In this study, A. pleuropneumoniae strain S4074 was used as a representative strain, which was grown at 37°C in tryptic soy broth (TSB; BD, United States) added with 10% FBS and 10% (vol/vol) nicotinamide adenine dinucleotide (NAD; Biofroxx, Germany). The wild-type S4074 and in-frame mutant strain ∆cpxRA were donated by Prof. Weicheng Bei (Li et al., 2018). Escherichia coli β2155 was cultured at 37°C in Luria-Bertani (LB) with 50 μg/mL diaminopimelic acid (DAP; Sigma, USA). The bacterial strains used in this study are listed in Table 1, and primers are described in Table 2. The recombinant plasmid pJFF224-PftsEX with IPTG-inducible promoter was electrically transferred to the ∆cpxRA mutant strains to regulate the ftsEX gene expression under the different concentrations of IPTG.

For scanning electron microscopy (SEM), wild-type S4074 and its mutant derivatives were harvested after they were cultured in TSB medium supplemented with 10% FBS and 10% NAD with or without the supplementation of 0.3 M KCl. Bacterial cells were fixed with 2.5% glutaraldehyde and deposited onto copper grids (200 mesh; Zhongjingkeyi, China). The copper grids were air-dried, mounted on the sample stub and coated with gold. Subsequently, strains were observed by the SEM (VEGA3; TESCAN, Czech) and the bacterial length was measured by Image J (NIH, United States).

WT and ∆cpxRA mutant strains were cultivated at 37°C shaking in TSB supplemented with 10% FBS and 10% NAD to an OD600 of 0.6, centrifuged (3,000 g, 5 min) to gather cells. Total RNA of strains was extracted using Total RNA Extractor kit (Sangon Biotech, China) according to the manufacturer’s instruction. DNA impurity was eliminated using DNase I kit (Vazyme, China) according to instruction. To determine the quality, the RNA was electrophoresed on a 1% agarose gel. cDNA was synthesized using reverse transcriptase from Vazyme. Quantitative Real-Time PCR was performed using Real-Time Quantitative PCR detecting System and SYBR qPCR Mix (Vazyme, China). 16S RNA was used as the endogenous control to normalize expression of target genes. The 2^−ΔΔCt method was used to calculate and analyze relative expression level of mRNA (Livak and Schmittgen, 2001). RT-PCR across the cdd-ftsX, ftsX-ftsE, ftsE-ftsY, and ftsY-rsmD junctions was performed as previously described (Liu et al., 2022a).

A ftsE-gfp fusion containing the ftsE promoter region and gfp gene, was cloned into the Xho I and Not I sites on the pJFF224-XN plasmid. Then, the ftsE-gfp reporter plasmid was electroporated into the wild-type S4074 and the ∆cpxAR mutant strain. A. pleuropneumoniae strains harboring a ftsE-gfp reporter plasmid were grown to an OD600 of 0.6 in TSB medium with or without the supplementation of 0.3 M KCl. The cells were harvested and resuspended in 1 mL of 10 mM PBS. Luminescence was measured in the Spectramax iD3 microplate reader with excitation at 485 nm and emission at 535 nm.

The purification of CpxR was conducted as previously described (Liu et al., 2022b). The E.coli BL21 containing the expression vector pET30a-CpxR was used to express His-CpxR protein. Expression of His-CpxR was induced by IPTG (0.5 mM) at 16°C overnight, and bacteria were harvested by centrifuging at 12000 rpm for 10 min. The cell pellet was resuspended in binding buffer (50 mM Na₃PO₄, pH 7.4, 500 mM NaCl, 30 mM imidazole), and lysed by sonication. The soluble fraction of the lysate was added to nickel-nitrilotriacetic acid (Ni-NTA) agarose column and mixed on Multipurpose Shaker QB-206 for 3 h. After being washed with binding buffer, the His-CpxR protein was eluted with elution buffer (20 mM Tris–HCl, pH 7.4, 500 mM NaCl, 500 mM imidazole). The purified His-CpxR was then stored at −80°C until use.

The promoter regions of target genes were amplified from the A. pleuropneumoniae S4074 genome and tagged with biotin using a EMSA Probe Biotin Labeling Kit (Beyotime, China). CpxR protein was phosphorylated by acetyl phosphate (Sigma, United States) (Pogliano et al., 1997). Biotin-labeled DNA probes (1 μM) were incubated at room temperature for 20 min with 0–4 pmol CpxR protein in binding buffer (Beyotime, China). The reaction mixtures were analyzed by 4% non-denaturing polyacrylamide gel electrophoresis and transferred to nylon membrane. Next, the band was detected using a Chemiluminescent EMSA Kit (Beyotime, China).

