Protocol to monitor activation of a two-component system in Dickeya dadantii during chicory leaf infection using Phos-tag gel

Summary

Quantifying the phosphorylation levels of proteins involved in bacterial signaling pathways is essential for understanding their role in stress adaptation and infection processes. Here, we present a protocol for the semi-quantitative assessment of the phosphorylation level of the transcriptional regulator CpxR during the infection process using Phos-tag gel. We describe steps for Dickeya dadantii infection of chicory leaves, followed by bacterial extraction, rapid acidic cell lysis, and subsequent protein analysis using Phos-tag SDS-PAGE and western blot.

For complete details on the use and execution of this protocol, please refer to Bontemps-Gallo et al.¹ and Cochard et al.²

Graphical abstract

Highlights

• •Instructions for infecting chicory leaves with Dickeya dadantii
• •Procedures for rapid acidic lysis to preserve phospho-aspartate bonds
• •Steps for resolving phosphorylated regulators using Phos-tag SDS-PAGE and western blot
• •Guidance on adapting the protocol for different response regulators and bacteria

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Quantifying the phosphorylation levels of proteins involved in bacterial signaling pathways is essential for understanding their role in stress adaptation and infection processes. Here, we present a protocol for the semi-quantitative assessment of the phosphorylation level of the transcriptional regulator CpxR during the infection process using Phos-tag gel. We describe steps for Dickeya dadantii infection of chicory leaves, followed by bacterial extraction, rapid acidic cell lysis, and subsequent protein analysis using Phos-tag SDS-PAGE and western blot.

Before you begin

To colonize new environments and hosts, bacteria must adapt to fluctuating physicochemical parameters. Two-component systems (TCS) are the primary mechanisms for sensing and responding to environmental stress. Typically, these systems (Figure 1) consist of a transmembrane sensor kinase that detects one or more stimuli, undergoes autophosphorylation via its intrinsic histidine kinase activity, and subsequently transfers the phosphate group to its cytoplasmic response regulator at an aspartate residue. However, directly detecting the phosphorylation state of response regulators has long been technically challenging. This difficulty stems from the intrinsic instability of the phospho-aspartate bond, which is highly labile and readily hydrolyzed during protein extraction and electrophoresis. As a result, phosphorylation is typically inferred indirectly by monitoring the expression of downstream target genes—an approach that requires prior identification of such targets and may not accurately reflect regulator activation under dynamic or in vivo conditions.

Figure 1.

Schematic representation of a two-component system

In response to a stimulus, the histidine kinase sensor becomes activated, autophosphorylates on a conserved histidine residue, and then transfers the phosphate group to a conserved aspartate residue on its cognate transcriptional regulator. The activated response regulator can then modulate the expression of target genes.

In 2008, Barbieri and Stock developed an analytical approach to investigate TCS phosphorylation, applying it to Escherichia coli in vitro and under laboratory culture conditions.³ Here, we present a refined and adapted protocol for Dickeya dadantii in the complex context of infection. This protocol focuses on the CpxAR two-component system, which is known to mediate envelope stress responses in enterobacteria⁴ and plays a role in the pathogenesis of various animal and plant pathogens.^1,5,6,7

By using Phos-Tag SDS-PAGE coupled with rapid acidic lysis of bacterial cells extracted from infected tissues, our method preserves and resolves phosphorylated forms of CpxR with enhanced sensitivity and specificity. Unlike indirect reporter-based assays, it does not rely on transcriptional readouts or require prior knowledge of CpxR target genes. This enables more reproducible and time-resolved assessment of regulator activation during host-pathogen interactions.

The procedure outlines the steps for infecting chicory leaves, extracting bacteria at specific time points, and performing rapid acidic lysis of bacterial cells. It includes detailed instructions for casting and running Phos-Tag SDS-PAGE gels, followed by Western blotting to assess phosphorylation status. Troubleshooting guidance is also provided to facilitate implementation in laboratories working on D. dadantii or related bacterial species.

Before you begin, ensure that all buffers and culture media are prepared according to the materials and equipment section. While preparing buffers the day before the experiment is preferable, some can be made in advance and appropriately stored. Note that Phos-Tag SDS-PAGE gels must be freshly prepared to ensure optimal separation of phosphorylated protein species.

