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. 2018 May 17;84(11):e00275-18. doi: 10.1128/AEM.00275-18

FIG 2.

FIG 2

Roles of DivL and CckA autophosphorylation in CtrA phosphorylation and RcGTA production. (A) Effect of the divL::aacC1 mutation on the CtrA∼P-dependent ghsAB promoter. The β-galactosidase (β-gal.) activities of SB1003, SB1003 divL::aacC1, and SB1003 ΔcckA containing the ghsAB promoter-lacZ reporter plasmid pXCA-ghsA are shown. (B) Amount of membrane-bound LH2 pigment in the culture supernatant of SBpG (WT), the RcGTA overproducer SBpG Δ280, and the SBpG Δ280-derived divL::aacC1 and ΔcckA double mutants. Representative curves are shown. (C) Effect of CtrA on divL promoter activity. The β-galactosidase activity from the chromosomally integrated divL promoter-lacZ reporter (pABW910) in SB1003, SB1003 ΔctrA, or the ΔctrA mutant (Δ) with nonphosphorylatable CtrA(D51A) or phosphomimetic CtrA(D51E) encoded on pD51A or pD51E, respectively, is shown. (D) Effect of the CckA(H399A) mutation on the CtrA∼P-dependent ghsAB promoter. The β-galactosidase activity from the chromosomally integrated ghsAB promoter-lacZ reporter (pABW848) in SB1003, SB1003 containing a ΔcckA mutation, and plasmid-encoded WT CckA (pRCckA) or ΔcckA and the predicted autophosphorylation-deficient CckA(H399A) (pCW104) is shown. (E) Effect of the CckA(H399A) mutation on RcGTA capsid production. The results for SB1003 or SB1003 containing a ΔcckA mutation and plasmid-encoded WT CckA (pRCckA) or ΔcckA and predicted autophosphorylation-deficient CckA(H399A) (pCW104) are shown. The immunoblot with proportionally equal loadings of the culture supernatant (extracellular) and cell pellet (intracellular) fractions was probed with anti-RcGTA capsid serum. Bars indicate means, and error bars indicate standard deviations for three biological replicates. *, a statistically significant difference, as follows: by a t test for the ghsAB promoter (P < 0.05) (A), by one-way ANOVA (F3,8 = 1,298; P < 0.0001) followed by Tukey's test (alpha = 0.05) (only selected significant results are indicated) (C), and by one-way ANOVA (F3,8 = 19.55; P = 0.005) followed by Tukey's test (alpha = 0.05) (D). n.s., not significant.