Residue L193P Mutant of RpoS Affects Its Activity During Biofilm Formation in Salmonella Pullorum

Abstract

The role of alternative sigma factor RpoS in regulating biofilm formation may differ in various Salmonella Pullorum strains. In this study, the biofilm-forming ability of two Salmonella Pullorum strains S6702 and S11923-3 were compared. The biofilm forming ability of S11923-3 was much stronger than that of S6702. After knocking out the rpoS gene, S11923-3ΔrpoS had significantly reduced biofilm while S6702ΔrpoS demonstrated similar biofilm compared with each parent strain. The analysis of RpoS sequences indicated two amino acid substitutions (L193P and R293C) between S6702 and S11923-3 RpoS. A complementation study confirmed that the expression of S11923-3 RpoS rather than S6702 RpoS could restore the biofilm-forming ability of ΔrpoS strains and the L193P mutation contributed to the restoration of the biofilm-forming ability. Further study indicated that RpoS with the L193P mutant had significantly improved expression level and binding activity to RNAP and csgD gene promoter, which increased the efficacy of the csgD gene promoter and biofilm-forming ability. Therefore, the L193P mutation of RpoS is critical for stronger biofilm formation of Salmonella Pullorum.

Keywords: Salmonella pullorum; biofilm; mutation; promoter; rpoS.

Figure 1

Effect of RpoS on biofilm formation in Salmonella Pullorum S6702 and S11923-3. (A) Crystal violet staining of bacteria grown in 96-well plates. Duplicate wells were used in each independent assay. (B) Quantification of crystal violet staining by measuring the optical density (OD₅₅₀). Means and standard deviations from three independent experiments are shown. (C) Morphology of colonies after growth on Congo red and calcofluor agar plates. (D) Field emission scanning electron microscope observation of bacteria. The bottom figure (bars = 1 μm) originated from the black boxes in the top figure (bar = 2 μm). (E) The mRNA levels of csgD, csgA, and bcsA genes determined by qRT-PCR. The mRNA level of each gene was normalized by the mRNA level of the gyrB gene. The bars represent the means of three independent assays. *P < 0.05.

Figure 2

Determination of biofilm formation of S6702ΔrpoS (A) and S11923-3ΔrpoS (B) complemented with two point mutants in residues 193 and 293 of RpoS. All of the strains were cultured in TSB medium first. After overnight cultivation, cultures were diluted in 1/10 TSB and grown in 96-well plates (100 μL/ well) at 28°C for 24 h without shaking. Discarding the supernatant and washing the wells gently with distilled water to remove non-adherent bacteria. After staining with 0.4% crystal violet for 20 min, washed the wells and the remaining crystal violet was solubilized with 100 μL of 25% acetone with anhydrous ethanol. Crystal violet staining quantification was tested by measuring the optical density (OD₅₅₀). Means and standard deviations from three independent experiments are shown. *P < 0.05.

Figure 3

Determination of RpoS expression by immunoblotting analysis in different S. Pullorum isolates (A), rpoS gene deletion mutants and complementary strains (B), and ropS gene substitution strains (C) during biofilm formation. All of the strains were cultured in TSB medium first. Overnight cultures were diluted in 1/10 TSB medium in small dishes at 28°C without shaking for 8 and 24 h. Supernatant was discarded and the scraped samples were collected. The samples were quantified to OD₆₀₀ = 1.0, then suspended in SDS sample buffer (Beyotime Biotechnology). The samples were resolved in 12% SDS polyacrylamide gels, transferred to PVDF membranes (Millipore), and analyzed by immunoblotting using anti-RpoS (NeoClone) and anti-RpoA (NeoClone) antibodies.

Figure 4

Determination of the binding ability of RpoS to RNAP and csgD promoter. (A) Immunoblotting analyses for binding activity of RpoS to RNAP. The purified RpoS protein (2.0 mg/mL) were incubated with his-tag Dynabeads (50 μL, Invitrogen). The beads were incubated with the clarified lysates including RNAP for 15 min. After being washed, the samples were resolved in SDS-PAGE, transferred onto PVDF membranes (Millipore), and probed with primary anti-RpoS, anti-RpoA, and anti-RpoB antibodies. (B) EMSA assays. The csgD promoter genes were PCR labeled using FAM-modified primer. The labeled DNA fragments and purified RpoS protein were incubated in the reaction system contained 4 μL 5 × binding buffer (Beyotime Biotechnology) and 200 ng of poly dI-dC (Sigma-Aldrich). The final mixtures were run on a 6% SDS-PAGE. The images were scanned and observed using a fluorescence imaging system (Typhoon FLA 9500, GE Healthcare). The data showed one representative experiment of three independent assays. (C) Determination of β-galactosidase activity. Plasmid pcsgD-lacZ was transformed into S6702, S11923-3, and their rpoS deletion mutants. The transformants were cultured in dishes at 28°C for 24 h and 48 h without shaking. After measuring the absorbance at 600 nm, the samples were mixed with reaction buffer and stop buffer. The absorbance was measured at 420 nm and the β-galactosidase activity was calculated. The bars represent the means of three independent assays. *P < 0.05.