Institutional permissions

Before initiating any work involving Dickeya spp., researchers must consult the relevant regulatory authorities to ensure compliance with national and international plant health and biosafety regulations. As Dickeya spp. are classified as quarantine pests in several countries, including under the European and Mediterranean Plant Protection Organization (EPPO) guidelines, specific permits or authorizations may be required.

Work involving genetically modified organisms (GMOs) is generally regulated by national laws and institutional guidelines. Researchers must ensure that they are aware of and comply with all applicable regulations.

In France, Dickeya dadantii is classified as a Biosafety Level 1 (BSL-1) organism, which does not require a specific biosafety permit for laboratory use. The possession and use of D. dadantii mutants in this study were declared to the French Ministry of Higher Education and Research (Ministère de l'Enseignement supérieur et de la Recherche), in accordance with national GMO regulations.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
CpxR (1:1,000)	Laboratory collection	N/A
Goat anti-rabbit IgG (1:10,000)	Cytiva	NA934-1ML
Bacterial and virus strains
Dickeya dadantii strain EC3937	Laboratory collection	N/A
Chemicals, peptides, and recombinant proteins
Bromophenol blue	Sigma	B8026
EDTA	Acros organic	14785-0010
MnCl2(4H2O)	Merck	M3634
SDS 20%	Euromedex	EU0660
Tris	Euromedex	26-128-3094-B
Ammonium persulfate (APS)	Merck	A3678
Tetramethylethylenediamine (TEMED)	Merck	T9281
Methanol	Carlo Erba	414942
Formic acid	Scharlau	Ac1085
NaOH	Carlo Erba	480507
BSA	Roche	10735086001
Glycerol anhydrous	Euromedex	EU6550
PBS solution 10X	Euromedex	ET330
Tween 20	Euromedex	2001-A
40% acrylamide/bis-acrylamide solution (37.5:1)	Bio-Rad	1610148
Phos-tag acrylamide, 10 mg	FUJIFILM Wako	AAL-107
TG-SDS 10X (Tris-Glycine-SDS)	Euromedex	EU0510
TG 10X (Tris-Glycine)	Euromedex	EU0550
LB (lysogeny broth) broth Lennox	BD Difco	240230
Granulated agar	BD Difco	214530
Color prestained protein standard, broad range	New England Biolabs	P7719
Critical commercial assays
ECL Prime western blotting detection reagent	GE Healthcare	28980926
Experimental models: Organisms/strains
Chicory leaves	Supermarket	N/A
Software and algorithms
GraphPad Prism 10	GraphPad Software	http://www.graphpad.com
Quantity One	Bio-Rad	1709602
ImageJ software	Schneider et al.8	https://imagej.net/ij/
Other
PowerPac basic power supply	Bio-Rad	1645050
Mini-PROTEAN tetra vertical electrophoresis cell for mini precast gels	Bio-Rad	1658004
Mini-PROTEAN tetra cell casting module	Bio-Rad	1658015
Trans-Blot Turbo transfer system	Bio-Rad	1704150
Trans-Blot Turbo Mini 0.2 μm nitrocellulose transfer packs	Bio-Rad	1704158
Inoculation loop, 10 μL, PS, blue, sterile	Sarstedt	86.1562.010
Sterile scalpel n°20 Swann-Morton	Swann-Morton	U20/0506
Tube 50 mL	Corning	430828
DiluPhotometer	Implen	OD600
Polystyrene semi micro cuvettes	Fisherbrand	FB55147
Petri dish, 92 × 16 mm, transparent, with ventilation cams	Sarstedt	82.1473.001

Materials and equipment

M63 Salt solution

Reagent	Final concentration	Amount
KH2PO4	0.1 M	13.6 g
(NH4)2SO4	15 mM	1.98 g
MgSO4	0.8 mM	96.3 mg
KOH	0.4%	4 g
FeSO4 (10 mg/mL)	20 μg/mL	2 mL
dH2O	N/A	Up to 1.000 mL
Total	N/A	1.000 mL

10% ammonium persulfate (10% APS)

Reagent	Final concentration	Amount
Ammonium persulfate	10% (w/v)	1 g
dH2O	N/A	Up to 10 mL
Total	N/A	10 mL

[Store the solution at 4°C. Use within 1 month.]

