Activation of a Cell Surface Signaling Pathway in Pseudomonas aeruginosa Requires ClpP Protease and New Sigma Factor Synthesis

Abstract

Extracytoplasmic function (ECF) sigma factors control expression of large numbers of genes in bacteria. Most ECF sigma factors are inhibited by antisigma proteins, with inhibition being relieved by environmental signals that lead to inactivation of the antisigma protein and consequent sigma factor activity. In cell surface signaling (CSS) systems in Gram negative bacteria antisigma activity is controlled by an outer membrane protein receptor and its ligand. In Pseudomonas aeruginosa one such system controls expression of genes for secretion and uptake of a siderophore, pyoverdine. In this system the activities of two sigma factors σ^FpvI and σ^PvdS are inhibited by antisigma protein FpvR₂₀ that binds to the sigma factors, preventing their interaction with core RNA polymerase. Transport of ferripyoverdine by its outer membrane receptor FpvA causes proteolytic degradation of FpvR₂₀, inducing expression of σ^FpvI- and σ^PvdS-dependent target genes. Here we show that degradation of FpvR₂₀ and induction of target gene expression was initiated within 1 min of addition of pyoverdine. FpvR₂₀ was only partially degraded in a mutant lacking the intracellular ClpP protease, resulting in an FpvR₂₀ subfragment (FpvR₁₂) that inhibited σ^FpvI and σ^PvdS. The translation inhibitor chloramphenicol did not prevent induction of an σ^FpvI-dependent gene, showing that degradation of FpvR₂₀ released pre-existing σ^FpvI in an active form. However, chloramphenicol inhibited induction of σ^PvdS-dependent genes showing that active σ^PvdS is not released when FpvR₂₀ is degraded and instead, σ^PvdS must be synthesized in the absence of FpvR₂₀ to be active. These findings show that sigma factor activation occurs rapidly following addition of the inducing signal in a CSS pathway and requires ClpP protease. Induction of gene expression that can arise from release of active sigma from an antisigma protein but can also require new sigma factor synthesis.

Keywords: ClpP protease; ECF sigma factor; antisigma; bacterial signal transduction; cell surface signaling; pyoverdine; regulated proteolysis; siderophore.

FIGURE 1

The pyoverdine signaling pathway. (A) In the absence of ferripyoverdine the activities of sigma factors σ^FpvI and σ^PvdS are inhibited by the antisigma protein FpvR₂₀. (B) Import of ferripyoverdine (Fe-PVD) causes a molecular rearrangement of the FpvA receptor protein, triggering a proteolytic cascade that degrades FpvR₂₀. σ^FpvI and σ^PvdS then become active, stimulating expression of the fpvA gene and of pyoverdine (pvd) synthesis genes, respectively. See text and (Llamas et al., 2014) for more detailed information. OM, outer membrane; CM, cytoplasmic membrane.

FIGURE 2

Activation of gene expression following addition of pyoverdine. Pyoverdine was added to Pseudomonas aeruginosa PAO1 pvdF bacteria (0 min) and samples were collected at intervals and analyzed by reverse transcription quantitative PCR (RT-qPCR). The amounts of pvdH, pvdL, and fpvA transcripts are shown relative to the reference genes clpX and oprL. Data are means of six technical replicates with standard deviation shown. Similar results were obtained when the experiment was repeated (Supplementary Figure S1).

FIGURE 3

Time-course of degradation of FpvR₂₀ and σ^PvdS following addition of pyoverdine. Pyoverdine was added to P. aeruginosa bacteria (0 min). Samples were collected at intervals and analyzed by Western blotting with antibodies against FpvR₂₀, σ^PvdS or RpoD (loading control). FpvR₂₀, σ^PvdS (full-size), PvdS₁₅ and RpoD are indicated. Times are shown in minutes. (A) PAO1 pvdF (B) PAO1 pvdF fpvA. Similar results were obtained when the experiment was repeated (Supplementary Figure S2).

FIGURE 4

Effect of clpP mutation on induction of gene expression. Pyoverdine was added (0 min) to P. aeruginosa PAO1 pvdF clpP (black triangles) and P. aeruginosa PAO1 pvdF clpP (minictx::tig-clpP) (open triangles). Samples were collected at intervals and analyzed by RT-qPCR. Data are means of six technical replicates with standard deviation shown. Equivalent data from strain PAO1 pvdF bacteria (Figure 2) (black squares) are included for comparison. (A) pvdH. (B) pvdL. (C) fpvA.

FIGURE 5

Effects of clpP mutation on degradation of FpvR₂₀ and σ^PvdS. Pyoverdine was added (0 min) to P. aeruginosa PAO1 pvdF bacteria containing a mutation in the clpP gene. Samples were collected at intervals and analyzed by Western blotting. Times are shown in minutes. (A) P. aeruginosa PAO1 pvdF clpP. (B) P. aeruginosa PAO1 pvdF clpP (minictx::tig-clpP). FpvR₂₀, FpvR₁₂, σ^PvdS (full-size), PvdS₁₅ and RpoD are indicated. FpvR₁₂ was not detected in P. aeruginosa PAO1 pvdF clpP (minictx::tig-clpP). Similar results were obtained when the experiment was repeated (Supplementary Figure S4).

FIGURE 6

Effect of chloramphenicol on induction of gene expression. Chloramphenicol (Cm) and then pyoverdine were added to P. aeruginosa PAO1 pvdF bacteria (0 min) and samples were collected at intervals. (A) Samples were analyzed by RT-qPCR for pvdH, pvdL, and fpvA. Open squares, Cm present; Black squares, Cm absent. Data are means of six technical replicates with standard deviation shown. (B) Samples were analyzed by Western blotting for σ^PvdS or FpvR₂₀ in the presence or absence of pyoverdine, as shown. FpvR₂₀, σ^PvdS (full-size), PvdS₁₅ and RpoD are indicated. Times are shown in minutes. Similar results were obtained when the experiment was repeated (Supplementary Figure S5).