A marker-free genetic manipulation method for G. parasuis strains developed by alternately culturing transformants at 37°C and 30°C

doi:10.21203/rs.3.rs-4705811/v1

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Research Article

A marker-free genetic manipulation method for G. parasuis strains developed by alternately culturing transformants at 37°C and 30°C

https://doi.org/10.21203/rs.3.rs-4705811/v1

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published 03 Sep, 2024

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Background

Glaesserella parasuis (G. parasuis) is a significant pathogen causing Glässer’s disease and substantial financial losses in swine production systems. However, studies on its gene function and pathogenesis are limited due to the lack of a broad marker-free knockout system. Currently, researchers mainly use antibiotic-resistant marked knockout mutants. The expression of antibiotic-resistance genes in bacterial cells can cause polar termination and unpredictable effects. Although there have been precedents in G. parasuis using gene targeting to construct marker-free mutant strains, this method is time-consuming, labor-intensive, and has an extremely low success rate. Therefore, there is an urgent need for an efficient and feasible marker-free genetic manipulation method for G. parasuis strains.

Results

In this study, we developed an optimized marker-free knockout system for G. parasuis based on a temperature-sensitive vector. By culturing the transformants at alternating temperatures of 37°C and 30°C, we used this system to knock out the Kan^R cassette from the marked mutant JS0135ΔnanH::Kan^R. To verify that temperature changes were key to this method, we successfully knocked out the nanH and apd genes in the CF7066 strain using this system. Subsequently, we found that knocking out the nanH gene severely reduces the growth vitality of the strains, while knocking out the apd gene can improve the adhesion rate of the strains. Finally, we observed that the expression of recombinant genes in the transformants was higher at 30℃ than at 37℃, but there was almost no difference in the expression of recombinant genes between 30℃ and 37℃ in wild strains. This difference may be due to a higher copy number of target plasmids at 30℃, leading to the upregulation of the expression of recombinant genes.

Conclusions

Overall, this newly developed gene knockout system for G. parasuis could be a valuable tool for the study of this organism.

G. parasuis

homologous recombination

temperature-sensitive plasmids

marker-free knockout

Glaesserella parasuis is a gram-negative bacterium that belongs to the Pasteurellaceae family [1]. This organism is known to cause Glässer’s disease, which is characterized by various symptoms, including fibrinous polyserositis, arthritis, meningitis, acute pneumonia, and septicemia[2–4]. Glässer’s disease, a significant bacterial disease of swine, has been reported on swine farms worldwide. [5–7]. In China, the isolation ratio of G. parasuis was up to 22.6% in weaned swine and 9.7% in healthy swine herds [5, 8]. Therefore, gaining insight into the pathogenesis of Glässer's disease and the interaction between G. parasuis and its host is crucial. To achieve this goal, it is essential to carefully evaluate specific phenotypes, and the development of a marker-free gene knockout system is necessary.

Most strains of G. parasuis possess stringent restriction-modification systems that impede the uptake of heterogeneous plasmids, particularly through electrotransformation [9, 10]. Consequently, allelic exchange knockout mutants of G. parasuis strains are often constructed using natural transformation with suicide plasmids. These plasmids contain antibiotic resistance genes flanked by homologous regions upstream and downstream of the target gene that is to be deleted. As a result, the resulting knockout mutants are marked with antibiotic resistance [9, 11–13].

While marked mutants are valuable for investigating the molecular pathogenesis of G. parasuis, they may also exhibit antibiotic resistance gene drift and other unpredictable polar effects. Moreover, these mutants are not suitable for use in commercial vaccines due to their presence of exogenous antibiotic resistance genes. In our preliminary research, we established a markerless gene knockout system in G. parasuis based on the Flp-FRT system; however, it still leaves FRT sites on the genome[14]. Thus, a marker-free knockout approach is necessary for studying G. parasuis pathogenesis and developing genetically engineered vaccines. A sacB-based counterselectable marker system has been employed to generate marker-free knockout mutants in G. parasuis; however, this method requires two natural transformations of two donor plasmids and is restricted to G. parasuis strains lacking spontaneous sucrose resistance [15].

