Avian pathogenic escherichia coli strain resistant to low-phosphorus environment and preparation method of avian pathogenic escherichia coli strain

Abstract

The invention belongs to the technical field of microorganisms and genetic engineering. The invention provides an avian pathogenic escherichia coli strain resistant to a low-phosphorus environment and a preparation method of the avian pathogenic escherichia coli strain. According to the strain, the integrity of an LPS map can be achieved under the phosphorus concentration of 20-200 [mu] M, the transcription levels of rmlB, rmlD, rmlA, fdtA, fetB, rmlC, wzy, wegR and wegS genes are up-regulated, and the transcription level of rfaH is up-regulated; the strain is named as DE17-delta phoB, the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.36537, the strain is preserved in the China General Microbiological Culture Collection Center, and the preservation time is November 7, 2025. It is found that PhoB is phosphorylated in a low-phosphorus environment, and an O-antigen gene cluster is regulated and controlled to participate in O-antigen synthesis, so that the low-phosphorus extreme environment is resisted; meanwhile, PhoB and RfaH are also found to regulate and control the O-antigen and maintain the transcription stability of the O-antigen so as to cope with environmental changes. The invention provides a corresponding research basis for prevention and treatment of avian pathogenic escherichia coli in the future.

Description

Technical Field

[0001] This invention belongs to the field of microbial and genetic engineering technology, specifically relating to a strain of pathogenic avian Escherichia coli tolerant to low phosphorus environments and its preparation method. Background Technology

[0002] Avian pathogenic Escherichia coli (APEC) is a subgroup of extraintestinal pathogenic Escherichia coli that primarily causes infectious diseases in poultry. APEC can infect various poultry species of all ages. Studies have shown that broilers are highly susceptible to APEC during their 4-6 week age, and laying hens face a continuous risk of APEC infection during their growth and egg-laying periods, especially around peak egg production, resulting in significant economic losses for the poultry industry.

[0003] APEC primarily infects poultry through the respiratory tract, but can also enter via the oral cavity, nasal cavity, or cloaca. The bacteria typically colonize the mucous membranes of the gastrointestinal tract, respiratory tract, and reproductive tract, but generally do not cause disease. However, as a primary pathogen or secondary to other diseases and environmental stressors, APEC can cause systemic infection. APEC transmission routes include fecal-oral or aerosol transmission; contaminated feed and water can spread the bacteria. APEC can also be vertically transmitted through hatching eggs. Initial infection occurs in the lungs and air sacs, as the lower respiratory tract is a key gateway for the bacteria to enter the bloodstream, and upper respiratory tract infection helps APEC evade the host's immune system. Simultaneously, multiple virulence factors, including adhesins, serum resistance-related genes, iron uptake systems, hemolysins, toxins, and invasion-related proteins, work synergistically, making respiratory colonization a crucial step in APEC pathogenesis. After invading the lungs, APEC spreads through the mucous membranes to multiple organs, causing systemic infection, sepsis, and organ damage.

[0004] APEC is considered to have zoonotic potential due to its high genetic correlation with human extraintestinal pathogenic Escherichia coli, especially urinary tract pathogenic Escherichia coli and neonatal meningococcal Escherichia coli, and the fact that some enteroassociated pathogenic strains can be transmitted through food, despite no reported infections. Studies have shown that APEC serotypes O2 and O18 can induce diseases similar to human extraintestinal pathogenic Escherichia coli in rodent meningitis models. Furthermore, UPEC can cause infection in the chicken reproductive tract, and the risk of APEC transmission in the food chain is of greater concern. Drug-resistant APEC strains can enter the food chain through retail chicken meat [16, 17], with emerging APEC strains (such as ST117) exhibiting significant pathogenicity. Therefore, research on the infection, pathogenicity, and prevention of this bacterium is of significant public health importance.

[0005] Phosphorus, the fifth most abundant element for life (after carbon at 50%, oxygen at 20%, nitrogen at 14%, and hydrogen at 8%), accounts for approximately 3% of bacterial dry weight and is essential for microbial growth and reproduction. Inorganic phosphate (Pi) is the preferred phosphorus source for bacteria. In organisms, phosphorus mainly exists in the +5 oxidation state, manifesting as inorganic phosphates or phosphate esters. In some organisms, phosphonates in the +3 oxidation state can also be found; these compounds have direct carbon-phosphorus (CP) covalent bonds. Phosphate derivatives are key hydrophilic components of amphiphilic phospholipids, constituting the cell membrane structure of all bacterial life. Through the formation of phosphodiester bonds with sugars such as ribose and deoxyribose, phosphate derivatives collectively construct the molecular backbone of DNA and RNA, thereby supporting the storage and transmission of genetic information. Furthermore, the core mechanism of bacterial energy metabolism relies on the energy released from the hydrolysis of anhydride bonds between phosphate groups in ATP and other nucleoside triphosphate molecules. Phosphate ions also participate in protein phosphorylation, chemically modifying specific amino acid residues such as histidine, aspartic acid, serine, threonine, and tyrosine to precisely regulate the biological activities of various proteins. As an abundant component of the cytoplasm, phosphates also function as biological buffers, playing a crucial role in maintaining pH homeostasis within bacteria.

[0006] Due to the essential biological functions of phosphate, bacteria must maintain an optimal concentration of intracellular Pi (Pi) pools. In bacteria such as *E. coli* and *Salmonella*, this concentration range is typically 1–10 mM. Through ATP synthesis, Pi is assimilated into biomolecules and subsequently participates in other metabolic transformations. Due to its negative charge, Pi primarily interacts with Mg (Mg) intracellularly. 2+ Metal ions form coordination complexes. Most *E. coli* strains have a biphasic life cycle, switching between a warm, anaerobic, high-density intestinal parasitic stage within the host and a complex and variable extraintestinal environment such as soil, water, or plants. Pi content varies significantly across different environments, and fluctuations in intestinal Pi concentration are influenced by dietary structure, different intestinal locations, and feeding times; environmental Pi levels are closely related to geographical features, geological conditions, and biological competition. To cope with extreme conditions of Pi limitation or excessive accumulation, bacteria have evolved sophisticated regulatory systems: activating efficient uptake mechanisms when environmental Pi is scarce, and initiating homeostasis maintenance programs when intracellular Pi is overloaded.

