As shown in Figure 7, the culture of JG1172 was dominated by filamentous cells, whereas JG1172 cells expressing the wild-type fliX gene had normal cell morphology. All fliX mutants, except fliX L85K (Figure 7), were able to restore the normal pattern of cell division in JG1172 cells. Once more, fliX
L85K displayed no activity in complementing a physiological defect in ΔfliX cells. Figure 7 Allele fliX L85K was unable to rescue the cell division defect of JG1172. Cells harvested https://www.selleckchem.com/products/bay-57-1293.html from overnight cultures were mounted on poly-L-lysine coated slides and examined by differential interference contrast (DIC) microscopy. Role of conserved FliX residues in interaction with FlbD Based on previous findings [35, 37] and the new evidence of this study (Figure 1), it has been conclusively established that FliX and FlbD bind each other both in vivo and in vitro. The above genetic analyses revealed that although the cellular contents of FliXΔ117-118 and FliXL85K were similar in a ΔfliX background, they exerted distinctive effects on FlbD activity. Their ability to interact with FlbD must have played a role. To test this idea, we performed an immunoprecipitation experiment in which cell extracts of Caulobacter were treated with agarose beads coated with anti-FlbD antibodies, and elutes
from the beads were probed with anti-FliX antibodies. Rebamipide As presented TH-302 price in Figure 8, mutants FliXR71A, FliXT130L, and FliXL136K interacted as well with FlbD as wild-type FliX did, if their cellular contents (Figure 4) were taken into consideration. In spite of the fact that FliXL85K, FliXΔ117-118, and FliX 1 were maintained at similar protein levels in JG1172 cells (Figure 4, lanes 13, 14, and 17), the precipitated amounts
of these proteins were dramatically different (Figure 8, lanes 6, 7, and 10). Abundant FliX 1 and a small amount of FliXΔ117-118 were obtained, whereas no detectable level of FliXL85K was observed. This indicates that FliX 1 has a strong association to FlbD, FliXΔ117-118 binds to FlbD with a low affinity, and FliXL85K no longer interacts with FlbD. Since a large amount of FliX 1 was successfully precipitated, the results of this experiment reflect a lack of interaction between FlbD and FliXL85K rather than a lack of FliXL85K protein in the cell extracts. Figure 8 Co-immunoprecipitation of FlbD and the FliX mutants. Extracts of JG1172 cells expressing various fliX alleles were incubated with agarose beads coated with anti-FlbD antibodies. Proteins bound to the bead complexes were detected using anti-FliX antibodies following SDS-PAGE electrophoresis. The immunoblot was developed to an extended period of time to Ilomastat visualize the band of FliXΔ117-118 (indicated by the arrow).