In the present study, transformants carrying temperature-sensitive (TS) donor plasmids were cultured alternately at 37°C and 30°C to ensure genomic DNA replication and plasmid maintenance. Our results demonstrated successful knockout of target genes in various G. parasuis strains, leading to the establishment of a comprehensive marker-free gene knockout system for this organism.

Bacterial strains and growth conditions

The bacterial strains used in the study are listed in Table 1. The Escherichia coli strain DH5α was grown in Luria–Bertani broth (LB; Difco, Detroit, MI, USA) or on LB agar. The G. parasuis strains were grown in tryptic soy broth (TSB; Difco) or on tryptic soy agar (TSA; Difco) supplemented with 10 µg/mL NAD and 5% bovine serum. When needed, media were supplemented with kanamycin (50 µg/mL) or gentamicin (20 µg/mL). The G. parasuis JS0135 (CCTCC AB 2021004) and CF7066 (CCTCC AB 2021003) strains were isolated in China and stored in our laboratory and at the China Center for Type Culture Collection. The original transformed recipient strain JS0135ΔnanH::Kan^R was generated through natural transformation with pK18-nHUD according to a previously published method [9]. The CF7066ΔnanH-N control mutant was constructed using a modified NgAgo system [16] (Table 1).

Cell culture

Porcine kidney 15 (PK-15) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) at 37°C with 5% CO₂.

Cloning and genetic manipulation

The plasmids used in the study are listed in Table 1. The primers for plasmid construction and mutant identification were designed based on the sequence of G. parasuis SH0165 (CP001321.1; Table S1). To construct the TS vector pSHG5, a fragment containing pUC ori and pA13 ori^TS was amplified from pSHK3^TS, and the gentamicin cassette was amplified from pSHK3-Gm [17, 18]. The resulting fragments were fused using a one-step cloning kit (Vazyme Biotech, Nanjing, China). For pSHG5-ΔnanH, the flanking homology arms of the deleted nanH allele from JS0135 (MT625975) were amplified using pK18-nHUD as a template and then ligated into pSHG5. For pSHG5-Δapd, the flanking homology fragments of the deleted apd allele were obtained from G. parasuis CF7066 (MK617354) and fused with the vector pSHG5 using overlapping polymerase chain reaction (PCR) and a one-step cloning kit. Genetic constructs and PCR fragments were sequenced using Sanger sequencing (TsingKe Biotech, Beijing, China; Table 1, Table S1, and Fig. S1).

Electrotransformation of G. parasuis

Electrotransformation of G. parasuis was performed using a modified method established by Lancashire et al. [19]. In brief, G. parasuis strains were grown on TSA plates at 37°C for 12–14 h. Bacterial cells were then harvested in sucrose/glycerol (SG) buffer (15% [v/v] glycerol, 272 mM sucrose), washed three times, and resuspended in 0.12 mL of SG buffer per sample. After electroporation (using a Bio-Rad MicroPulser Electroporator [Bio-Rad Laboratories, Hercules, CA, USA]; 2.5 kV, 0.2 cm cuvettes) with 5 µg of plasmid DNA, the cells were suspended in 1 mL of TSB medium, grown at 37°C for 2 h, and then spread on TSA plates supplemented with 20 µg/mL gentamicin.

Construction of marker-free knockout mutants in G. parasuis

To generate crossover mutants, recipient strains were electroporated with donor plasmids and grown on gentamicin plates at alternating temperatures of 37°C and 30°C every 12 hours. After several passages, single or double crossovers colonies were screened by PCR using primers targeting the flanking regions of upstream and downstream homologous arms in the genome. Once a colony with a double crossover event was identified, it was cultured at 42°C to cure the plasmid. To generate JS0135ΔnanH and CF7066ΔnanH, the double-crossover colony was further cultured at 42°C until the optical density at 600 nm (OD₆₀₀) reached 0.5 and then spread on gentamicin-free TSA plates to isolate cells of different genotypes. For CF7066Δapd, double-crossover colonies were directly streaked on TSA plates at 42°C overnight and subsequently identified by PCR.