[0007] Adapting APEC to a low-phosphorus environment would be helpful in controlling pathogenic E. coli in poultry. However, there are currently few reports on how to modify APEC to adapt to a low-phosphorus environment. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a low-phosphorus-tolerant avian pathogenic Escherichia coli strain and its preparation method. The obtained strain maintains LPS spectrum integrity at phosphorus concentrations of 20-200 μM, and simultaneously achieves resistance to pathogenic Escherichia coli under low-phosphorus conditions. rmlB, rmlD, rmlA, fdtA, fetB, rmlC, wzy, wegR, wegS Gene transcription levels are upregulated, and upregulated rfaH Transcription level.

[0009] To achieve the aforementioned technical objectives, the present invention provides the following technical solution: A low-phosphorus-tolerant avian pathogenic Escherichia coli strain, which maintains LPS spectrum integrity at phosphorus concentrations of 20-200 μM, enabling [further monitoring / control]. rmlB, rmlD, rmlA, fdtA, fetB, rmlC, wzy, wegR and wegS Gene transcription levels are upregulated, and upregulated rfaH Transcription level; the strain undergoes Δ phoB Gene deletion mutation.

[0010] The strain was named: Escherichia coli DE17-Δ phoB The accession number is CGMCC No.36537, and it is deposited at the China General Microbiological Culture Collection Center on November 7, 2025.

[0011] Specifically, the phoB The nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0012] A method for preparing a low-phosphorus-tolerant pathogenic Escherichia coli strain in birds, the method comprising the following steps: (1) Using the DE17 strain genome and pKD3 plasmid as templates, amplification was performed respectively. phoB The upstream and downstream homologous arms of the gene and the antibiotic resistance gene were fused and amplified using the overlap PCR method. (2) The homologous arm containing the antibiotic resistance fragment was electroporated into DE17 competent cells, positive clones were screened for antibiotic resistance, and then competent cells were prepared. (3) Eliminate the chloramphenicol resistance gene in the positive clone to obtain the result.

[0013] Optionally, the antibiotic includes chloramphenicol.

[0014] Optionally, the receptive state is prepared using the electro-receptive state method.

[0015] Preferably, the electric shock receptive state method includes the following steps: (1) Resuscitate the streaked strain from a -80℃ freezer and incubate at 37℃ overnight; (2) Pick a single colony and inoculate it into 5 mL of LB liquid medium. When the bacteria grow to the OD value... 600 When the concentration is 0.8, transfer it to 100 mL of LB liquid medium at a ratio of 1:100; (3) When bacteria grow to OD 600 When the concentration is 0.6, let it stand on ice for 30 minutes, while pre-cooling sterile 10% glycerol in advance; (4) Centrifuge at 5000 rpm and 4℃ for 8 min to collect the bacterial culture, and wash twice with sterile 10% glycerol. (5) Finally, resuspend the bacterial cells in 1 mL of sterile 10% glycerol, dispense into 100 μL / tube, and store at -80℃.

[0016] The beneficial effects of this invention are: This invention develops a low-phosphorus-tolerant strain of pathogenic Escherichia coli in birds. This strain maintains LPS spectrum integrity at phosphorus concentrations of 20-200 μM, and simultaneously achieves resistance to pathogens under low-phosphorus conditions. rmlB, rmlD, rmlA, fdtA, fetB, rmlC wzy, wegR, wegS Gene transcription levels are upregulated, and upregulated rfaH Transcriptional level. This invention discovered that under low phosphorus conditions, PhoB is phosphorylated and regulates the O-antigen gene cluster to participate in O-antigen synthesis, thus resisting the extreme low phosphorus environment. It was also found that both PhoB and RfaH regulate O-antigen, maintaining O-antigen transcriptional stability to cope with environmental changes. This invention provides a research foundation for future prevention and control of pathogenic Escherichia coli in birds. Attached Figure Description

