Figure 4
The function of PP4397 in motility control is dependent on its c-di-GMP binding ability. Relative swimming motility of the double PP2258/PP4397 null derivative of P. putida KT2701 on 0.3% soft agar LB plates supplemented with carbenicillin. (A) Strains harbouring either a vector control (Vec. Cont.; pVI2300) or lacIQ/Ptac expression plasmids for derivatives of PP4397: c-di-GMP binding proficient wild type PP4397 (WT, pVI2301), or c-di-GMP binding deficient mutants R127A (pVI2302) or G162A (pVI2303). (B) Strains harbouring either a vector control (Vec. Cont.; pVI2300) or lacIQ/Ptac expression plasmids for FLAG-tagged derivatives of PP4397: c-di-GMP binding proficient wild type PP4397 (WT, pVI2304), or c-di-GMP binding deficient mutants R127A (pVI2305) or G162A (pVI2306). Graphed values are averages with standard deviations calculated from three independent colonies. Experiments were normalized by setting the values of the double mutant harbouring the vector control as 1 and P-values calculated with two-tailed student t-test (***P < 0.001). Images of representative swim rings are shown above the graphed values. The insert in panel B shows Western analysis of the FLAG-tagged PP4397 derivatives present in 10 µg of crude extract from the same cells. The cropped Western analysis image is derived from the same gel and is shown alongside molecular size markers in Fig. S4A.