Detection of NanH and Apd using western blotting

The expression of NanH and Apd in the wild-type and deletion strains was assessed by western blotting using anti-NanH and anti-Apd mouse sera obtained from recombinant proteins, respectively. Newly harvested bacterial cells were subjected to sodium dodecyl sulfate‒polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. After blocking and washing, the membranes were incubated with 1:5000 of antisera specific for recombinant Apd and NanH in TBST buffer (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.4) containing 5% skim milk. The membranes were then exposed to horseradish peroxidase-labeled goat anti-mouse IgG (ABclonal, Inc., Wuhan, China) at a 1:5000 dilution in TBST buffer containing 5% skim milk, and the bound antibodies were visualized using Clarity Western ECL Substrate (Bio-Rad Laboratories).

Bacterial growth curve assays

The strains were activated on TSA plates for three generations, and then single colonies were inoculated into 5 mL of TSB media, cultured with shaking at 37°C/200 rpm for 12 h, and transferred to fresh TSB media at a 1:100 dilution. Next, 200 µL of bacterial solution was added to each well of the special detection plate suitable for the Bioscreen automatic growth curve analyzer. The OD₆₀₀ of the bacterial solution was measured every 30 minutes, and the growth curve was recorded and plotted.

PK-15 cell adhesion and invasion assay

G. parasuis CF7066, JS0135 and the mutants were streaked on TSA plates and cultured at 37°C for 16 h. The bacterial cells were then harvested on TSB media, and the OD₆₀₀ was adjusted to 1.0. PK-15 cells were grown to confluent monolayers in 24-well plates and infected with G. parasuis at a multiplicity of infection (MOI) of 0.02. The plates were centrifuged at 800 × g for 10 min to transport bacteria to the surface of the monolayer and incubated at 37°C for 2 h. Thereafter, 100 µL of medium and 900 µL of ice water were added to each well, and the plates were incubated for 15 min at 4°C. The cells were then vigorously washed three times with PBS to eliminate nonspecific bacterial attachment and incubated for 15 min at 4°C. Then, the cells were disrupted by repeated pipetting to liberate bacteria. Serial dilutions of the cell lysate were plated onto TSA and incubated for 48 h at 37°C. Adhesion and invasion rate = CFU cell-associated/(CFU in medium + CFU cell-associated). All assays were repeated at least three times in independent experiments.

Quantitative reverse-transcription PCR (qRT‒PCR)

Total RNA was extracted from the bacterial cells using the HiPure Bacterial RNA Kit (Magen, Guangzhou, China), and 1 µg of RNA was reverse transcribed into cDNA using the HiScript II Q RT SuperMix Kit (Vazyme Biotech, Nanjing, China) according to the manufacturer’s protocol. qRT‒PCR was performed using a ChamQ Universal SYBR qPCR Master Mix Kit (Vazyme Biotech, Nanjing, China) with the primers listed in Supplementary Table S1, and the results were normalized to those for GAPDH. The G. parasuis harboring plasmid pSHG5-Δapd was streaked on TSA plates, divided into two groups and cultured at 37°C or 30°C. Bacterial cells were harvested, and the OD₆₀₀ was adjusted to 1.0. For each bacterial solution group, 1 µL of the sample was used for qRT‒PCR. All assays were repeated at least three times in independent experiments.

Table 1

Strains and plasmids used in this study.
Strains or plasmids	Genotype or characterization	Source or reference
Escherichia coli
DH5α	F⁻, φ 80dlacZΔM15, Δ(lacZYA-argF) U169, deoR, recA1, endA1, hsdR17 (rK⁻,mK⁺), phoA, supE44, λ⁻, thi − 1, gyrA96, relA1	TaKaRa Bio Inc., Dalian, China
Glaesserella parasuis
SH0165	Wild type, serotype 5	[20]
JS0135	Wild type, serotype 4	This laboratory
JS0135ΔnanH::Kan^R	ΔnanH::Kan^R, generated via natural transformation of pK18-nHUD	This laboratory
CF7066ΔnanH-N	ΔnanH, constructed by NgAgo deletion system	This study
JS0135ΔnanH	ΔnanH, constructed by temperature-sensitive system	This study
CF7066	Wild type, serotype 5	This laboratory
CF7066ΔnanH	ΔnanH, generated via temperature-sensitive system	This study
CF7066Δapd	ΔapD, generated via temperature-sensitive system	This study
Plasmids
pK18 mobsacB	pUC ori, Kan^R, suicide vector of G. parasuis	[21]
pK18-nHUD	pUC ori, Kan^R, ΔnanH, from pK18 mobsacB, containing upstream and downstream regions of nanH to be deleted	This study
pSHK3-Gm	Gm^R, pUC ori, pYC93 ori, shuttle vector of G. parasuis	[18]
pSHK3^TS	Kan^R, pUC ori, pA13 ori^TS, temperature vector of G. parasuis	[17, 22]
pSHG5	Gm^R, pUC ori, pA13 ori^TS	This study
pSHG5-ΔnanH	Gm^R, pUC ori, pA13 ori^TS, ΔnanH	This study
pSHG5-ΔapD	Gm^R, pUC ori, pA13 ori^TS, ΔapD	This study