Experimental data were analyzed by Student’s two-tailed t-tests using GraphPad Prism 7.0 (GraphPad lnc.), and shown as mean ± standard deviation (SD). Statistical significance was assumed at a p value of <0.05.

To explore the role of CpxAR in bacterial adaptation to osmotic stress in A. pleuropneumoniae, we tested the growth traits of the wild-type S4074 and its cpxRA mutant strain grown in solid or liquid medium with or without the supplementation of 0.3 M KCl or 0.3 M NaCl. When the cells were grown in solid medium, the growth of the ∆cpxRA strain was markedly reduced with the supplementation of 0.3 M KCl, but similar to that of the WT strain with the supplementation of 0.3 M NaCl or normal medium (Figure 1A). Optical density and colony forming units showed that the growth rate of the ∆cpxRA strain was significantly lower than that of the WT strain when grown with the supplementation of 0.3 M KCl (Figures 1B,C). These findings suggested that the growth defect of the mutant strain ∆cpxRA was significantly increased compared with that of WT and C∆cpxRA strains under high-K⁺ stress. These results suggested that CpxAR contributes to A. pleuropneumoniae survival under high potassium stress.

To further investigate how the inactivation of cpxRA genes affected the growth of A. pleuropneumoniae under high-K⁺ stress, we used scanning electron microscope (SEM) to observe the bacterial morphology of the WT, ∆cpxRA and C∆cpxRA strains when they were grown with or without the supplementation of 0.3 M KCl. When grown with the supplementation of 0.3 M KCl, the cell length of ∆cpxRA strain showed a 2-fold increase on average compared to the wild-type and C∆cpxRA strains (Figures 2A,B). In addition, we found that the cell length of ∆cpxRA strain was also longer than that of the wild-type and C∆cpxRA strains without K⁺ stress, but the difference was much smaller than that under K⁺ stress (Figures 2A,B). Together, these findings suggested that CpxAR regulates cell division to help A. pleuropneumoniae cope with potassium stress.

To gain insight into the mechanism of CpxAR affecting the cell division of A. pleuropneumoniae, we compared the transcript levels of cell division genes in the WT and ∆cpxRA strains by qRT-PCR. As shown in Figure 3A, the relative transcript levels of ftsE and ftsX were significantly downregulated in the ∆cpxRA strain with or without the supplementation of 0.3 M KCl, but ftsY was not (Figure 3A). However, there were no significant changes in the cell division genes ftsL, ftsI, ftsW, ftsQ, ftsZ, ftsB, ftsA, zipA, ftsH, and ftsK in the ∆cpxRA strain compared with the WT strain (Supplementary Figure S1).These results suggested that CpxAR regulates the expression of the cell division genes ftsE and ftsX in A. pleuropneumoniae.

The cell division genes ftsY, ftsE, and ftsX are adjacent on the A. pleuropneumoniae chromosome (Figure 3B). To characterize the ftsYEX locus, we performed RT-PCR across the cdd-ftsX, ftsX-ftsE, ftsE-ftsY, and ftsY-rsmD junctions. The RT-PCR analysis indicated that the ftsY, ftsE, and ftsX genes are co-transcribed as a single mRNA and comprise an operon. To verify whether the ftsEX operon contributes to A. pleuropneumoniae survival under high-K⁺ stress, the growth of ∆cpxRA/PftsEX was assayed. When the expression of FtsE and FtsX increased with the increase of IPTG concentration, the growth defects of the ∆cpxRA strain were significantly rescued (Figure 3C). These observations indicated that the ftsEX operon plays an important role in A. pleuropneumoniae response to high-K⁺ stress.

Here, the prediction results (BDGP)¹ showed that the ftsYEX operon has two putative promoter regions, respectively, located upstream of ftsY and ftsE. To explore the mechanism by which CpxR regulates the ftsYEX operon expression, we tested whether CpxR-P binds to the promoter regions of ftsY and ftsE using gel shift analysis. EMSA analysis showed that CpxR was capable to bind with the promoter regions of ftsE and rpoE (positive control), but not to the promoter regions of ftsY and rpoD (negative control) (Figure 3D). In addition, EMSAs showed that phosphorylated CpxR did not bind to the promoter sequence of ftsE when 200X of unlabeled probes were added (Figure 3E). To examine the link between CpxR and the expression of the ftsEX operon, the GFP reporter strains for ftsE gene promoter were constructed. As shown in Figures 3F,G, fluorescence intensities and confocal microscopy analysis showed that the transcrition activity of the promoter was significantly decreased in ∆cpxRA strain with or without high-K⁺ stress. Together, these data demonstrated that CpxAR directly regulates the expression of ftsEX operon.