[0017] Figure 1 The results of Western blotting to detect changes in PhoB expression in DE17 at different Pi concentrations and grayscale analysis were obtained. Figure 2 For different Pi concentrations phoB Experimental results analyzing gene transcription (A), intracellular alkaline phosphatase content under different culture conditions (B), and PhoB protein expression (C and D); Figure 3 Results of experimental analysis of the transcriptional level of O-antigen gene clusters in a low-phosphorus environment; Figure 4 For Δ phoB Results of the identification experiment of missing and supplemented strains, Part A is Δ phoB strain, part B is CΔ phoB The bacterial strain, part C is for Western blotting verification, and part D is for intracellular alkaline phosphatase content verification. phoB 、CΔ phoB In Part A, lane 1: ΔphoB Lane 2: DE17; Lane 3: Negative control; In Part B, lanes 1-8: CΔ phoB Lane 9: Negative control; Figure 5 For Δ phoB Growth capacity test results; Figure 6 DE17 and Δ at different Pi concentrations phoB 、CΔ phoB LPS spectrum analysis; Part A: LPS spectra of DE17 cultured at different Pi concentrations using silver staining; Part B: ΔP spectrum analysis of DE17 cultured at different Pi concentrations using silver staining. phoB LPS spectrum; Part C: Silver staining identification of low phosphorus, high phosphorus, and LB medium Δ phoB LPS spectrum; Part D: Western blotting identification of low phosphorus, high phosphorus, and LB medium Δ phoB LPS map; Figure 7 Δ under low phosphorus conditions phoB Experimental results of transcriptional level analysis of genes related to O-antigen synthesis; Figure 8 Δ under LB conditions phoB Experimental results of transcriptional level analysis of genes related to O-antigen synthesis; Figure 9 Figure 1 shows the results of expression, purification, and polyclonal antibody preparation of recombinant RfaH protein. Part A shows PCR identification of the protein expression plasmid pSL2185; Part B shows SDS-PAGE verification of RfaH protein; Part C shows Western blotting verification of RfaH protein; Part D shows Western blotting identification of RfaH polyclonal antibody. Specifically, in Part A: Lane 1: pSL2185; Lane 2: Negative control; in Part B: Lane 1: Broken precipitate; Lane 2: Broken supernatant; Lanes 3-4: Elution with 50 mmol / L imidazole; Lanes 5-8: Elution with 300 mmol / L imidazole; in Part D: Lane 1: Whole bacterial protein; Lane 2: Negative control. Figure 10 Figure showing the experimental results of EMSA detection of the binding ability of RfaH to the promoter region of the O-antigen gene cluster; Figure 11 To build rfaH Figure 1 shows the results of PCR identification experiments for the deleted and complemented strains; where part A represents the Δ... rfaH Part B is CΔ rfaH Strains; Part C is for Western blotting verification Δ rfaH 、CΔ rfaH Part D is Δ rfaHLPS spectral analysis; Part A: Lane 1: Δ rfaH Lane 2: DE17; Lane 3: Negative control; Part B: Lanes 1-7: CΔ rfaH Lane 8: Negative control; Figure 12 For Δ rfaH Growth capacity test results (image); Figure 13 Δ in low phosphorus (purple) and LB (blue) environments rfaH Figure 1; Experimental results of transcriptional level analysis of genes related to O-antigen synthesis; Figure 14 Figure 1 shows the results of Western blotting detection of RfaH expression in DE17 at different Pi concentrations (Part A) and grayscale analysis (Part B). Figure 15 For Western blotting to detect Δ at different Pi concentrations phoB Figures showing the experimental results of changes in RfaH expression levels (Part A) and grayscale analysis (Part B); Figure 16 Figure showing the experimental results of bacterial two-hybrid assay to detect the interaction between PhoB and RfaH; Figure 17 Figure 1 shows the experimental results of EMSA detection of the binding ability of PhoB to the promoter region of rfaH (Part A) and competitive EMSA detection of the binding ability of PhoB and RfaH to O-antigen (Part B). Detailed Implementation

[0018] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are still within the scope of protection of the present invention.

[0019] Example 1

[0020] Preparation of a low-phosphorus-tolerant pathogenic Escherichia coli strain in birds: I. Preparation of bacterial strains 1.1 Strains, test animals and culture conditions Strain: O2 serotype APEC Escherichia coli DE17 (hereinafter referred to as DE17, kindly provided by Researcher Han Xiangan of Shanghai Veterinary Research Institute). E. coli DH5α and E. coli - Rosetta Purchased from Qingke Biotechnology.

[0021] Genome: The DE17 genome is preserved and used by this laboratory.

[0022] Culture conditions: DE17, E. coli DH5α, and E. coli-Rosetta were cultured in LB medium at 37°C and 180 rpm.

[0023] Plasmids and culture conditions: pKD46, pKD3, pCP20, pSTV28 (respectfully provided by Researcher Han Xiangan of Shanghai Veterinary Research Institute). pKD46 and pCP20: 30°C, ampicillin resistance; pKD3 and pSTV28: 37°C, chloramphenicol resistance.

[0024] Experimental animals and breeding conditions: Hy-Line Brown chickens were purchased from Hangzhou Xiaoshan Breeding Chicken Co., Ltd. They were provided with purified water and feed daily and housed in separate cages within the animal facility. The animal experiments were approved by the Animal Welfare and Ethics Committee of Zhejiang Agriculture and Forestry University, under approval number ZAFUAC202477.

