The bacterial strains and plasmids, as well as primers used in this study, are listed in Tables 1,2. A. pleuropneumoniae cells were grown in tryptic soy broth (TSB; Solarbio, China) or on TSB agar plates supplemented with 10 μg/ml NAD (Sigma-Aldrich, United States) and 10% fetal bovine serum (FBS; Every Green, China). E. coli cells were grown in LB medium supplemented with 50 μg/ml diaminopimelic acid (Sigma-Aldrich, United States) or relevant antibiotics (chloramphenicol, 5 μg/ml; kanamycin, 50 μg/ml). The ΔcspC, ΔcspD and CΔcspC strains were generated using the suicide plasmid pEMOC2 and the shuttle plasmid pJFF224-XN as described earlier (Liu et al., 2022a). In order to induce gene expression, plasmid pJFF224-PcspC which expressed the cspC gene under the control of IPTG-inducible promoter was constructed, and electrically transferred to the mutant strain ΔcpxAR.

Bacterial cells grown overnight in TSB medium at 37°C were subcultured 1:1000 in fresh TSB broth. Cells grown up to the exponential phase (OD600; 0.6) were exposed to 4°C for a series of times. Total RNA was extracted from bacterial cultures using the Bacteria Total RNA Isolation Kit (Sangon Biotech, China). The synthesis of complementary DNA (cDNA) was achieved using the HiScript II Q RT SuperMix for qRT-PCR (Vazyme, China). For quantitative RT-PCR, the reactions were performed using SYBR qPCR Mix (Vazyme, China) and run in the CFX96 Real-Time System (Bio-Red, United States). Data were normalized using the 16S rRNA as internal control, and calculated using the 2^−ΔΔCt method.

A cspC/cspD-lacZ fusions containing the promoter region of cspC/cspD and the lacZ gene, and cloned into the Xho I and Not I sites of the plasmid pJFF224-XN. Then, the recombinant plasmid was introduced into the wild-type and the ∆cpxAR mutant strain. All strains were grown overnight in TSB broth at 37°C, diluted 1:1,000 in fresh TSB broth, and grown at 20°C for a series of times. The collected culture was assayed for β-Galactosidase activity by using a β-galactosidase (β-GAL) Activity Assay Kit (Micromethod; Sangon Biotech) according to the manufacturer’s specification.

The His6-CpxR protein was expressed using E. coli BL21 (DE3)-containing pET30a-CpxR and purified by using a Ni-nitrilotriacetic acid (Ni-NTA) resin affinity chromatography (Qiagen, Germany). The purified protein was phosphorylated by acetyl phosphate (Sigma, United States) according to previously described procedures (Li et al., 2018). DNA probes were amplified and purified and then labeled using a Biotin Labeling Kit (Beyotime, China). Then, the phosphorylated CpxR and labeled probes were used for protein-DNA EMSAs as described previously (Cheng et al., 2021). Labeled DNA probe (1 μM) and various concentrations of phosphorylated CpxR protein (0–4 pmol) were incubated at 24°C for 20 min in reaction buffer (50 mM Tris–HCl, pH 8.0, 2.5 mM MgCl₂, 100 mM KCl, 0.2 mM DTT, 10% glycerol, 2 μg salmon sperm DNA). For competition experiments with unlabeled DNA probes, a 100-fold molar excess was preincubated with phosphorylated CpxR protein. The reaction mixtures were loaded on a 4% non-denaturing polyacrylamide electrophoresis in 0.5 × Tris-borate-EDTA (TBE) buffer. The bands of labeled probes were subsequently transferred to nylon membrane (Beyotime, China), and detected using the Chemiluminescent EMSA Kit (Beyotime, China).

To measure cold growth, the A. pleuropneumoniae strain S4074 and its mutant derivatives were cultured in TSB medium overnight at 37°C, then diluted 1:100 (~1 × 10⁷ CFU/ml) into fresh TSB broth and incubated at 20°C for 48 h. Every 4 h for 48 h, samples were serially diluted and plated on TSB agar, and the OD600 was measured.

The −10 and − 35 promoter regions, and transcriptional atart site (TSS) of the cold shock gene cspC were, respectively, predicated by BPROM (Prediction of bacterial promoters)¹ and Berkeley Drosophila Genome Project (BDGP).² All data were analyzed using two-tailed Student’s t-tests (GraphPad Prism version 7.0, GraphPad Software, inc., San Diego, United States), and presented as mean ± standard deviation (SD). p < 0.05 was considered statistically significant.