Knockout of the Kan^R cassette in JS0135ΔnanH::Kan^R

Considering the potential resistance to the knockout of endogenous genes, we first attempted to knock out the exogenous Kan^R gene in JS0135. Compared to the parent strain JS0135ΔnanH::Kan^R, the KanR knockout strain did not exhibit growth defects and was more suitable for subsequent mutant screening. Therefore, we used a more efficient natural transformation method, as described above, to replace the nanH gene with Kan^R in the G. parasuis strain JS0135, and we obtained the marked mutant JS0135ΔnanH::Kan^R.

To knock out the Kan^R gene, the donor plasmid pSHG5-ΔnanH carrying the homologous arms of nanH was electroporated into JS0135ΔnanH::Kan^R. However, JS0135 has strong resistance to electroporation due to its abundant restriction-modification systems, which results in the generation of only one transformant without recombination after multiple attempts (Fig. 1a and b). Notably, the colonies grew better at 37°C, but the temperature-sensitive plasmid pSHG5-ΔnanH was fully functional below 31°C. To meet different temperatures, after being incubated for 2 days on gentamicin TSA plates at 37°C, the transformants were streaked on fresh gentamicin TSA plates and cultured alternately at 37°C and 30°C for one passage (24–48 h). Newly grown single colonies were picked and cultured in the same way for another generation; after five generations, we found that most of the colonies exhibited multiple genotypes (Fig. 1c). The bands seen in the gel can be interpreted as four genotypes of JS0135 mutants: the 3239 bp band corresponded to the genotype of the parent strain (ΔnanH::Kan^R) without recombination; the 2330 bp band corresponded to the genotype of the knockout mutant (ΔnanH) resulting from double crossover with the parent strain; and the 6650/10061 bp bands corresponded to the single/twice crossover genotype where the plasmid could integrate into the parent strain genome via the upstream or downstream homologous arms (Fig. 1a and c). Interestingly, a faint band near the single crossover band (6650 bp) was found after inverting the image colors, which was speculated to correspond to plasmid integration into the knockout mutant genome (Fig. S2).

To further screen for the knockout mutant, we selected colony No. 4, which showed a relatively bright band for the double-crossover genotype. Following inoculation into gentamicin-free TSB and culture to the logarithmic growth phase, the cells were spread on gentamicin-free TSA plates at 42°C. Finally, we successfully isolated Kan^R knockout colonies as the only genotype; 90% of the colonies exhibited the Kan^R cassette knockout genotype (Fig. 1d). The double and single crossover PCR fragments were purified and identified through Sanger sequencing (Fig. 1e and Fig. S3).

Figure 1. Knockout of the Kan^R cassette from the marked mutant JS0135ΔnanH::Kan^R. (a) Schematic diagram illustrated the process for generating the marker-free mutant JS0135ΔnanH from the marked mutant JS0135ΔnanH::Kan^R. SCO, the single crossover genotype with one entire plasmid integrated into the genome. (b) Only one transformant grew, and PCR analysis using primers nanH-F1/R1 flanking the upstream and downstream homologous arms of nanH (3239 bp) showed that this transformant had no genotype change compared to JS0135ΔnanH::Kan^R. The nanH knockout mutant, CF7066ΔnanH-N, was generated by the NgAgo gene deletion system and served as the positive control. C, negative control. (c) Multiple genotypes (2330, 3239, 6650, and 10061 bp) were observed in progeny colonies after culturing alternately at 30°C and 37°C for five generations and were then screened by colony PCR analysis. Colony No. 4 was selected to culture at 42°C for two passages using the process described above. (d) The marker-free nanH knockout mutants (2330 bp) were detected in 90.0% (18/20) of colonies. (e) Sanger sequencing was conducted to confirm the antibiotic-resistance marker had been eliminated.