[0025] 1.2 Test Reagents Table 1. List of reagents required for the experiment

[0026] Reagent preparation: MOPS medium: (1) MOPS salt solution: 83.72 g sodium 3-(N-morpholine)-2-hydroxypropanesulfonate and 7.17 g trimethylglycine were added to 300 mL ddH2O and shaken until completely dissolved. The pH was adjusted to approximately 7.4 using hydrochloric acid. (2) 10 mM ferrous sulfate solution: 1.52 g of ferrous sulfate heptahydrate was diluted to 100 mL of ddH2O, shaken until completely dissolved, and added to MOPS salt solution to prepare a 400 mL MOPS / Tricine / FeSO4 mixed solution for later use; (3) 1.9 M ammonium chloride solution: 5.1 g of ammonium chloride was diluted to 50 mL of ddH2O, shaken until completely dissolved, and set aside for use; (4) 20 mM calcium chloride solution: Add 0.22 g ammonium chloride to 100 mL ddH2O, shake until completely dissolved, and set aside for use; (5) 2.5 M magnesium chloride solution: 25.4 g magnesium chloride hexahydrate was diluted to 50 mL ddH2O, shaken until completely dissolved, and set aside for use; (6) 276 mM potassium sulfate solution: 4.8 g potassium sulfate was diluted to 100 mL ddH2O, shaken until completely dissolved, and set aside for use; (7) 5 M sodium chloride solution: 58.5 g of sodium chloride was diluted to 200 mL of ddH2O, shaken until completely dissolved, and set aside for use; (8) 10×MOPS salt solution: Take 50 mL of the above 1.9 M ammonium chloride solution, 10 mL of 276 mM potassium sulfate solution, 0.25 mL of 20 mM calcium chloride solution, 2.1 mL of 2.5 M magnesium chloride solution, and 100 mL of 5 M sodium chloride solution and add them to 400 mL of MOPS / Tricine / FeSO4 buffer solution and bring the volume to 1000 mL. Filter the solution using a vacuum filter at 0.22 μM, aliquot and store at 4°C in the dark. (9) MOPS medium trace element storage solution: 0.62 g boric acid, 0.09 g molybdate, 0.32 g cobalt chloride hexahydrate, 0.09 g copper sulfate pentahydrate, 0.4 g manganese dichloride and 0.124 g zinc sulfate heptahydrate were diluted to 500 mL ddH2O, shaken until completely dissolved, filtered through a vacuum filter at 0.22 μM, dispensed and stored in a refrigerator at 4℃ protected from light; (10) 1 M dipotassium hydrogen phosphate solution: 8.709 g dipotassium hydrogen phosphate was diluted to 100 mL ddH2O, shaken until completely dissolved, and then autoclaved. (11) 10% glucose solution: 5 g glucose was diluted to 50 mL ddH2O, shaken until completely dissolved, and filtered at 0.22 μM. (12) 0.01% thiamine solution: 0.01 g thiamine was diluted to 10 mL ddH2O, shaken until completely dissolved, and filtered at 0.22 μM. (13) 50 μM Pi concentration MOPS medium: 50 mL 10×MOPS salt solution, 25 μL 1 M dipotassium hydrogen phosphate solution, 50 μL MOPS medium trace element storage solution, 50 μL 0.01% thiamine solution to a final volume of 500 mL ddH2O, store at 4℃ protected from light. (Add 1 M dipotassium hydrogen phosphate solution according to the experimental requirements for different Pi concentrations).

[0027] 1.3 Preparation and Procedure of Reverse Transcription PCR and Quantitative Real-Time PCR Reaction Systems Table 2 HiScript III All-in-one RT SuperMix Perfect for qPCR Reaction System and Procedure

[0028] Table 3. Reaction system of 2×Taq Pro Universal SYBR qPCR Master Mix

[0029] Table 4. Reaction program for 2 × Taq Pro Universal SYBR qPCR Master Mix

[0030] 1.4 Western blotting detection of target protein (1) Western blotting was performed after SDS-PAGE electrophoresis; (2) Use a rapid wet transfer apparatus to perform rapid protein transfer. Put 10 mL of equilibration buffer into the WB reaction box. (3) Activate the PVDF membrane with methanol, and then place it in the equilibration solution for 1 min; (4) Fill the electrophoresis gel box with SDS-PAGE electrophoresis solution, carefully remove the electrophoresed gel, and soak it in the electrophoresis gel box for about 1 minute to prevent the gel from drying out; (5) Open the transfer clamp and lay it flat on the experimental table. Place a dry sponge pad in the transfer clamp. (6) Remove the membrane from the equilibration solution, lay it flat on a dry sponge pad, spread the gel on the membrane, and remove air bubbles with a transfer roller; (7) Lay a dry sponge pad on the gel and close the transfer clamp to conduct the transfer test; (8) After the transfer is completed, block with TBST solution containing 5% skim milk powder for 2 h; (9) Then wash 3 times with 1×TBST, 5 min each time; (10) Incubate the corresponding primary antibody (dilution ratio 1:1000) at 4°C overnight, and repeat step (12); (11) Incubate the corresponding secondary antibody (dilution ratio 1:5000) at room temperature for 45 min, and repeat step (12); (12) HRP chemiluminescent solution was incubated in the dark for 2 min, and immunoblotting was performed using a chemiluminescence imager.

[0031] 1.5 Determination of Inorganic Phosphate Ion Concentration (1) Aseptic operation: Take the organs and tissues of 7-day-old and 6-month-old Hy-Line Brown chickens, grind them at low temperature using a homogenizer, and take the homogenized liquid. (2) The phosphate concentration of the homogenate was determined using an inorganic phosphorus detection kit (ferrous sulfate molybdenum blue colorimetric method) (Shanghai Yuanye Biotechnology Co., Ltd.); (3) Measure according to the instructions. OD 640 The phosphate concentration was calculated by comparing it with the standard phosphate curve.

[0032] 1.6 Extraction of total bacterial RNA RNA extraction was performed using the Trizol method, and the specific steps are as follows.

[0033] (1) Take 2 mL of each strain cultured at different Pi concentrations for 12 h, and centrifuge at 8000 rpm at 4℃ for 2 min; (2) Resuspend using DEPC H2O and repeat centrifugation; (3) After resuspending with 150 μL of 20 mg / mL lysozyme, let stand at room temperature for 45 min; (4) Add 1 mL Trizol, shake to mix, and let stand in the dark for 5 min; (5) In a fume hood, add 200 μL of chloroform, shake to mix, and let stand in the dark for 3 min; (6) Obvious stratification was observed. Centrifuge at 12,000 rpm at 4℃ for 15 min. (7) Take 500 μL of supernatant into the EP tube of the DEPC water treatment system; (8) Add 500 μL of isopropanol, shake to mix, let stand in the dark for 10 min, and then centrifuge at 12000 rpm at 4℃ for 15 min. (9) Gently aspirate the supernatant with a pipette tip, add 500 μL of 75% ethanol, shake, and centrifuge at 7500 rpm at 4℃ for 15 min; (10) Repeat step 9; (11) Gently aspirate the supernatant with a pipette tip, dry for 10 min, and dissolve in 30 μL DEPC H2O; (12) Once RNA extraction is complete, subsequent experiments can be performed or the RNA can be stored in a -80°C freezer.

