Ser/Thr protein kinase Stk1 phosphorylates the key transcriptional regulator AlgR to modulate virulence and resistance in Pseudomonas aeruginosa

Figures & data

Figure 1. Deletion of stk1 leads to reduced production of PCN and PVD, and attenuated twitching motility.

(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.

Figure 2. Effect of the deletion of stk1 on biofilm production and EPS secretion.

(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.

Table 1. MIC of the indicated antibiotics for the strains PAO1, Δstk1, Δstk1:stk1 and PAO1-algR^S143A.

Strains	MICs (μg/mL)
	CAZ	GEN	TOB	CIP
PAO1	1	1	0.5	0.25
Δstk1	8	8	2	0.5
Δstk1:stk1	1	1	0.5	0.25
PAO1-algR^S143A	4	8	1	0.25

Figure 3. AlgR is a substrate of Stk1 and its Ser143 can be phosphorylated by this kinase.

(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 AlgR^S143A 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).

Figure 4. Both the deletion of stk1 and the mutation of algR^S143A led to increased biofilm production, EPS secretion and antibiotic resistance.

(a) Biofilm production of PAO1, Δstk1 and PAO1-algR^S143A 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-algR^S143A stain was detected by Congo red staining. A representative image from three experiments was presented.

Figure 5. Deletion of stk1 and mutation of algR^S143A both resulted in decreased PCN production and attenuated twitching motility.

(a) Representative pigment colour profiles of bacterial cultures to show the difference in PCN production between PAO1, Δstk1 and PAO1-algR^S143A strains after 12 h of cultivation. (b) Determination of PCN production of PAO1, Δstk1 and PAO1-algR^S143A 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-algR^S143A 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.

Figure 6. Determination of the relative expression of algR and several algR-regulated genes in PAO1, Δstk1 and PAO1-algR^S143A by qPCR.

czcR, algD and pilV are genes controlled by algR, which are also key genes in the regulation of PCN production, biofilm formation and twitching motility, respectively. Data are from three independent experiments performed in triplicate. *** P<0.001 and **** P<0.0001.

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.