Xing-Yong Wu, Xue-Miao Li, Cheng-Li Liu, Shen Tian, Yan-Shuang Wang, An-Yang Li, Qiao Zhu, Qian-Feng Xia?

1. School of Tropical Medicine, Hainan Medical Universuty, Haikou 571199, China

2. Key Laboratory of Translational Medicine of Ministry of Education, Hainan Medical University, Haikou 571199, China

Keywords:Burkholderia pseudomallei relA Gene knockout Biological properties

ABSTRACT Objective: The relA gene,a gene associated with the synthesis of rigorous response signaling molecule (p)ppGpp in Burkholderia pseudomallei, was knocked out and its effects on the biological functions of bacterial growth, motility and biofilm formation were investigated.Methods: The plasmids with trimethoprim resistance (TPR) were modified by enzyme digestion and enzyme ligation method. The TPR fragment was linked to the suicide plasmid pK18mobSacB containing SacB sucrose killing gene by Bgl Ⅱ enzyme digestion site,and trimethoprim resistance was obtained Plasmid(TPR-pK18mobSacB); The relA gene of Burkholderia pseudomallei HNBP001 was knocked out by homologous recombinant gene knockout method, and the relA mutant was obtained. The growth, motility and biofilm phenotypes of Burkholderia pseudomallei HNBP001 were compared before and after the deletion of relA gene. Results: The relA mutant was successfully constructed. The growth rate,motility and biofilm formation of ΔrelA decreased. Conclusion: The modified plasmid TPRpK18mobSacB can improve the knockout efficiency of HNBP001 strain, which can be widely used in the gene knockout of Burkholderia pseudomallei, and provide convenience for the laboratory to study the gene function of Burkholderia pseudomallei at the molecular level; The mutant of the relA gene inhibits the growth, motility and biofilm formation of HNBP001.?Corresponding author:XIA Qian-feng, Professor, Doctoral Supervisor.

1. Introduction

Burkholderia pseudomallei (B. pseudomallei), widely distributed in tropical and subtropical areas, is the causative agent of melioidosis.Hainan is a high incidence area of melioidosis in China[1, 2].It is very important to study the pathogenic mechanism of B. Pseudomallei from the molecular level, because its virulence is strong and the pathogenic mechanism is not clear [3]. At present, the gene editing technology of B. pseudomallei. is not perfect, but the homologous recombinant gene knockout technology of prokaryotes is an important means to study the gene function of bacteria. It can lay a foundation for improving the molecular research level of B.pseudomallei by developing efficient gene knockout on this basis.

(P)ppGpp is a rigorous response mechanism of bacteria to various adverse environmental stresses. This strong response is caused by the stress signaling system mediated by guanosine tetraphosphate(ppGpp) and guanosine pentaphosphate (pppGpp) under nutrient deficiency stress, and this mechanism may also promote antibiotic tolerance[4, 5]. (P)ppGpp is synthesized by GDP or GTP via relA and spoT enzymes. RelA enzyme plays a major role in synthesis, while spoT enzyme is a bifunctional enzyme with synthase and hydrolase activities[6]. (P)ppGpp affects the growth, adaptation, motility,tolerance, virulence and biofilm formation and development of bacteria[7].

This study explored a more effective and low threshold gene knockout method for B. pseudomallei by constructing ΔrelA mutant of HNBP001, and its effects on the growth, swimming and biofilm of B. pseudomallei were investigated.

2. Materials and Methods

2.1 Strain culture and construction of recombinant plasmid

Burkholderia pseudomallei HNBP001 was provided by our research group and its genome sequence was uploaded to NCBI[8]. DH5α competent cells and S17-1 competent cells were purchased from Beijing ZoMan Biotechnology Co., Ltd. Except for the screening of gene knockout transforters on a plate containing 15% sucrose(without NaCl) and culture at room temperature, the rest were cultured in LB medium at 37℃, and AGAR and corresponding concentration of antibiotics were added as required. pSCB2-sgRNA plasmid is derived from (Addgene plasmids # 129463; http://n2t.net/addgene:129463; RRID: Addgene_129463)[9]; The recombinant plasmid TPR-pK18mobSacB was constructed and donated by professor Liu Chengli of our laboratory.