[0034] 1.7 Reverse Transcription PCR (1) Quantification of bacterial RNA concentration; (2) RNA was reverse transcribed into cDNA using the One Step RT-PCR Kit (Nanjing Novizan Biotechnology Co., Ltd.). The reverse transcription system and procedure are detailed in Table 2. (3) Once the cDNA is prepared, it can be used for subsequent experiments or stored in a -20℃ refrigerator.

[0035] 1.8 Real-time quantitative PCR (1) Dilute the cDNA at a ratio of 1:5 using DNase-free ddH2O; (2) On ice, in the dark, add the samples in the order of the reaction systems in Table 3; (3) The real-time fluorescence quantitative PCR machine (Bio-Ray Biomedical Products Co., Ltd.) was used for the process. The reaction procedure was carried out according to Table 4.

[0036] 1.9 Alkaline phosphatase assay (1) Take 2 mL of each strain cultured at different Pi concentrations for 12 h, centrifuge and resuspend in sterile physiological saline, and repeat twice; (2) The resuspension of each strain was ultrasonically disrupted, and then protein quantification was performed; (3) The alkaline phosphatase content of each strain was determined using an alkaline phosphatase kit (Nanjing Jiancheng Bioengineering Institute); (4) Measure according to the instructions. OD 510 The intracellular alkaline phosphatase content of each strain was calculated by comparing it with the standard alkaline phosphatase curve.

[0037] 1.10 Preparation of competent cells for avian pathogenic Escherichia coli The competent states were prepared using the electro-sensor method, and the specific steps are as follows.

[0038] (1) Resuscitate the streaked strain from a -80℃ freezer and incubate at 37℃ overnight; (2) Pick a single colony and inoculate it into 5 mL of LB liquid medium. When the bacteria grow to the desired size... OD 600 When the concentration is 0.8, it is transferred to 100 mL of LB liquid medium at a ratio of 1:100; (3) When bacteria grow to OD 600 When the concentration is 0.6, let it stand on ice for 30 minutes, while pre-cooling sterile 10% glycerol in advance; (4) Collect the bacterial culture by centrifuging at 5000 rpm and 4℃ for 8 min, and resuspend in sterile 10% glycerol and wash twice. (5) Finally, resuspend the bacterial cells in 1 mL of sterile 10% glycerol, dispense into 100 μL / tube, and store at -80℃.

[0039] 1.11 phoB Construction of gene deletion strains phoB The gene nucleotide sequence is shown in SEQ ID No. 1, as follows: .

[0040] Using Red homologous recombination phoB The specific steps for constructing gene-deleted strains are as follows.

[0041] (1) Target fragment amplification: Using the DE17 strain genome and pKD3 plasmid as templates, primers were used to amplify the target fragments. phoB -UF / UR amplification phoB upstream homologous arm of the gene, using primers phoB -DF / DR amplification phoB Each downstream homologous arm of the gene is approximately 500 bp. Using the pKD3 plasmid as a template, primers were used... phoB- CF / CR amplified a 500 bp chloramphenicol resistance gene fragment, and then fused the above three fragments using the overlap PCR method to obtain the targeted amplification; (2) Electroporation and PCR identification: 1 µg of the amplified targeting fragment was electroporated into DE17 (pKD46) competent cells. After mixing by pipetting, the cells were incubated on ice for 30 min. The cells were then placed in a pre-chilled 2 mm electroporation cuvette and electroporated at 2500 V, 200 Ω, and 25 µF. LB liquid medium was added, and the cells were incubated at 37°C and 200 rpm for 1 h. The incubator was then plated onto chloramphenicol-resistant LB solid medium and incubated overnight at 37°C. Single colonies were prepared into bacterial suspensions using sterile PBS. phoB -UF / CR primers were used for PCR identification. The PCR reaction system is shown in Table 5. After successful PCR verification and sequencing, the culture was purified, preserved, and competent cells were prepared. (3) Knockout of chloramphenicol resistance gene: Subsequently, 2 μg of pCP20 plasmid was electroporated into competent cells of mutant strain containing chloramphenicol gene, plated on ampicillin-resistant LB solid medium, and cultured overnight at 28°C; single clones were selected from the plates and primers were used. phoB -outF / phoB -outR was used for PCR identification. After obtaining positive clones, they were passaged at 42℃ with the plasmid removed. After PCR identification and DNA sequencing confirmed their correctness, the deletion strain was named Δ. phoB .

[0042] Table 5 Primers used for strain construction

[0043] This bacterium is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; its name is DE17-ΔphoB, accession number is CGMCC No. 36537, and the deposit date is November 7, 2025. 1.12 Construction of replacement strains Plasmid replacement was used to construct replacement strains in order to build... phoB Taking replanting as an example, the specific steps are as follows.

[0044] (1) Using the bioinformatics website (https: / / www.fruitfly.org / seq_tools / promoter.html) to... phoB Predicting promoter regions in genes; (1) Using the DE17 genome as a template, amplify the genome containing... phoB Gene promoter and CDS sequence; (2) Using the pSTV28 plasmid vector, construct the complement plasmid pSTV28- phoB ; (3) Perform plasmid extraction for replenishment; (4) 2 μg of replenishment plasmid was electroporated into Δ phoB Competent cells of the deletion strain were obtained after verification by PCR and identification by sequencing. phoB The reintroduced strain was named CΔ phoB .

[0045] 1.13 Growth curve determination of mutant strains (1) Resuscitate the streaked strain from a -80℃ freezer and incubate at 37℃ overnight; (2) Select a single colony and inoculate it into 5 mL of LB liquid medium. Wait for the bacterial cells to settle. OD 600 When the value reaches approximately 0.8, it is transferred to 100 mL of LB liquid medium at a ratio of 1:100 and incubated in a constant temperature shaker at 37°C. (3) The growth curve was defined as 0 h after the transfer, and the bacterial culture was measured every 1 h thereafter. OD 600 Measurements were taken for up to 12 hours. (4) Record the data and draw the growth curve.

