Figures & data
(a) The PCN production was detected in the wild-type PAO1, the Δstk1 mutant and the complemented Δstk1:stk1 strains. The PCN was quantified by measuring the OD520 value. (b) Detection of PVD production in PAO1, Δstk1 and Δstk1:stk1 strains. (c) Detection of the percentage of Fe3+ uptake in PAO1, Δstk1 and Δstk1:stk1 strains. (d) Detection of twitching motility zone of PAO1, Δstk1 and Δstk1:stk1 strains. Cultures on plates were stained with 0.5% crystal violet and the diameters of the twitching zones were measured to quantify the twitching motility. Data are presented as the mean ± standard deviation (SD) of three independent experiments performed in triplicate. ** P<0.01, *** P<0.001 and **** P<0.0001.
(a) Detection of biofilm production in PAO1, Δstk1 and Δstk1:stk1 strains using the crystal violet staining method. Biofilms were quantified by measuring OD590/OD600 values. Data are from three independent experiments performed in duplicate. ** P<0.01 and *** P<0.001. (b) Detection of EPS secretion in PAO1, Δstk1 and Δstk1:stk1 strains. The EPS secreted by each strain were stained with Congo red. Three replicates of each experiment were carried out and representative images were presented.
(a) The modelled 3D structure of AlgR and its Ser143 site. The 3D structure of AlgR was homology modelled using Phyre V2.0 software, and 246 residues (2-246, 98% coverage) were modelled with 100.0% confidence based on the template c4cbvE_. Template PDBTitle: X-ray structure of full-length ComE from Streptococcus pneumoniae. The structure of AlgR was generated by PyMol and the S143 (including side chain hydroxyl groups) is shown in yellow. (b) In vitro assays of AlgR phosphorylation catalysed by Stk1. Purified kinase Stk1 and the substrate AlgR or AlgRS143A were used for the phosphorylation reaction, i.e. incubation at 30 °C for 1.5 h or the indicated time. ATP was added to the phosphorylation reaction system as a phosphate group donor. Western blot (WB) was used to measure the phosphorylation level of AlgR using an anti-phospho-Ser pan antibody. SDS-PAGE was used as a loading control (LC).
(a) Biofilm production of PAO1, Δstk1 and PAO1-algRS143A strains was detected by using the crystal violet staining method. Biofilm quantification was performed by measuring the OD590/OD600 values. * P<0.05 and ** P<0.01. (b) EPS secretion of PAO1, Δstk1 and PAO1-algRS143A stain was detected by Congo red staining. A representative image from three experiments was presented.
(a) Representative pigment colour profiles of bacterial cultures to show the difference in PCN production between PAO1, Δstk1 and PAO1-algRS143A strains after 12 h of cultivation. (b) Determination of PCN production of PAO1, Δstk1 and PAO1-algRS143A strains using a chloroform-hydrochloric acid extraction method at 12, 24, 36 and 48 h after incubation. The PCN was quantified by measuring the OD520 value. (c) Representative images showing the twitching motility of PAO1, Δstk1 and PAO1-algRS143A strains. (d) Determination of twitching motility of PAO1, Δstk1 and Δstk1:stk1 strains. Cultures on plates were stained with 0.5% crystal violet and the diameters of the twitching zones were measured to quantify the twitching motility. Data are from three independent experiments performed in duplicate. ** P<0.01, *** P<0.001 and **** P<0.0001.
Supplemental material
Supplemental Material
Download MS Word (566.7 KB)Data availability statement
The data that support the findings of this study are openly available in figshare at https://doi.org/10.6084/m9.figshare.25551474.v3, reference number 25,551,474.