Knockout of the nanH gene in wild-type CF7066

A previous study by our laboratory failed to screen mutants at 30°C based on a traditional temperature-sensitive plasmid method (unpublished). However, this time, we successfully screened the mutants and observed many recombinant genotypes. We speculated that the key to the success of this method was the alternate culture temperature of the transformants. To further confirm that alternating culture temperatures at 37°C and 30°C contributed to the accumulation of recombinants, we electroporated CF7066 with the pSHG5-ΔnanH plasmid and cultured it on gentamicin plates at 37°C/30°C, with alternating temperatures every 12 hours. Among 21 transformants picked randomly, colonies No. 1 and 14 had a single crossover genotype (5929 bp), and the other transformants were wild type (2518 bp) (Fig. 2a and b).

As expected, colony No. 1 (Fig. 2b), which was subjected to alternating streaking at 37°C and 30°C, gave rise to the nanH knockout genotype (1610 bp) eight passages later (Fig. 2c). Colony No. 21 (Fig. 2c) showed that the brightest band corresponding to the ΔnanH genotype was further cultured, and the nanH knockout colonies were successfully isolated using the method described above (Fig. 2d). As a result, the nanH knockout genotype was found in 7.6% (8/105) of the colonies (Fig. 2d and Fig. S4). Sanger sequencing confirmed the knockout of nanH (Fig. 2e).

Most colonies in the CF7066ΔnanH screen showed restoration of the wild-type genotype (Fig. 2d and Fig. S4), in contrast to those in the JS0135ΔnanH generation, in which most colonies exhibited the knockout genotype (Fig. 1d). This may be due to the growth advantage conferred by the dominant genotype: JS0135ΔnanH was the dominant growth strain compared to JS0135ΔnanH::Kan^R because the expression of Kan^R had no effect on its growth on nonselective plates but only increased the growth burden; similarly, due to the lack of nanH, which may play an essential role in strain metabolism, CF7066 was the dominant growth strain compared to CF7066ΔnanH. During the subculture process, this growth advantage continued to increase.

Open reading frame (ORF) knockout contributes to the screening of mutants

To further confirm our hypothesis, we attempted to knock out the apd gene of CF7066 using the same method. To avoid difficulties in screening knockout colonies due to growth disadvantages, we improved the design of the donor plasmid pSHG5-Δapd by placing the upstream and downstream homologous arms outside the ORF of apd. In this way, even if the transformant undergoes gene recombination resulting in the knockout of apd in the genome, the protein Apd can still be expressed from the plasmid.

The plasmid pSHG5-Δapd was then electroporated into CF7066 and cultured on gentamicin plates at 37°C. Among the 22 transformants tested by colony PCR analysis, 21 produced a band corresponding to the wild-type genome (3958 bp). However, colony No. 17 produced a band indicative of the knockout genotype (1448 bp; Fig. 3a and b). After streaking this transformant on gentamicin-free plates at 42°C for one passage, seven progeny colonies were identified as the apd knockout genotype (Fig. 3c). Subsequently, colony No. 8 was streaked on gentamicin-free plates at 42°C for a second passage, and the apd knockout genotype appeared in 100% of the progeny colonies tested (Fig. 3d and Fig. S5). Sanger sequencing was performed to further confirm the deletion of apd (Fig. 3e).

During the process, the plasmid genotypes of the transformants were also detected by PCR using primers targeting the plasmid backbone that flanked the upstream and downstream homologous arms of apd (Fig. 3a). Of the 22 transformants tested, 14 contained pSHG5-Δapd carrying the deleted apd allele (1130 bp), while six possessed the plasmid carrying the wild-type apd allele (3660 bp) (Fig. 3b). This result suggested that it might be necessary to examine the homology arms on the plasmid in the primary transformants to ensure that it has not been swapped with the genome. After colony No. 8 was cultured on gentamicin-free plates for one passage, 72.7% of the progeny colonies were cured (Fig. 3c and Fig. 3d). Compared to the previous two mutants JS0135ΔnanH and CF7066ΔnanH, the screening of CF7066Δapd required only two passages, which considerably reduced the time required for mutant construction.