CRITICAL: Ammonium persulfate is toxic, and users should handle them with care.

5 mM Phos-tag

Reagent	Final concentration	Amount
Phos-tag (MW = 595)	5.0 mmol/L	10 mg
Methanol	3% (v/v)	0.10 mL
dH2O	N/A	Up to 3.3 mL
Total	N/A	3.3 mL

[Store the solution at 4°C in the dark. Use within 4 months. Storage at −80°C improves stability for over a year]

CRITICAL: Methanol is toxic, and users should handle them with care.

10 mM MnCl2 solution

Reagent	Final concentration	Amount
MnCl2 (H2O)4 (MW = 198)	10 mM	0.10 g
dH2O	N/A	Up to 50 mL
Total	N/A	50 mL

[Store at Room Temperature (20°C). Solution remains viable for weeks to months, no need to make fresh each time]

Tris-HCl (0.5 M, pH 6.8)

Reagent	Final concentration	Amount
Tris	0.5 M	60.55 g
dH2O	N/A	Up to 1,000 mL
Total	N/A	1,000 mL

[Store at Room Temperature (20°C). Solution remains viable for weeks to months, no need to make fresh each time.]

Adjust the pH to the desired value using hydrochloric acid (HCl).

Tris-HCl (1.5 M, pH 8.8)

Reagent	Final concentration	Amount
Tris	1.5 M	181.7 g
dH2O	N/A	Up to 1,000 mL
Total	N/A	1,000 mL

[Store at Room Temperature (20°C). Solution remains viable for weeks to months, no need to make fresh each time.]

Adjust the pH to the desired value using hydrochloric acid (HCl).

10% SDS-PAGE resolving gel

Reagent	Final concentration	Amount
dH2O	N/A	2.325 mL
40% Acrylamide/bis-acrylamide (37.5:1)	10%	1.25 mL
Phos-Tag 5 mM	35 μM	35 μL
MnCl2 10 mM	70 μM	35 μL
1.5 M Tris-HCl, pH8.8	375 mM	1.25 mL
20% SDS	0.1% (w/v)	25 μL
10% APS	0.2% (w/v)	50 μL
TEMED	0.08% (v/v)	4 μL
Total	N/A	5 mL

CRITICAL: Acrylamide/bis-acrylamide solution, APS, TEMED, and Phos-Tag are toxic and users should handle them with care.

Note: The TEMED should be added last and immediately before pouring the gel.

4% SDS-PAGE stacking gel

Reagent	Final concentration	Amount
dH2O	N/A	1.290 mL
40% Acrylamide/bis-acrylamide (37.5:1)	4%	0.2 mL
0.5 M Tris-HCl, pH 6.8	125 mM	0.5 mL
20% SDS	0.1 (w/v)	10 μL
10% APS	0.2% (w/v)	20 μL
TEMED	0.08% (v/v)	2 μL
Total	N/A	2 mL

CRITICAL: Acrylamide/bis-acrylamide solution, APS, TEMED, and Phos-Tag are toxic and users should handle them with care.

Note: The TEMED should be added last and immediately before pouring the gel.

TG-SDS running buffer (Tris-Glycine-SDS)

Reagent	Final concentration	Amount
TG-SDS 10X	1 x	100 mL
dH2O	N/A	900 mL
Total	N/A	1000 mL

[Store at 4°C. Solution remains viable for weeks to months, no need to make fresh each time.]

1 M formic acid

Reagent	Final concentration	Amount
Formic acid (100%)	1 M (v/v)	1 mL
dH2O	N/A	20 mL
Total	N/A	21 mL

[Store at Room Temperature (20°C). Solution remains viable for weeks to months, no need to make fresh each time]

50% glycerol

Reagent	Final concentration	Amount
glycerol (100%)	50% (w/v)	50 g
dH2O	N/A	50 mL
Total	N/A	100 mL

[Store at Room Temperature (20°C). Solution remains viable for weeks to months, no need to make fresh each time]

4x SDS-PAGE loading buffer

Reagent	Final concentration	Amount
0.5 M Tris-HCl, pH6.8	50 mM	80 μL
20% SDS	2%	200 μL
50% Glycerol	10%	200 μL
Bromophenol Blue	0.02%	0.2 mg
dH2O	N/A	520 μL
Total	N/A	1 mL