[0046] 1.14 Identification of LPS patterns of mutant strains The specific steps for identifying LPS spectra using the silver staining method are as follows.

[0047] (1) Take 2 mL of each strain cultured at different Pi concentrations for 12 h, centrifuge at 5000 rpm for 5 min, and resuspend and wash the bacterial cells with 1×PBS; (2) Add 150 μL of lysis buffer, resuspend and boil in a water bath for 10 min, then centrifuge at 12000 rpm for 10 min at room temperature; (3) Take 30 μL of supernatant and mix it with 270 μL of loading buffer. Add 3 μL of proteinase K to the mixture and shake it in a shaker at 37°C for 1 h. (4) Perform SDS-PAGE electrophoresis on the samples and stain with silver nitrate for subsequent experiments; (5) Remove the separating gel and fix it in a fixative solution on a shaker at room temperature for 4 h, then wash with ddH2O for 15 min. Repeat the washing three times; (6) Oxidize with periodic acid solution on a shaker at room temperature for 7 min, then wash with ddH2O for 15 min. Repeat washing 3 times; (7) Stain with silver staining solution on a shaker at room temperature for 10 min, then wash with ddH2O for 15 min. Repeat washing 3 times; (8) Add an appropriate amount of color developing solution, shake slowly until a black band appears, and then record and photograph the test results.

[0048] 1.15 Data Processing and Analysis All experiments were performed in triplicate, with three biological replicates. Results were analyzed using GraphPad 8.0 software. t - Test, statistical analysis, data expressed as mean ± SD * indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001, layout and graphic design were performed using Adobe Illustrator 2021 software.

[0049] II. Experimental Results 2.1 Increased expression of PhoB at 20-200 µM Pi concentrations Wild-type strains were cultured in MOPS medium with different concentrations of phosphorus ions. Whole-cell proteins from different culture conditions were then extracted and subjected to SDS-PAGE, followed by Western blotting with a prepared PhoB polyclonal antibody. The results showed that the PhoB expression level of DE17 cultured in MOPS with concentrations of 20-200 µM Pi was significantly higher than that in LB culture, while there was no significant difference in PhoB expression level between DE17 cultured in MOPS with concentrations of 4 mM-20 mM Pi and that in LB culture. Figure 1 Part A). Gray-scale statistical analysis results show that PhoB protein expression was significantly increased in the 20-200 µM phosphorus ion concentration stress compared to 4 mM-20 mM and LB. P <0.001) Figure 1 Part B of the middle section.

[0050] 2.2 The average Pi concentration in the respiratory organs of 7-day-old Hy-Line Brown chickens was below 50 μM. Since Pi concentrations of 20-200 µM all showed an increase in PhoB expression levels of DE17, in order to simulate the phosphate concentrations that DE17 might encounter during real-world infection, the average phosphate concentrations of various organs in Hy-Line Brown chickens at 7 days old (peak infection period) and 6 months old (peak egg production period) of the natural host were detected using the ferric sulfate molybdenum blue method. The results showed that the average Pi concentration in various organs of 7-day-old chicks was lower than that of 6-month-old adult chickens. In 7-day-old chicks, the average Pi concentration in air sacs (3.74 μM), trachea (11.97 μM), lungs (15.47 μM), spleen (7.64 μM), and cecum (36.24 μM) was all below 50 μM. Meanwhile, in 6-month-old adult chickens, the Pi concentration in air sacs (13.49 μM) and trachea (11.97 μM) was below 50 μM, and the average Pi concentration in lungs was 52.82 μM. Therefore, 50 μM was selected as the low-phosphorus environment and 4 mM as the high-phosphorus environment for subsequent experiments.

[0051] 2.3 Under low phosphorus conditions phoB Transcriptional translation levels and bacterial alkaline phosphatase levels increased Alkaline phosphatase, as the most significant indicator of PhoB activation and phosphorylation, indirectly represents the regulatory role of PhoB activated by a low-phosphorus environment. Since the results indicate that 50 μM Pi is the critical value for phosphate ion concentration in the respiratory tract, the transcription, translation, and phosphorylation levels of PhoB at 50 μM Pi were measured to ensure that PhoB exerts its regulatory function at 50 μM Pi in subsequent experiments. The results showed that at a concentration of 50 μM Pi, compared to 4 mM Pi and normal LB culture medium, [the following parameters were observed]. phoB The transcriptional level is upregulated (Log2 fold change > 2), and it also acts as a downstream regulated factor. waaH The transcriptional level was increased (Log2 foldchange>3), and at 4 mM Pi and normal LB medium, phoB and waaH There was no significant difference in transcriptional levels. Figure 2 In Part A of the study, the intracellular alkaline phosphatase content of bacteria increased significantly. P <0.001)( Figure 2 In part B of the study, the protein expression level of PhoB was significantly increased. Figure 2 In the middle C section, the protein grayscale analysis results showed that the expression level of PhoB protein was significantly increased ( P <0.001)( Figure 2 (Part D) shows that PhoB is activated and exerts its regulatory function under 50 μM Pi culture conditions.

[0052] 2.4 The transcriptional level of O-antigen-related genes increases under low phosphorus conditions. To investigate whether the O-antigen participates in resisting low phosphorus conditions, total RNA was extracted from bacteria grown in low-phosphorus and normal LB media for 12 h. The transcription levels of genes related to the O-antigen gene cluster were measured using quantitative real-time analysis. The results showed that, compared to normal LB culture, the transcription levels of 13 genes in the O-antigen gene cluster were significantly higher. rmlB - rmlD - rmlA - fdtA - fdtB - wzx - wegP - rmlC - wzy - wegQ - wegR - wegS The transcriptional level was upregulated (Log2 fold change > 2) under low phosphorus culture conditions, serving as a positive control. phoB and waaH The transcriptional levels of the genes were significantly upregulated compared to those under normal LB culture conditions (Log2 fold change > 4). Figure 3 This indicates that the O-antigen gene cluster is involved in resisting low phosphorus environments.

