were measured, except for R1Δ4 and R1Δ2–4. Visual inspection clearly demonstrated that all deletion strains were motile without detectable swimming defects. The wild type strains showed in a 4 s observation interval a reversal rate of 10% (R1) and 25% (S9) in the unstimulated state.