Fig 1.
The GacSA-RetS-PA1611-LadS signaling network.
(A) Current model for the regulatory elements influencing the expression of two sRNAs, RsmY and RsmZ. See text for details. (B) The multicomponent signal transduction system made of the LadS hybrid HK and the GacS/GacA TCS. In the presented model sustained by results obtained in the present study, this multicomponent signal transduction system made of the LadS hybrid HK and the GacS/GacA TCS forms a multiple-input system probably reflecting the variability of environmental conditions P. aeruginosa is faced with and may result in a range of gradations of chronic infection. IM (Inner Membrane), P (Periplasm), C (Cytoplasm).
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 (OD600nm≈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 (OD600nm≈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.
Fig 3.
Role of the GacS/GacA TCS 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, PAKΔgacS or PAKΔgacA strains. (A) Activities of the rsmY–lacZ (left panel, blue bars) and rsmZ–lacZ (right panel, brick-red-colored bars) transcriptional chromosomal fusions were monitored after 6 hours of growth (OD600nm≈4) and corresponding β-galactosidase activities are expressed in Miller units and correspond to mean values (with error bars) obtained from three independent experiments. (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 *, **, *** and ns referred to p<0.05, p<0.01 and p<0.001 and nonsignificant difference, respectively. (C) Transcript levels of PelA (violet bars), VgrG1b (T6SS) (green bars) and ExoS (T3SS) (royal blue bars) were monitored by RT-qPCR in PAK, PAKΔgacS and PAKΔgacA strains with the pBBRladS plasmid containing the ladS HK gene and the pBBRMCS4 corresponding empty cloning vector and fold induction was presented for the two mutant strains as compared to the PAK strain. Moderated t-tests were performed; *, ** and *** referred respectively to p<0.05, p<0.01 and p<0.001.
Fig 4.
Interactions between H1 domains of the LadS hybrid HK, the GacS unorthodox HK and the RetS hybrid HK using pull-down and two-hybrid experiments.
(A) Pull-down experiments. N-terminal FLAG or Strep versions of the H1 domain of GacS, LadS and RetS HKs were constructed in pBBRMCS3 and pCR2.1 vectors, respectively, and expressed in E. coli. Cell lysates were immunoprecipitated using anti-Strep antibody-coupled beads, and FLAG and Strep derivatives were further detected using StrepTactin Alkaline Phosphatase conjugate (upper panel) and anti-FLAG antibody detection (lower panel). (B) In two-hybrid experiments, the ladSH1, retSH1 and gacSH1 DNA regions were cloned into the two-hybrid pUT18C or pKT25 vectors and corresponding vectors were co-transformed in BTH101 cells that were further streaked on LB plates containing X-gal. A blue color of colonies reflects interaction between chimeric proteins, while white color attests to the absence of interaction. The interactions were further quantified by measuring the corresponding ß-galactosidase levels expressed in Miller units (values and standard deviations of 3 independent clones below corresponding colonies).
Fig 5.
H1 and D1 domain involvement of the LadS hybrid HK in the LadS signaling pathway.
(A) The pBBRladSH1D1 plasmid containing the ladSH1D1 cytoplasmic DNA region of the LadS hybrid HK fused to a C-terminal His-tag, the pBBRladSH1H→QD1 and pBBRladSH1D1D→A variant plasmids and the pBBRMCS4 corresponding empty cloning vector were conjugated in the PAK strain. Production of the corresponding cytoplasmic versions of LadS was checked in whole cell extracts using western blot and a monoclonal anti-His antibody. Numbers on the left side are molecular weight standards (kDa) (upper panel). Activity of the rsmY–lacZ (blue bars) and rsmY–lacZ (brick-red-colored bars) transcriptional chromosomal fusions were monitored after 6 hours of growth (OD600nm≈4) and 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 (middle panel). Production of the H1-T6SS VgrG1 protein was detected in whole cell extracts using western blot with an anti-VgrG1 polyclonal antibody. Numbers on the left side are molecular weight standards (kDa) (lower panel). (B) Biofilm production in glass tubes of PAK, PAKΔladS and of point chromosomal mutants PAKladSH1H→QD1 and PAKladSH1D1D→A was presented (upper panel) and quantified after crystal violet-staining and extraction (lower panel). Corresponding levels of biofilm production represented by mean values and standard deviations were 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. (C) Transcript levels of RsmY (blue bars), RsmZ (brick-red-colored bars), VgrG1b (T6SS) (green bars) and ExoS (T3SS) (royal blue bars) were monitored in PAK, PAKΔladS and in point chromosomal mutants PAKladSH1H→QD1 and PAKladSH1D1D→A strains using RT-qPCR. Fold induction was presented for the three mutant strains as compared to the PAK strain. Moderated t-tests were performed; *, ** and *** referred respectively to p<0.05, p<0.01 and p<0.001. (D) Activity of the pelA–lacZ (violet bars) transcriptional chromosomal fusion was monitored after 6 hours of growth (OD600nm≈5) and corresponding β-galactosidase activities are expressed in Miller units and correspond to mean values (with error bars) obtained from three independent experiments. Statistical tests were performed and ** referred to p<0.01.
Fig 6.
Involvement of the H2 domain of the GacS unorthodox HK in the LadS signaling pathway.
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.
Fig 7.
In vitro transphosphorylation assays.
(A) For transphosphorylation assay between LadS or GacS variants and GacSH2 variants or HptA protein, 2 mM of LadSH1D1 or LadSH1D1D→A recombinant proteins were incubated with [γ-32P] ATP and GacSH2 (lanes 1 and 2), GacSH2H→Q (lanes 3 and 4) or HptA (lanes 5 and 6) at room temperature for 20 min (see Materials and Methods) then separated in an SDS-polyacrylamide gel in duplicate. (B) Transphosphorylation assay between the LadSH1D1D→A or GacSH1 and LadSD1 or GacsD1 domains with or without the GacSH2 domain. Two mM of LadSD1 or GacSD1 recombinant proteins were incubated with [γ-32P] ATP and LadSH1D1D→A or GacSH1 (left panel) together with the GacSH2 domain (right panel) at room temperature for 20 min. In both experiments mixtures of proteins were separated in an SDS-polyacrylamide gel in duplicate. Numbers on the left side are molecular weight standards (kDa). Locations of the recombinant proteins are indicated by arrowheads. For each experiment presented in panels A and B, one gel was detected by western blot using an anti-penta-His antibody (upper panel) while the other was autoradiographied (lower panel).
Fig 8.
Transphosphorylation kinetic between the LadSH1D1 or GacSH1D1 and GacSH2 domains.
Two mM of LadSH1D1 or GacSH1D1 and GacSH2 recombinant proteins were incubated with [γ-32P] ATP at room temperature. The reaction was stopped at different time points (see Materials and Methods) and the samples were separated in an SDS-polyacrylamide gel and autoradiographied.
Table 1.
Strains used in this study.
Table 2.
Plasmids used in this study.