[Store at - 20°C]

5 N NaOH

Reagent	Final concentration	Amount
NaOH	5 N (w/v)	2 g
dH2O	N/A	Up to 10 mL
Total	N/A	10 mL

[Store at Room Temperature (20°C). Solution remains viable for weeks to months, no need to make fresh each time]

0.5 M EDTA-HCl (pH 8.0)

Reagent	Final concentration	Amount
EDTA	0.5 M	146.12 g
dH2O	N/A	Up to 1,000 mL
Total	N/A	1,000 mL

[Store at Room Temperature (20°C). Solution remains viable for weeks to months, no need to make fresh each time.]

Adjust the pH to the desired value using hydrochloric acid (HCl).

Transfer buffer (Tris-Glycine)

Reagent	Final concentration	Amount
Tris-Glycine 10X	1 x	100 mL
dH2O	N/A	900 mL
Total	N/A	1000 mL

[Store at Room Temperature (20°C). Solution remains viable for weeks to months, no need to make fresh each time.]

EDTA Transfer buffer

Reagent	Final concentration	Amount
0.5 M EDTA-HCl pH 8.0	1 mM	100 μL
Tris-Glycine transfer buffer	1 x	50 mL
Total	N/A	50 mL

[Prepare a fresh solution before each use.]

PBS-Tween buffer (PBS-T)

Reagent	Final concentration	Amount
10 x PBS	1x	100 mL
Tween 20	0.05%	500 μL
dH2O	N/A	900 mL
Total	N/A	1,000 mL

[Store at Room Temperature (20°C). Solution remains viable for weeks to months, no need to make fresh each time]

5% BSA in PBS-Tween buffer

Reagent	Final concentration	Amount
BSA	5% (w/v)	5 g
PBS- Tween	1x	100 mL
Total	N/A	100 mL

[Prepare a fresh solution before each use.]

Step-by-step method details

Chicory leaf infection with Dickeya dadantii

Timing: 5 days

This step describes the process from bacterial pre-culture to the inoculation of D. dadantii on chicory leaves.

Note: We inoculated chicory leaves on different days for practical experimental reasons to collect samples at 1-, 2-, and 3-days post-inoculation on the same day. This approach prevents any issues related to sample conservation and potential loss of phosphorylation.

Note:Cichorium intybus var. foliosum, also known as Brussels chicory or witloof chicory, was purchased from a supermarket in a “ready-to-use” form.

Note: It is recommended to include a negative control by inoculating either the medium used to resuspend the bacteria (such as M63 salt medium, LB, or physiological water), or a non-virulent bacterial strain (such as the cpxR mutant).

• 1.Using a sterile loop, streak in a zigzag pattern following the quadrant method on a Petri dish containing LB solidified with 1.5% agar to isolate colonies from an initial frozen stock.

CRITICAL: Avoid completely thawing the frozen bacterial tube. Freeze-thaw cycles significantly reduce the survival of D. dadantii from the frozen stock store at −80°C.

• 2.Incubate overnight (∼12 h) at 30°C to allow bacterial growth.
• 3.Inoculate 5 mL of LB medium in a 50 mL tube with bacteria from the streaked Petri dish prepared in step 1.
• 4.Place the tube on a shaker at 180 rpm and incubate overnight (∼12 h) at 30°C.
• 5.Measure the turbidity of the culture with a spectrophotometer (OD_600nm) of the overnight bacterial culture by taking 1 mL sample.

Note: Depending on the spectrophotometer used, it may be necessary to perform a 1:10 dilution to ensure the turbidity falls within the instrument's reading range.

• 6.Centrifuge the 1 mL of culture at 4,000 g for 10 min at room temperature (20°C).
• 7.Discard the LB culture medium and ensure complete removal.
• 8.Resuspend the pellet in M63 salt solution to adjust the solution to a final OD_600nm = 2 (corresponding to 2 × 10⁹ bacteria/mL).

Note: It is possible to resuspend the bacteria in LB medium or physiological serum (0.9% NaCl) without affecting the infection development of the wild-type strain.

