The Sia System and c-di-GMP Play a Crucial Role in Controlling Cell-Association of Psl in Planktonic P. aeruginosa

Abstract

Many bacterial species use the secondary messenger, c-di-GMP, to promote the production of biofilm matrix components. In Pseudomonas aeruginosa, c-di-GMP production is stimulated upon initial surface contact and generally remains high throughout biofilm growth. Transcription of several gene clusters, including the Sia signal transduction system, are induced in response to high cellular levels of c-di-GMP. The output of this system is SiaD, a diguanylate cyclase whose activity is induced in the presence of the detergent SDS. Previous studies demonstrated that Sia-mediated cellular aggregation is a key feature of P. aeruginosa growth in the presence of SDS. Here, we show that the Sia system is important for producing low levels of c-di-GMP when P. aeruginosa is growing planktonically. In addition, we show that Sia activity is important for maintaining cell-associated Psl in planktonic populations. We also demonstrate that Sia mutant strains have reduced cell-associated Psl and a surface attachment-deficient phenotype. The Sia system also appears to posttranslationally impact cell-associated Psl levels. Collectively, our findings suggest a novel role for the Sia system and c-di-GMP in planktonic populations by regulating levels of cell-associated Psl.

Keywords: Pseudomonas aeruginosa; biofilm formation; c-di-GMP.

FIG 1

The Sia signaling network impacts attachment of P. aeruginosa to surfaces. (A) The Sia signaling network regulates SiaD diguanylate cyclase activity. In response to an unknown environmental signal, SiaA dephosphorylates SiaC. SiaC then binds SiaD, activating SiaD DGC activity. SiaB is a kinase that competitively binds SiaC and rephosphorylates it. Once rephosphorylated, SiaC will no longer bind SiaD, and DGC activity is shut off. Figure created in BioRender.com (29). (B) Static biofilm formation of sia mutant strains. Biofilm biomass produced by each strain was measured by crystal violet staining and normalized to wild type (PAO1). Presented as mean and standard deviation. N, 3 biological replicates, *P < 0.05. (C) Adherence of sia mutant strains to glass. Cells were incubated on a glass coverslip, rinsed and attached cells were immediately quantified by microscopy. Presented as mean and standard deviation. N, 3 biological replicates, *, P < 0.05.

FIG 2

The Sia signaling network impacts the surface adherence of planktonic cells through control of cell-associated Psl. (A) Adherence of matrix mutant strains to glass. Cells were incubated on a glass coverslip, rinsed and attached cells were immediately quantified by microscopy. (B) Representative immunoblot for Psl produced by sia mutants, extracted from midlog planktonic cells (OD_600nm = 0.5). RNAP served as a loading control. (C) Quantification of relative Psl production calculated using blots in 2B. Psl band intensity was normalized to RNAP levels and then compared to wild-type (PAO1) cell-associated Psl. (D) Representative immunoblot for Psl from PAO1 and ΔsiaD throughout planktonic growth. Negative-control samples (ΔpslD) were processed at OD_600nm = 2. At each time point 1 × 10⁹ cells were processed. RNAP served as a loading control. (E) Growth curve of strains evaluated in 2D, in Lennox Broth. The time points collected for 2D are marked on the curve. (F) Quantification of relative cell-free and cell-associated Psl production calculated using blots in 2D. Psl band intensity was normalized to RNAP levels and then compared to PAO1 at OD_600nm = 0.3. Presented as mean and standard deviation. N, 3 biological replicates, *, P < 0.05.

FIG 3

The Sia system impacts levels of cell-associated Psl through SiaD diguanylate cyclase activity. (A) C-di-GMP levels of by diguanylate cyclase mutant strains. C-di-GMP was extracted from midlog planktonic cells (OD_600nm = 0.5). Presented as mean and standard deviation. N = 5 biological replicates, *, P < 0.05. (B) C-di-GMP levels of the siaD^E142A catalytic mutant strain. C-di-GMP was extracted from midlog planktonic cells (OD_600nm = 0.5). Presented as mean and standard deviation. N = 3 biological replicates, *, P < 0.05. (C) Adherence of the siaD^E142A mutant strain to glass. Cells were incubated on a glass coverslip, rinsed and attached cells were immediately quantified by microscopy. Presented as mean and standard deviation. N, 3 biological replicates, *P < 0.05. (D) Representative immunoblot for Psl produced by the siaD^E142A mutant, extracted from midlog, planktonic cells (OD_600nm = 0.5). RNAP served as a loading control. (E) Quantification of relative Psl production calculated using blots in 3D. Psl band intensity was normalized to RNAP levels and then compared to wild-type (PAO1) cell-associated Psl. N, 3 biological replicates, *, P < 0.05.

FIG 4

Planktonic levels of cell-associated Psl are not impacted by DGCs than SiaD. (A) Representative immunoblot for Psl produced by DGC transposon insertion mutants, extracted from midlog planktonic cells (OD_600nm = 0.5). RNAP served as a loading control. (B) Quantification of cell-free and (C) cell-associated Psl production calculated using blots in 4A. Psl band intensity was normalized to RNAP levels and then compared to the wild-type control (MPAO1). Presented as mean and standard deviation. N, 3 biological replicates, *, P < 0.05.

FIG 5

Overproduction of c-di-GMP causes an increase in cell-associated Psl levels. (A) C-di-GMP levels of cells overexpressing the heterologous cyclase Ml1419c in P. aeruginosa. C-di-GMP was extracted from midlog planktonic cells (OD_600nm = 0.5). Presented as mean and standard deviation. N, 5 biological replicates, *, P < 0.05. (B) Representative immunoblot for Psl produced by cells overexpressing siaD or ml1419c, extracted from midlog, planktonic cells (OD_600nm = 0.5). RNAP served as a loading control. (C) Quantification of relative Psl production calculated using blots in 5B. Psl band intensity was normalized to RNAP levels and then compared to the wild-type vector control (PAO1 VC) cell-associated Psl. Presented as mean and standard deviation. N, 3 biological replicates, *, P < 0.05, VC=vector control.

FIG 6

The Sia system regulates cell-associated Psl production posttranslationally. (A) q-RT-PCR of pslA-containing transcripts relative to rpoD transcript levels. RNA was extracted from midlog planktonic cultures (OD_600nm = 0.5). (B) Expression of the psl operon was driven by the P_BAD promoter to uncouple any potential transcriptional control of the Psl promoter. These strains were analyzed for potential effects on Psl protein translation. Representative immunoblots are shown for Psl, PslB, and PslG. RNAP served as a loading control. (C) Quantification of relative Psl production calculated using blots in 6B. Psl band intensity was normalized to RNAP levels and then compared to wild-type (PAO1 P_BADpsl) cell-associated Psl. (D) Adherence of psl overexpression strains to glass. Cells were incubated on a glass coverslip, rinsed and attached cells were immediately quantified by microscopy. Presented as mean and standard deviation. N, 3 biological replicates, *, P < 0.05.