Figure 7. CleA is activated by c-di-GMP binding but not phosphorylation.

(a) Schematic of the domain structure of CleA with mutations that interfere with potential phosphorylation control (green) or c-di-GMP binding (red). The domains are labeled as in Figures 2, 3 and 4. (b-c) CleA is activated by c-di-GMP binding. Single cell analysis of reversal frequencies and mean speed of C. crescentus wild type andΔcleA mutants harboring plasmid driven cleA alleles as indicated in (a). cleA alleles were expressed from a xylose-dependent promoter and were induced with 0.1% xylose for 3 hr before imaging. Experiments in (b) and (c) include the analysis of >850 cells for each strain.

Figure 7—figure supplement 1. CleA and CleD are activated by c-di-GMP binding but not phosphorylation.

(a) Functionality of CleA variants. Analysis of reversal frequencies and mean speed of strains harboring different cleA alleles. Box plots are as in Figure 5—figure supplement 1b,c. All strains harbored a deletion in pilA encoding the major pilin subunit. Median reversal frequencies and speeds are from trajectories of over 950 cells (pMT687 (WT) = 1139 cells; ΔcleA pMT687 = 1486 cells; ΔcleA pMT687-CleA = 1158 cells; ΔcleA pMT687-CleA_RR153AA = 1134 cells; ΔcleA pMT687-CleA_R167A = 967 cells; ΔcleA pMT687-CleA_D67A = 1095 cells; ΔcleA pMT687-CleA_R111A = 1848 cells) combined from two independent experiments. (b) CleD is activated by c-di-GMP binding. Spreading in semisolid agar plates was analyzed as described in the legend of Figure 5a. Strains analyzed are indicated below the graph. Different cleD alleles were expressed from a plasmid-born xylose-dependent promoter in aΔcleDΔpdeA background as indicated. The schematic on the right indicates the position of cleD mutations interfering with potential phosphorylation control (green) or c-di-GMP binding (red). The data include five biological replicates. Bars are average spreading areas with standard deviations indicated.