Biological characteristics of the three mutants

Target gene knockout was confirmed through Western blotting. In the wild-type strains JS0135 and CF7066, NanH expression was detectable but absent in the JS0135ΔnanH and CF7066ΔnanH knockout mutants (Fig. 4a and b). Similarly, Apd was expressed in the wild-type CF7066 strain but not in the mutant CF7066Δapd strain (Fig. 4c). These results provided further evidence that the TS plasmid-based procedure generated marker-free deletion mutants of G. parasuis.

The phenotype, growth and adhesion of the strains were also detected. The growth curve presented in Fig. 4d shows that the deletion of apd had no significant effect on the growth of CF7066, but the deletion of nanH significantly inhibited the growth of CF7066 and JS0135. Furthermore, the deletion of apd led to a significant improvement in the bacterial adhesion rate of CF7066 compared to that of the wild-type strain, while the absence of nanH in CF7066 and JS0135 did not result in any obvious differences compared to that in their respective wild-type strains (Fig. 4e). This could be attributed to the diverse roles of nanH and apd in bacterial biological functions, which is worthy of further exploration. Moreover, the differences in growth trends among the mutants may be one of the reasons why apd could be knocked out more quickly and easily than nanH.

Plasmid copy number significantly affects the expression of recombination-related proteins

The optimal temperature for G. parasuis growth and DNA replication is approximately 37°C, rather than 30°C. Therefore, we speculated that 37°C may be more conducive to homologous DNA recombination. We first evaluated the expression of recombinant genes (recA, recB, recC, recD, and ruvA) in wild-type CF7066 at 30°C and 37°C by RT‒qPCR, and the results revealed minimal differences in the expression of each gene at different temperatures (Fig. 5a). However, in the transformant CF7066 (pSHG5-Δapd), the expression of each recombinant gene was significantly upregulated at 30°C (Fig. 5b). Specifically, the expression of recC was the most obviously upregulated, which was approximately 7 times greater than that at 37°C (Fig. 5b). Moreover, the RT‒qPCR results of the bacterial solution indicated that the plasmid copy number at 30°C was more than twice that at 37°C (Fig. 5c). This suggested that the plasmid copy number was greater at 30°C, resulting in greater expression of the recombinant genes. However, the slow growth of colonies at 30°C is not conducive to recombination, and our previous study had never successfully screened mutants at 30°C. In the window of temperature alternation, a high copy number of plasmids induces high expression of recombination-related genes, while bacterial chromosomes maintain efficient replication; therefore, DNA recombination is more likely to occur during temperature alternation.

In previous studies, we attempted to use the method described by Taturn and Briggs [22] to screen for double-crossover strains. In brief, the transformants were first subjected to selection at 42°C to cure the plasmids, allowing only the single-crossover colonies to grow under selection pressure; then, the single-crossover colonies were propagated on gentamicin-free plates at 30°C to screen for the knockout strains. However, this method failed in our study because homologous DNA recombination is infrequent during normal cellular metabolism, and target gene knockout confers a growth disadvantage to the resulting mutants. As a result, the single-crossover strains were more inclined to revert to the wild type. Moreover, single-crossover strains can only recombine once after the plasmids are cured, making screening difficult.

To increase the likelihood of obtaining double-crossover genotypes, we developed a strategy for culturing transformants alternately at 37°C and 30°C. Our approach is based on the hypothesis that DNA damage occurs spontaneously during normal cellular metabolism and that double-strand DNA breaks (DSBs) are the most severe type of DNA damage. Homologous recombination is now a well-known pathway for repairing DSBs [23, 24] and occurs mainly during DNA replication[25–28]. Therefore, we propose that a greater copy number of plasmids in cells would lead to more frequent DSBs and consequently activate the repair mechanism more frequently, thus increasing the probability of homologous recombination. Additionally, previous work has shown that recombination is partially dependent on DNA replication [29, 30] and that blocking DNA synthesis by high temperatures reduces the frequency of recombination [30]. We believe that DNA double-strand unwinding before DNA replication provides opportunities for homology searching and DNA strand invasion, which is a key reaction involved in homologous recombination [31]. G. parasuis grows significantly more slowly at 30°C, demonstrating that DNA synthesis decreases, and the TS plasmid fails to replicate at or above 42°C in G. parasuis [32]. Consequently, we surmised that culturing transformants alternately at 37°C and 30°C could ensure that both the genome and plasmids replicate efficiently, thereby improving the frequency of recombination.

