Fig 2. Biofilm production, Pel EPS expression, H1-T6SS production and T3SS expression in the LadS signaling pathway.

The pBBRladS plasmid containing the ladS HK gene (dark bars) and the pBBRMCS4 corresponding empty cloning vector (light bars) were conjugated in the PAK, PAKΔrsmY, PAKΔrsmZ or PAKΔrsmYΔrsmZ strains. (A) 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. (B) Activity of the pelA–lacZ transcriptional chromosomal fusion was monitored in the same strains with the pBBRladS plasmid containing the ladS HK gene (dark violet bars) and the pBBRMCS4 corresponding empty cloning vector (light violet bars) after 4 hours of growth (OD_600nm≈3.5). Corresponding β-galactosidase activities are expressed in Miller units and correspond to mean values (with error bars) obtained from three independent experiments. (C) Production of the H1-T6SS VgrG1s proteins was detected in whole cell extracts using western blot with an anti-VgrG1 polyclonal antibody. Numbers on the left side correspond to molecular weight standards (kDa). (D) Activity of the exoS–lacZ transcriptional chromosomal fusion was monitored in the same strains with the pBBRladS plasmid containing the ladS HK gene (dark royal blue bars) and the pBBRMCS4 corresponding empty cloning vector (light royal blue bars) after 6 hours of growth (OD_600nm≈4). Corresponding β-galactosidase activities are expressed in Miller units and correspond to mean values (with error bars) obtained from three independent experiments. Wilcoxon-Mann-Whitney tests were performed and *, **, *** and ns referred to p<0.05, p<0.01 and p<0.001 and nonsignificant difference, respectively.