[0053] 2.5 Successfully built phoB Missing and Replaced Plants In order to study phoB The function of genes and in-depth research phoB The regulatory role of genes on O-antigen; this invention constructs a Δ phoB Gene deletion strains and complemented strains. Δ gene deletion was constructed using Red homologous recombination technology. phoB Gene deletion strains, using phoB -UF and phoB -DR primers amplified a band of 1053 bp, and DE17 a band of 1743 bp, successfully constructing the Δ... phoB ( Figure 4 Part A of the study involved PCR amplification of the promoter region using the DE17 genome as a template. phoB The fragment was 879 bp. The PCR-purified fragment was ligated with the pSTV28 plasmid by enzyme digestion. The successfully constructed complement plasmid was named pSL2186, and then electroporated to Δ phoB The competent cells, identified as 968 bp using primer M13, were confirmed to be of expected size by sequencing, and CΔ was successfully constructed. phoB ( Figure 4 Part B). The protein levels of the deleted and complemented strains were verified using PhoB polyclonal antibody, and the results are shown in Δ. phoB No obvious band was observed at the PhoB protein site, while DE17 and CΔ phoBThe expected bands were observed at all locations of the PhoB protein, demonstrating... phoB Deletion and replacement strains were successfully constructed. Figure 4 Part C), and Part 2.3 demonstrates that PhoB phosphorylation in a low-phosphorus environment mediates an increase in downstream alkaline phosphatase levels. Therefore, the alkaline phosphatase content of the deletion strain under low-phosphorus conditions was detected. The results show that Δ phoB Compared to wild-type plants, the intracellular alkaline phosphatase content was significantly reduced under low phosphorus conditions. P <0.001)( Figure 4 Part D of the middle section), proof phoB The missing and supplemented strains were successfully constructed.

[0054] 2.6 phoB The absence of [something] does not affect in vitro growth capacity. To detect Δ phoB To investigate whether it would affect the growth capacity of bacteria in vitro, the growth curves of each strain were measured over 12 hours. The results showed... phoB Gene deletion does not affect the growth of DE17 in LB medium. Figure 5 ).

[0055] 2.7 Δ under low phosphorus conditions phoB Compared to DE17, it restored the integrity of the LPS map. In section 2.4 of this embodiment, the present invention discovered that the transcription level of the O-antigen gene cluster significantly increased under low phosphorus conditions. How exactly does the O-antigen change? Therefore, silver staining was used to detect DE17 and Δ... phoB LPS spectra at different Pi concentrations were analyzed to observe changes in the number and chain length of O-antigens. The results showed that the number of O-antigen bands at 20-200 μM Pi differed from that at 4-80 mM Pi, especially with the absence of medium-length O-antigen units. Figure 6 (Part A), while Δ phoB The number of O-antigen bands was not significantly different between 20-200 μM Pi and 4-20 mM Pi, LB. Compared with DE17, it restored the integrity of the LPS spectrum under low phosphorus conditions, especially the medium-length O-antigen units. Figure 6 (Part B), and this invention also addresses the low-phosphorus and high-phosphorus research standards established in Part 2.2. phoB The missing and filled LPS spectra were verified by silver staining and Western blotting. The results show that silver staining ( Figure 6 (Part C) and Western blotting ( Figure 6 Both the middle D section and the middle D section demonstrate that under low phosphorus culture conditions, DE17 and CΔ phoBThere is a phenomenon of deletion in medium-length O-antigen units, while Δ phoB restored medium-length O-antigen units, and there was no significant difference between high-phosphate culture and normal LB culture.

[0056] 2.8 Under low-phosphate conditions, Δ phoB the transcriptional level of genes related to O-antigen synthesis and rfaH the transcriptional level increased Since it was found in part 2.7 of this example that Δ phoB restored medium-length O-antigen units under low phosphate, then did the transcriptional level change? Therefore, fluorescence quantitative detection was used to detect the transcriptional level of genes related to O-antigen synthesis in Δ phoB under low-phosphate conditions. As shown by the results, Δ phoB compared with DE17 under low-phosphate conditions rmlB , rmlD , rmlA , fdtA , fetB , rmlC , wzy , wegR , wegS the gene transcriptional level was up-regulated (1 < Log2 fold change < 2), and it was experimentally found that fepE the gene controlling O-antigen chain length and rfaH another regulatory factor controlling O-antigen Figure 7 had their transcriptional levels up-regulated (

[0057] 2.9 Under normal LB conditions, the transcriptional level in Δ phoB increased rfaH Since it was found in part 2.8 of this example that the transcriptional level in Δ phoB increased under low-phosphate conditions, then this experiment detected the transcriptional level of genes related to O-antigen synthesis in normal LB medium. As shown by the results, Δ<000043 * 1 *> phoB compared with DE17, there was no significant change in the transcriptional level of the O-antigen gene cluster under LB culture, but fepE the gene controlling O-antigen chain length and rfaH another regulatory factor controlling O-antigen Figure 8 had their transcriptional levels significantly up-regulated (Log2 fold change > 2) ( phoB ). This shows that in the normal growth environment of bacteria, Δ rfaH does not cause a change in the transcriptional level of the O-antigen gene cluster, which is consistent with the conclusion that PhoB dephosphorylation does not regulate downstream genes in LB, and phoB the change in transcriptional level also shows that rfaH after the deletion of rfaH it will compensatorily cause the transcriptional level of rfaH to be up-regulated. It should be noted that there seems to be some repeated or unclear tags in the original text (such as rfaH being used multiple times in a way that might be an error in the original). The translation is done based on the best understanding of the text with the given tags.