• 9.Using a sterile scalpel, make a 1 cm incision on the surface of the chicory leaf (Figure 2A).
• 10.Inoculate 5 μL of bacterial suspension (containing 10⁷ bacteria) directly onto the incision.
• 11.Place the inoculated leaves in a dew chamber at 30°C.
• 12.Incubate for the desired period (e.g., 24, 48, 72 h) (Figure 2B).

Figure 2.

Infection of endive leaves by Dickeya dadantii

(A) Schematic representation of an endive leaf showing tissue area scarification on the left and the macerated area to be sampled on the right.

(B) Representative image illustrating the development and progression of tissue maceration on endive leaves following infection with D. dadantii, as described in the experimental protocol.

Preparation of Phos-tag gel

Timing: 3 h

This step describes the preparation of an SDS-PAGE gel containing Phos-Tag for separating phosphorylated proteins. The gel must be prepared fresh on the day of the experiment. The following protocol describes the preparation of a 10% polyacrylamide gel containing 35 μM Phos-Tag acrylamide and 70 μM MnCl₂ (see troubleshooting 1, troubleshooting 2).

• 13.Prepare the resolving gel solution and vortex briefly to mix.
• 14.Immediately pour the resolving gel mixture into the 1 mm gel cassette
• 15.Carefully overlay with 1 mL of MQ water to ensure a smooth gel interface.
• 16.Allow polymerization for 1 h at room temperature (20°C).
• 17.After polymerization, pour off the overlay water.
• 18.Rinse the surface with MQ water to remove any unpolymerized acrylamide.
• 19.Using an absorbent filter paper, thoroughly dry the inside of the gel cassette to remove excess moisture.
• 20.Prepare the stacking gel solution.
• 21.Pour the stacking gel solution on top of the polymerized resolving gel.
• 22.Carefully insert the gel comb to create wells.
• 23.Allow the staking gel to polymerize fully (∼30 min) before proceeding with electrophoresis.
• 24.Place the gel in the electrophoresis tank and carefully pour the cold TG-SDS running buffer into the tank until it reaches the recommended fill line.

Sample preparation for Phos-tag analysis

Timing: 10 min

This step describes the process of protein extract preparation starting from infected chicory leaves.

CRITICAL: From leaf collection to lysis, the entire procedure must be completed within less than 10 min. Keep all materials on ice and work swiftly to preserve phosphorylation states

• 25.Using a sterile scalpel, carefully cut macerated tissue from the chicory leaves and transfer it into 20 mL of M63 salt solution (Figure 2A).
• 26.Vortex vigorously for 30 sec.
• 27.Filter the suspension to remove any plant debris.
• 28.Measure the turbidity (OD_600nm) of the filtrate using a spectrophotometer.
• 29.Collect an appropriate volume of the filtrate containing 1.5 × 10⁸ bacteria (an equivalent of 1 mL at OD_620nm = 0.15 of D. dadantii cells).
• 30.Centrifuge the collected volume at 4,000 g for 5 min.
• 31.Carefully remove and discard the supernatant.
• 32.Immediately lyse the bacterial pellet by adding 12.7 μL of 1 M formic acid.

CRITICAL: Acid lysis, unlike other lysis methods such as mechanical disruption (e.g., FastPrep), preserves protein phosphorylation, as previously described by Barbieri et al.³

• 33.Solubilize the lysed sample by adding 5 μL of 4x SDS-PAGE loading buffer.
• 34.Neutralize the solution by adding 2.8 μL of 5 N NaOH.

Note: Each sample is processed sequentially, from the lysis step to the addition of NaOH (steps 32 to 34), before proceeding to the next sample.

Protein migration in Phos-tag SDS-PAGE and western blot analysis

Timing: 2 days

This step describes the process from sample loading into the Phos-Tag SDS-PAGE gel to the completion of a Western blot.

• 35.Quickly load the entire sample volume (20.5 μL) into the Phos-Tag SDS-PAGE prepared earlier in this protocol.

CRITICAL: Samples are not heated before loading to preserve protein phosphorylation.

• 36.Load the 5 μL of the PageRuler Prestained Protein Ladder into the gel.

Note: The protein ladder is used to monitor protein separation based on molecular weight. However, some protein ladders, such as the PageRuler Prestained Protein Ladder, contain phosphorylated bands (e.g., the red band at ∼70 kDa). As a result, the migration pattern of the molecular weight marker may be altered by Phos-Tag, meaning the apparent molecular weight may not accurately correspond to the actual size of the protein of interest.