2.2 Main reagents and instruments

Genome Extraction Kit, Tiangen; PCR product Purification Kit and Plasmid extraction reagent, OMEGA. PrimeSTAR Max DNA Polymerase , TaKaRa. Restriction endonuclease (Bgl II 、BamHⅠ、Xbal Ⅰ、Hind Ⅲ) and T4 DNA ligase ,NEB. YEAST,OXOID; RYPTONE, OXOID; Sucrose, XiLONG Science; Sodium chloride, XiLONG Science; 50× TAE Buffer, Sangon Biotech; Petri dish, biosharp; 96 - well plates, CELL TER; Multifunctional enzyme marker, CELL TER; Gel imaging analysis system, JunYi; A2 Biosafety cabinet, Haier; PCR amplification instrument, Eppendorf.

2.3 Primers

The relA gene of HNBP001 was identified by sequence alignment with pseudomallei K96243 genome of B. pseudomallei on NCBI.NCBI gene coordinates are located at 2140463-2142700(B.pseudomallei K96243,2317104 - 2319341 ) on chromosome 1.The primers were designed according to the relA gene sequence of HNBP001(GenBank: CP038806.1) in GenBank. PrimersΔrelALF/ΔrelA-LR and ΔrelA-RF/ΔrelA-RRwere used to amplify the upstream and downstream homologous arms of relA gene,respectively. The primersΔrelA-VF/ΔrelA-VR were used to verify the gene deletion strains of relA. Primers TPR-F/ TPR-R were used to amplify TPR resistant fragments. Primer sequences are shown in Table 1, all synthesized by Sangon Biotech (Shanghai) Co., Ltd.

2.4 Plasmid modification of pK18mobSacB

Using pSCB2-sgRNA plasmid containing TPR resistant fragment as template, a pair of primers TPR-F/TPR-R containing Bgl II restriction site were designed according to the sequence provided by the manufacturer, and the TPR resistant fragment containing promoter was amplified by TaKaRa high-fidelity enzyme.The amplified gene products were purified and digested byrestriction enzyme Bgl II to isolate the TPR resistant fragment and pK18mobSacB plasmid. The digested product was purified and ligated by T4 DNA ligase. After ligating, DH5α competent cells were transformed to expand and enhance the plasmid. After screening for TPR resistance, the plasmid was extracted and verified by enzyme digestion. The transformed plasmid was named pK18mobSacB. PCR amplification conditions: Denaturation at 98℃ for 10 s, annealing at 59℃ for 5 s, extension at 72℃ for 10 s, 30 cycles.

Table 1 Primer sequence and restriction site (restriction site indicated by underscore)

2.5 Construction of deleted strains

Take 100 uL of bacterial solution coated with double-antibody plates containing trimethoprim and gentamicin (B. pseudomallei strain has natural gentamicin resistance) for screening. A second round of screening for successfully conjugated colonies was performed on plates containing 15% sucrose (without NaCl) to promote vector loss. Obtainment of deletion strains was confirmed by PCR validation using the respective validation primers. PCR amplification conditions: 30 cycles of denaturation at 98℃ for 10 s,annealing at 68℃ for 5 s, and extension at 72℃ for 10 s.

2.6 Growth curve determination

Single colonies of HNBP001 and its ΔrelA deletion strains grown in fresh LB medium were picked and cultured in LB liquid medium to log phase. The bacterial liquid was diluted with LB liquid medium to OD600=0.05, and 200 μL of the diluted bacterial liquid was taken into a 96-well plate, with three replicate wells for each sample, and placed in a multi-function microplate reader (Biotek) at 37℃ and 180 rpm orbit. The cells were cultured with shaking for 48 h, and the absorbance at OD600 nm was automatically measured once every 2 h in a continuous dynamic manner.

