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. 2016 May 13;12(5):e1006032. doi: 10.1371/journal.pgen.1006032

Fig 6. Involvement of the H2 domain of the GacS unorthodox HK in the LadS signaling pathway.

Fig 6

The pBBRladS plasmid containing the ladS HK gene and the pBBRMCS4 corresponding empty cloning vector were conjugated in the PAKΔgacS strain in which the gacSH2 or gacSH2H→Q gene versions or the corresponding suicide vector were chromosomally integrated at the Tn7 site. (A) RsmY (blue bars) and RsmZ (brick-red-colored bars) transcript levels were monitored using RT-qPCR and fold induction was presented in the strains PAKΔgacS::miniTn7gacSH2 (gacSH2) and PAKΔgacS::miniTn7gacSH2H→Q (gacSH2H→Q) as compared to the PAKΔgacS::miniTn7 strain (miniTn7). (B) Biofilm production in glass tubes was illustrated (upper panel) and quantified after crystal violet-staining (lower panel). Corresponding levels of biofilm production represent mean values and standard deviations obtained from three independent experiments. Wilcoxon-Mann-Whitney tests were performed; ** and ns referred to p<0.01 and nonsignificant difference. C. PelA (violet bars) and ExoS (royal blue bars) transcript levels were monitored using RT-qPCR and fold induction was presented in the strains PAKΔgacS::miniTn7gacSH2 (gacSH2) and PAKΔgacS::miniTn7gacSH2H→Q (gacSH2H→Q) as compared to the PAKΔgacS::miniTn7 strain (miniTn7). (D) Production of the H1-T6SS Hcp1 proteins was detected in whole cell extracts using western blot with an anti-Hcp1 polyclonal antibody. Numbers on the left side are molecular weight standards (kDa). Moderated t-tests were performed and *, **, *** and ns referred to p<0.05, p<0.01 and p<0.001 and nonsignificant difference, respectively. (E) Transcript levels of RsmY (blue bars), RsmZ (brick-red-colored bars), VgrG1 (green bars), PelA (violet bars) and ExoS (royal blue bars) were monitored using RT-qPCR. Fold induction was presented in the strains PAK, PAKΔladS, PAKgacSH1H→Q, PAKgacSH1H→QΔladS, PAKgacSH1H→QH2H→Q and PAKgacSH1H→QH2H→QΔladS in order to disable autophosphorylation of the GacSH1 domain and the functionality of the GacsH2 domain, respectively. Moderated t-tests were performed and *, **, *** and ns referred to p<0.05, p<0.01 and p<0.001 and nonsignificant difference, respectively.