• 37.Run the gel at 4°C under constant voltage (150 V) using a standard running TG-SDS buffer.
• 38.Continue electrophoresis for 10 to 40 min after the migration front (bromophenol blue dye) exits the gel.

Note: The migration time should be adjusted based on the desired resolution of protein separation. For CpxR, electrophoresis is stopped when the band corresponding to a molecular weight of 35 kDa reaches the bottom of the gel. Gel running time typically ranges from 2 to 2.5 h at 150 V in a cold room (4 °C), depending on the acrylamide concentration and gel size.

• 39.Incubate the gel with gentle agitation in EDTA transfer buffer for 10 min.

Note: The EDTA incubation step chelates Mn²⁺ ions present in the gel, which would otherwise interfere with protein transfer.

• 40.Wash the gel in the transfer buffer for 10 min.
• 41.Transfer proteins onto a 0.2 μm nitrocellulose membrane using the Trans-Blot Turbo Blotting system (Bio-Rad) with the preprogrammed protocol (2.5 A, up to 25 V, 7 min).

Note: During the development of this protocol, we tested classical transfer approaches, including tank transfer systems and semi-dry methods. However, only the use of the Trans-Blot Turbo system combined with precut Blotting Transfer Packs allowed for reproducible detection of protein phosphorylation levels.

• 42.Block the membrane in blocking buffer (5% BSA in PBS-T buffer) at 4°C overnight (∼12 h) with gentle shaking.
• 43.Incubate the membrane with the anti-CpxR polyclonal antibodies at a dilution of 1:1,000 in PBS-T buffer supplemented with 2% BSA.
• 44.Incubate at room temperature (20°C) for 1 h 30 min with gentle shaking.
• 45.Wash the membrane three times with PBS-T buffer, 10 min each wash.
• 46.Incubate the membrane with anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP) at a dilution of 1:10,000 in PBS-T buffer.
• 47.Incubate at room temperature (20°C) for 1 h.
• 48.Wash the membrane three times with PBS-T buffer.
• 49.Apply the ECL reagent to the membrane (according to the manufacturer’s instructions.).
• 50.Incubate for 5 min in the dark.
• 51.Remove excess ECL reagent by gently blotting the edges of the membrane with filter paper.
• 52.For Western blot imaging, place the membrane in the Fusion Solo 4M (Vilber) using the mode dedicated to chemiluminescence detection.

Note: Western blot imaging can be performed using different approaches. A more traditional method relies on radiographic films followed by chemical baths for development and detection. However, the reagents involved are toxic. Today, chemiluminescence imaging systems such as the ChemiDoc Imaging System (Bio-Rad), the ImageQuant 800 (Cytiva), or the Solo 4M (Vilber) are commonly used. These systems provide a safer alternative and allow for optimized exposure times, thereby improving signal detection and quantification.

Expected outcomes

The fraction of the phosphorylated proteoform relative to the total pool of the transcriptional regulator serves as a direct indicator of the two-component system’s activation level. Applied to the CpxAR system, targeting the CpxR regulator, this approach enabled us to observe CpxAR activation during the infection of chicory leaves (Figure 3), highlighting its crucial role in plant colonization.¹ Conversely, when this protocol was applied to the EnvZ-OmpR system, targeting the OmpR regulator, we observed that this system was not activated during plant infection.² These two examples from our laboratory demonstrate that the protocol can be easily adapted to different transcriptional regulators.

Figure 3.

Detection of CpxR and CpxR∼P proteoforms

Phosphorylation levels of CpxR in the D. dadantii wild-type strain were assessed before inoculation (in vitro culture), and at 1-, 2-, and 3-days post-inoculation in endive leaves. The cpxR mutant was included as a negative control

(A) Uncropped Phos-Tag acrylamide gel showing the separation of CpxR and its phosphorylated form (CpxR-P), detected by western blot. This result is representative of three independent experiments.

(B) Box-and-whisker plots (Tukey method) show the percentage of phosphorylated CpxR-P relative to total CpxR in the wild-type strain. Data are from at least three independent experiments. Statistical analysis was performed using the One-way ANOVA with Tukey multiple comparisons tests. ∗∗p = 0.004643.