In conclusion, we successfully established a marker-free knockout system for G. parasuis by electrotransforming TS donor plasmids. Our screening process revealed that culturing transformants alternately at 37°C and 30°C promoted homologous recombination, making the production and screening of double crossover mutants more feasible. Our findings also showed that simultaneous single exchange events based on the two different homologous arms of the target gene could occur, resulting in the insertion of two plasmids into the genome. This marker-free knockout system is simple and efficient, requiring no transformation efficiency, and the plasmid used for transformation is small and requires no additional elements, which is particularly important for G. parasuis due to its plentiful and stringent restriction-modification systems. Additionally, the promotion of crossover and recombination observed in the TS mutation system through alternate cultivation at 37°C and 30°C may have potential applications in other microorganisms.

G. parasuis: Glaesserella parasuis ; TS: temperature-sensitive; SG buffer: sucrose/glycerol buffer; ORF: Open reading frame.

Data availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files). All vectors generated in this study can be obtained from the corresponding author upon reasonable request.

Funding

This work was funded by the National Key Research and Development Program of China (2022YFD1800902) and the Major Science and Technology Projects of Hubei Province (2021ABA005).

Authors’ contributions

Conceived and designed the experiments: J.X. and X.X. Performed the experiments: J.X. and Y.W. Contributed reagents/materials: X.X., D.W., Y.S., X.C., H.C. and H.Z. Wrote the paper: J.X., Y.W., H.Z., and X.X. All authors have reviewed the manuscript.

Ethics approval and consent to participate

This article does not contain any studies with human participants or animals performed by any of the authors.

Consent for publication

The authors provide consent for publication.

Competing interests

The authors declare no competing interests.

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You are reading this latest preprint version

A marker-free genetic manipulation method for G. parasuis strains developed by alternately culturing transformants at 37°C and 30°C

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Methods

Bacterial strains and growth conditions

Cell culture

Cloning and genetic manipulation

Electrotransformation of G. parasuis

Construction of marker-free knockout mutants in G. parasuis

Detection of NanH and Apd using western blotting

Bacterial growth curve assays

PK-15 cell adhesion and invasion assay

Quantitative reverse-transcription PCR (qRT‒PCR)

Results

Knockout of the Kan^R cassette in JS0135ΔnanH::Kan^R

Knockout of the nanH gene in wild-type CF7066

Open reading frame (ORF) knockout contributes to the screening of mutants

Biological characteristics of the three mutants

Plasmid copy number significantly affects the expression of recombination-related proteins

Discussion

Conclusion

Abbreviations

Declarations

Data availability

Funding

Authors’ contributions

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

A marker-free genetic manipulation method for G. parasuis strains developed by alternately culturing transformants at 37°C and 30°C

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Methods

Bacterial strains and growth conditions

Cell culture

Cloning and genetic manipulation

Electrotransformation of G. parasuis

Construction of marker-free knockout mutants in G. parasuis

Detection of NanH and Apd using western blotting

Bacterial growth curve assays

PK-15 cell adhesion and invasion assay

Quantitative reverse-transcription PCR (qRT‒PCR)

Results

Knockout of the KanR cassette in JS0135ΔnanH::KanR

Knockout of the nanH gene in wild-type CF7066

Open reading frame (ORF) knockout contributes to the screening of mutants

Biological characteristics of the three mutants

Plasmid copy number significantly affects the expression of recombination-related proteins

Discussion

Conclusion

Abbreviations

Declarations

Data availability

Funding

Authors’ contributions

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Knockout of the Kan^R cassette in JS0135ΔnanH::Kan^R