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. 2020 Aug 10;202(17):e00345-20. doi: 10.1128/JB.00345-20

FIG 3.

FIG 3

CckN controls development by regulating CtrA activity. (a) β-Galactosidase assays were performed on wild-type (RH50), ΔdivJ ΔpleC (RH1103), and ΔdivJ ΔpleC ΔcckN (RH1111) strains harboring lacZ fusions to CtrA-dependent (PsciP, PCC1128, PCC2199, and PtacA) and CtrA-independent (PCC3574) promoters grown in complex medium (PYE) and normalized to the wild type (WT) (100%). Error bars indicate SD; n = 4. (b) The protein and the phosphorylation levels of CtrA were measured in wild-type (RH50), ΔdivJ ΔpleC (RH1103), and ΔdivJ ΔpleC ΔcckN (RH1111) strains and normalized to the WT (100%). The CtrA protein levels normalized to the MreB levels were determined by Western blotting. The CtrA phosphorylation levels were determined in vivo as described in Materials and Methods. The CtrA∼P/CtrA ratios were obtained by dividing black values by yellow values. Error bars indicate SD; n = 3. (c) Bacteriophage sensitivity assays were performed with CbK and Cr30 on wild-type (RH50), ΔpleC (RH217), ΔcckN (RH1106), ΔdivJ ΔpleC (RH1103), and ΔdivJ ΔpleC ΔcckN (RH1111) strains on PYE agar plates. (d) β-Galactosidase assays were performed on wild-type (RH50), ΔpleC (RH217), ΔcckN (RH1106), ΔdivJ ΔpleC (RH1103), ΔdivJ ΔpleC ΔcckN (RH1111), and ΔdivJ ΔpleC cckNH47A (RH1800) strains harboring PpilA::lacZ or PhvyA::lacZ fusions grown in complex medium (PYE) and normalized to the WT (100%). Error bars indicate SD; n = 3. (e) Buoyancy was evaluated by mixing 550 μl of Ludox LS colloidal silica (30%) with 1 ml of wild-type (RH50), ΔpleC (RH217), ΔcckN (RH1106), ΔdivJ ΔpleC (RH1103), and ΔdivJ ΔpleC ΔcckN (RH1111) strains grown in PYE. Then, the mix was centrifuged for 15 min at 9,000 rpm. (f) Cr30-dependent transduction efficiency was measured by transducing Cr30 lysates (LHR73 or LHR75) in wild-type (RH50), ΔpleC (RH217), ΔcckN (RH1106), ΔdivJ ΔpleC (RH1103), ΔdivJ ΔpleC ΔcckN (RH1111), and ΔdivJ ΔpleC cckNH47A (RH1800) strains grown in complex medium (PYE) and normalized to the WT (100%). Error bars indicate SD; n = 2. Means were statistically compared using a two-way ANOVA (a, b, and d) or a one-way ANOVA (f), followed by Tukey’s multiple-comparison test; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.