A successful result is characterized by the presence of two well-resolved bands corresponding to the two proteoforms of the regulator of interest, with consistent intensity across biological replicates. Band intensity typically falls within a linear detection range, ensuring accurate quantification. Negative controls (mutant lacking the regulator) consistently show no detectable signal, confirming antibody specificity. The signal was reproducible across at least three independent experiments. When samples were extracted from plants, the standard deviation for normalized band intensities was below 6.4% (5.38% at Day 3, 6.39% at Day 2, and 3.55% at Day 1). In vitro (before inoculation), variability was minimal, with a standard deviation below 1% (0.75% in our case).

Quantification and statistical analysis

Timing: ∼15 min

Western blot signals were quantified using ImageJ software. Blot images were first converted to 8-bit grayscale format, and regions of interest corresponding to protein bands were selected using a uniform rectangular selection box. For each band, the integrated density (area × mean gray value) was measured. Background correction was performed by subtracting the integrated density of a nearby blank area from each band’s value. The corrected values were exported and analyzed using GraphPad Prism.

Limitations

Our protocol reliably allows for the reproducible determination of the ratio between phosphorylated and non-phosphorylated protein forms. The main limitation in applying this method lies in the time elapsed between bacterial extraction from the host and sample loading onto the gel. Our approach addresses this by enabling this critical step to be performed in under 10 min.

This gel-based approach requires a highly specific antibody, free from non-specific bands. If no antibody is available for the regulator of interest, the protein can be tagged (e.g., with c-Myc), and detection can be achieved using a monoclonal antibody against the tag in the Western blot. Antibody specificity is verified by standard SDS-PAGE and Western blot analysis on both the wild-type strain and the regulator mutant, to confirm the presence of a single band corresponding to the cytoplasmic regulator of interest and the complete absence of any nonspecific bands.

Lastly, we anticipate that this protocol can be readily adapted for regulators other than CpxR and for use in different models. However, some steps may require optimization. For instance, extracting bacteria from tissues that are not macerated or disorganized may prove more challenging. Likewise, bacterial lysis efficiency may vary depending on the species and cell wall properties, requiring further optimization. We recommend conducting preliminary pilot experiments using bacterial cultures before applying the method in an infection context.

Troubleshooting

Problem 1

The phosphorylated and non-phosphorylated forms are not well resolved.

Potential solution

Several parameters can be optimized to improve band separation. These include increasing the migration time, adjusting the Phos-Tag concentration, and/or the acrylamide concentration depending on the protein’s molecular weight. Additionally, although our protocol uses Mn²⁺, Zn²⁺ can also be used as an alternative. This necessitates empirical testing for each protein of interest.

Problem 2

The gel did not polymerize properly.

Potential solution

In the rare event of polymerization failure (e.g., incomplete gelation or abnormal band migration), we systematically verified the freshness and concentration of APS and TEMED, ensured proper pH of the gel solution, and confirmed that the Phos-tag reagent had not degraded.

Resource availability

Lead contact

Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Sébastien Bontemps-Gallo (sebastien.bontemps-gallo@cnrs.fr).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to and will be answered by the technical contact, Edwige Madec (edwige.madec@univ-lille.fr).

Materials availability

This study did not generate any new reagents. The anti-CpxR antibody was produced in our laboratory and is available upon request, subject to stock availability.

Data and code availability

The published articles include all datasets analyzed during these studies.

Acknowledgments

The authors thank Peggy Gruau for her assistance in preparing this manuscript. The study was funded by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (Inserm), the Institut Pasteur de Lille, and the Université de Lille. This project has received financial support from the CNRS through the MITI interdisciplinary programs and the Agence National de la Recherche (ANR-21-CE15-0047). Figures were created using BioRender.

Author contributions

Conceptualization: E.M., J.-M.L., and S.B.-G. Investigation and data analysis: E.M., J.-M.L., and S.B.-G. Writing – original draft: E.M. and S.B.-G. Writing – review and editing: E.M., J.-M.L., and S.B.-G. Funding acquisition: S.B.-G.

Declaration of interests

The authors declare no competing interests.

Declaration of AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used ChatGPT 4o to improve language and readability. After using this service, the authors reviewed and edited the content as needed and take full responsibility for the publication’s content.