To investigate whether CpxAR is involved in the cold adaptation of A. pleuropneumoniae, we tested the growth rates of the WT, ∆cpxAR and C∆cpxAR strains when exposed to cold stress (20°C). Under cold stress, the growth rate of the mutant strain ∆cpxAR was significantly lower than that of the WT and C∆cpxAR strains (Figures 1A,B). Our previous study found that the growth rate of ∆cpxAR was also lower than that of the WT at 37°C, and the difference was obviously much smaller than that at 20°C (Li et al., 2018). The qRT-PCR analysis showed that the mRNA level of the cpxA and cpxR genes were upregulated under cold stress in the WT strain (Figures 1C,D). These results suggest that CpxA/CpxR plays an important role in cold adaptation of A. pleuropneumoniae.

Analysis of the A. pleuropneumoniae genome revealed that it contains two Csp proteins (CspC and CspD) which have a high amino acid identity with E.coli CspA (Figure 2A). Each protein possesses a conserved cold shock domain (CSD) that harbors two nucleic acid-binding motifs RNAP1 and RNAP2.

To elucidate the mechanism of CpxA/CpxR affecting APP cold adaptability, β-Galactosidase assay was performed to examine the link between CpxR and the cold shock genes cspC and cspD. As shown in Figures 2B,C, the promoter activity of cspC-lacZ under cold stress were significantly elevated in the WT strain, and decreased in the ∆cpxAR mutant compared with the former, but the the cspD gene was not. These findings indicated that CpxA/CpxR regulates the expression of the cold shock gene cspC which is cold inducible in A. pleuropneumoniae.

The cold shock gene cspC is adjacent to the gene bioB and proQ in the A. pleuropneumoniae chromosome (Figure 2D). In order to describe the cspC gene, we performed RT-PCR across the bioB-cspC and cspC-proQ junctions. The RT-PCR analysis indicated that the cspC gene is transcribed separately from the proQ gene.

To investigate whether CspC and CspD are involved in cold stress, we generated the ∆cspC and ∆cspD mutant strains, and the C∆cspC complemented strain, and tested the growth rates of the WT, ∆cspC, ∆cspD and C∆cspC strains at 20°C and 37°C. Under cold stress (20°C), the ∆cspC mutant exhibited growth defects, whereas the WT, ∆cspD and C∆cspC strains displayed normal growth (Figures 3A,B). When the bacteria were cultured at 37°C, the growth property of ∆cspC mutant was similar to those of the WT and C∆cspC strains (Figures 3C,D). The growth of ∆cpxAR/PcspC in liquid media was then assayed. Clearly, when CspC was produced with IPTG at 0.1 mM and above, the growth defect of the ∆cpxAR mutant strain was significantly rescued (Figures 3E,F). These results suggested that CspC is essential for cold growth in A. pleuropneumoniae.

Since CpxR is a response regulator, we examined the interaction between the recombinant CpxR protein and the putative cspC promoter region using EMSA. As shown in Figure 4A, incubation of biotin-labeled cspC with CpxR protein led to the formation of DNA-protein complexes in a protein concentration-dependent manner. Meanwhile, no CpxR-DNA complex was observed with adding excess amounts of unlabeled probes, suggesting that the CpxR could bind specifically to the cspC promoter region (Figure 4B).

Previous studies found that the CpxR binding site has a conserved sequence GTAAA-(N)_4–8-GTAAA (Bruna et al., 2018; Jia et al., 2022). In this study, we found that a 16-nt region (5’-GGAAAACATCGATAAA-3′) was relatively consistent with the characteristics of conserved sequence. As shown in Figure 4C, CpxR-P was unable to bind to a mutant cspC putative promoter region in which the 16-nt region was deleted from the putative CpxR-P binding box. In order to describe the CpxR binding site in detail, the cspC promoter region and transcription start site were, respectively, analyzed by BPROM and BDGP. As shown in Figure 4D, a putative −10 AGAATTGC box, −35 TGGAGA box and TSS were detected, and, respectively, located 30 bp, 50 bp and 12 bp upstream of the start codon.