2.7 Biofilm assay

The bacterial liquid was homogenized to OD600=0.1 in the same way as the growth curve determination method. Crystal violet staining method [10]: Take 200 μL of the diluted bacterial solution into a 96-well plate, carefully remove the supernatant after culturing 3 replicate wells for 48 h, and then wash the 96-well plate with 200 μL of 1xPBS for 3 times After drying, 200 μL of 1% crystal violet was stained for 30 min; then washed twice with 200 μL of 1xPBS,dried, incubated with 200 μL of 30% acetic acid for 20 min, and the absorbance was measured by OD600. Glass test tube observation method: respectively take 2 mL of diluted bacterial solution in glass test tubes, and observe after culturing for 48 hours.

2.8 Motility assay

The bacterial solution was homogenized to OD600=0.1 with the same growth curve determination method, and 2 μL of bacterial solution was drawn into 0.3% agar LB medium for upright culture,three replicates, and the bacterial swimming circle was observed and measured after culturing at 37℃ for 24 h. diameter.

2.9 Experimental data processing

Statistical analysis was performed using GraphPad Prism version 9.0 software and paired t-test statistical method, * indicates a significant difference at P＜0.05.

3. Results

3.1 Construction of TPR-pK18mobSacB vector plasmid

In order to improve the gene knockout efficiency of HNBP001 strain, we inserted the trimethoprim-resistant fragment into the suicide plasmid pK18mobSacB through the Bgl II single enzyme cleavage site. The plasmid map of pK18mobSacB is shown in Figure 1A. The TPR-pK18mobSacB plasmid is shown in Figure 1B. The successfully constructed TPR-pK18mobSacB recombinant plasmid was verified by digestion with Bgl II restriction endonuclease, and the size of the enzyme-cut band was correct, as shown in Figure 1C,and sequencing was performed. The results showed that the TPRpK18mobSacB vector plasmid was successfully constructed.

3.2 Construction of ΔrelA recombinant vector plasmid

Figure1 Plasmid profile and electrophoresis of TPR-pK18mobSacB digestion verification

Figure2 Homologous recombination pattern and electrophoresis results of mutan

In order to construct the ΔrelA deletion strain of HNBP001, we first PCR amplified the upstream and downstream homology arms (L,R) of the relA gene, and then inserted into the TPR-pK18mobSacB vector plasmid by restriction enzyme, and screened by TPR resistance, as shown in Figure 2A. The successfully constructed ΔrelA recombinant vector plasmids BamH I and Hind III were verified by enzyme digestion, and the size of the recombinant vector plasmid was verified by enzyme digestion to meet expectations. The result showed that the recombinant vector plasmid was successfully constructed, as shown in Figure 2B, the red arrow indicates the band For connecting upper and lower arms.

3.3 Construction of ΔrelA deletion strain

The method of homologous recombination to construct a ΔrelA deletion strain is shown in Figure 2A. The recombinant vector plasmid was transformed into S17-1 competent cells and conjugated with HNBP001. The first round of screening was carried out with double-anti-trimethoprim and gentamicin for transformant screening,and the second round of screening was carried out with 15% sucrose plate. Deletion strains that lose the recombinant vector plasmid.Deletion strains were verified with the verification primers relA-VF/relA-VR, as determined by the correct band size, as shown in Figure 2C.

3.4 The effect of knocking out relA on the growth of HNBP001

Bacterial growth and metabolism play an important role in adapting to external environmental stress. When bacteria encounter adverse environments (such as antibiotics, ultraviolet rays, temperature, etc.),in order to survive, bacteria will reduce their growth rate to adapt to adversity to survive. To explore whether relA has an effect on the growth of HNBP001, we compared the growth of HNBP001 wild strain and its DrelA-deficient strain in nutrient-rich LB medium for 48 h, as shown in Figure 3A. OD600 nm turbidity measurement showed that there was no significant difference in the growth rate between HNBP001 and ΔrelA-deficient strains before about 8 h in log phase, and the growth rate of ΔrelA-deficient strains was lower than that of HNBP001 after that. The results showed that when ΔrelA was depleted, the level of (p)ppGpp synthesis was reduced,thereby inhibiting bacterial growth. Studies have found that the increase and decrease of (p)ppGpp levels can significantly inhibit the growth of E. coli, increasing (p)ppGpp levels will limit ribosome synthesis, and decreasing (p)ppGpp levels will limit the expression of metabolic proteins [11] .

3.5 The effect of relA on the mobility of HNBP001

Previous studies have shown that (p)ppGpp can regulate the synthesis of flagella, and bacteria realize their motility through flagella [7]. By comparing the mobility of the wild strain and its DrelA-deficient strain in 0.3% soft agar LB semi-solid medium for 24 h, we observed that the mobility of the ΔrelA-deficient strain was lower than that of HNBP001, as shown in Figure 3B and C. It indicated that the deletion of relA gene could inhibit the motility of HNBP001.

Figure3 Growth curve and motility

3.6 The effect of relA on the formation of HNBP001 biofilm

Figure4 Biofilm

Biofilm is closely relAted to bacterial resistance. As a global regulator, many studies have shown that (p)ppGpp can participate in regulating bacterial biofilm formation [12]. As shown in Fig. 4A, after culturing for 48 h, it was observed that the air-liquid interface biofilm of the ΔrelA-deficient strain was thinner and relAtively looser than that of HNBP001. The results showed that the biofilm formation decreased and became more fragile after the deletion of the relA gene. The crystal violet staining method observed an obvious crystal violet staining ring at the gas-liquid interface, as shown in Figure 4B; the quantitative measurement results were shown in Figure 4C, the biofilm of the ΔrelA-deficient strain was lower than that of HNBP001. It indicated that relA gene deletion had inhibitory effect on the biofilm formation of HNBP001.

4. Discussion

Melioidosis is a serious and potentially fatal disease. Its complex clinical manifestations and resistance to many antibiotics seriously threaten human life and health. So far, there is no vaccine for clinical application [13, 14].We improved the efficiency of B.pseudomallei gene knockout by modifying the sucrose lethal plasmid pK18mobSacB, and the desired gene deletion strain can be obtained in about two weeks. The reagents and instruments required for the entire gene knockout experiment are not demanding and can be widely used in laboratories studying B. pseudomallei.We validated the method by knocking out the stringent response signaling molecule (p)ppGpp synthesis-relAted gene relA, and determined its biological phenotype. Stringency response is a stress signaling system and an adaptive mechanism for prokaryotes to cope with adverse living conditions, allowing bacteria to survive under conditions of nutrient starvation and other stresses, including antibiotic pressure [15].(p)ppGpp signaling may be an important component of the regulatory network of B. pseudomallei virulence gene expression and stress adaptation, and its deletion mutant may be a promising candidate for live attenuated vaccines [16]. When bacteria encounter adversity, in order to survive, they will produce tolerant bacteria that are more tolerant to the environment, which can respond to the unfavorable environment by reducing growth rate,movement and increasing the formation of biofilm [17].

Under the action of antibiotics, slow-growing resistant bacteria can avoid the stimulating and killing effects of antibiotics due to the reduction of basal metabolic activity [18]. We found that the ΔrelA deletion strain was lower in logarithmic late and stationary phase than the wild strain. This is relAted to the reduction of (p)ppGpp levels after deletion of the relA gene, which limits the expression of metabolic proteins [11]. The (p)ppGpp synthase gene is involved in regulating the movement of Vibrio alginolyticus [19]. Our results show that motility is reduced when ΔrelA is depleted, which may be relAted to the requirement for (p)ppGpp participation in bacterial flagella synthesis [19]. Biofilm formation is an important strategy for bacteria to survive in hostile environments or during infection in vivo. Biofilm is closely relAted to bacterial resistance. As a global regulator, many studies have shown that (p)ppGpp can participate in regulating bacterial biofilm formation [12]. Compared with HNBP001, the amount of biofilm formation in the ΔrelA deletion strain was decreased.

In conclusion, our engineered TPR-pK18mobSacB provides a favorable tool for the study of B.pseudomallei gene function. In the characterization of the relA gene function, we found that knocking out the relA gene inhibited the growth, motility and biofilm formation of HNBP001, laying a foundation for further research on the related mechanism of the relA gene.