[0058] It is generally believed that when the environmental Pi concentration is <4 μM, the Pi-binding protein PstS acts as the main sensor, triggering a conformational change in PhoR, causing it to dissociate from the inhibitory complex. PhoR restores histidine kinase activity and transfers a phosphate group to the aspartic residue of the response regulatory protein PhoB via autophosphorylation, forming phosphorylated PhoB (PhoB~P). Subsequently, PhoB~P forms a dimer, binds to the DNA sequence upstream of the Pho regulator gene, and recruits RNA polymerase (RNAP) to initiate transcription. When the environmental Pi concentration is >4 μM, the Pst transport system forms a complex (PstSCAB-PhoU) with PhoR histidine kinase / phosphatase and PhoU protein, inhibiting the autophosphorylation activity of PhoR. At this time, PhoR functions as a PhoB phosphatase, continuously dephosphorylating PhoB and preventing its activation. However, in the early stages of this invention, when using 4 μM as a low-phosphorus environment for stress, it was found that the DE17 strain could not tolerate such a low Pi concentration, resulting in growth inhibition. This suggests that different strains have strain-specific tolerance limits to low phosphorus. Therefore, this experiment detected the expression level of PhoB under different Pi concentrations and normal LB culture. The results showed that the expression level of PhoB in DE17 cells cultured with 20-200 µM Pi in MOPS was significantly higher than that of PhoB cells cultured with 4-20 mM Pi concentration and LB culture. P The value <0.001 indicates that PhoB is involved in environmental stress of 0-200 µM Pi.

[0059] Because bacteria typically express multiple isolated two-component regulatory systems that regulate different physiological processes, studies have shown that bacterial two-component systems exhibit cross-regulation. This cross-regulation allows bacteria to respond to environmental changes in a more integrated and coordinated manner, enhancing the adaptability and flexibility of their signal transduction networks. For example, the absence of the YycFG two-component system in Bacillus subtilis affects the PhoPR-dependent phosphate-limiting response, such as its inability to be properly activated. phoA and phoPR The expression, unable to suppress tagAB and tagDEF Transcription. Additionally, YycF can directly bind to... phoPR Promoter region. The existence of this mechanism indicates that bacterial response mechanisms are far more complex than expected, involving multi-level regulation and signal integration. Therefore, it is speculated that in DE17 culture at 20-200 µM Pi MOPS, the expression level of PhoB is increased due to tandem transmission of other phosphokinases.

[0060] This invention, through inorganic phosphate detection, found that the average Pi concentration in the respiratory tract of 7-day-old Hy-Line Brown chickens, the natural host of APEC, was below 50 µM Pi. Furthermore, this invention detected Pi concentrations below 50 µM Pi. phoB The transcriptional, translational, and indirect phosphorylation levels. phoA The gene-encoded alkaline phosphatase (AP), as a member of the Pho regulator, is widely used as an indicator to monitor the expression level of the Pho regulator. Results showed that in culture under 50 µM Pi phosphate ion stress... phoB The transcription and translation levels of PhoB were significantly increased, and the intracellular alkaline phosphatase content was also significantly increased. However, there was no significant difference between PhoB at a concentration of 4 mM Pi and under normal LB conditions, indicating that PhoB is phosphorylated in response to low phosphorus stress at a concentration of 50 µM Pi. For the DE17 strain, 50 µM Pi represents a low-phosphorus environment, while 4 mM Pi represents a high-phosphorus environment. Current research on the PhoB / PhoR two-component system focuses primarily on enteropathogenic Escherichia coli (APEC), due to fluctuations in phosphate concentrations in the gut. Studies have shown that healthy adults typically have a phosphate concentration of 15 to 30 mM in their gut. This level of in vivo phosphate should inhibit the expression of the Pho regulator. Since APEC infects chickens via the respiratory route, this invention hypothesizes that APEC encounters a low-phosphorus environment in the early stages of infection, thereby allowing PhoB to exert its biological effects.

[0061] This invention examined the transcriptional level of the O-antigen gene cluster under low phosphorus conditions. The results showed a significant upregulation of the O-antigen gene cluster transcription level (Log2 fold change > 2), indicating that PhoB phosphorylation regulation of the O-antigen gene cluster participates in resistance to low phosphorus environments. Furthermore, LPS mapping analysis revealed the deletion of O units in the short layer of the O-antigen. Bacteria can precisely allocate the synthesis sites of short and long LPS chains to ensure the spatial polarity of outer membrane phospholipids and LPS, thereby enhancing bacterial survival in harsh environments. This invention demonstrates that under low phosphorus conditions, Δ phoB The short-chain phenotype was restored, while the O-antigen gene transcription level was upregulated by 1–2 times. Furthermore, in Δ phoB middle rfaH Transcriptional levels were upregulated compared to the wild-type strain. This indicates that when... phoB When missing, ​ The compensatory upregulation partially makes up for the demand for O-antigen synthesis, enhancing the polar transcription of the O-antigen gene cluster. However, in normal LB medium, Δ ​ The transcription level of O-antigen was not significantly different from that of DE17. ​ The significantly elevated transcription levels further illustrate the influence of Δ ​ middle ​The compensatory upregulation of the O-antigen gene cluster, coupled with the fact that the strain was not subjected to extreme environmental stress, resulted in no significant change in the transcriptional level of the O-antigen gene cluster, which also confirms that the LPS profile remained unchanged during LB culture.

[0062] In summary, this invention has discovered that DE17 can activate the PhoB / PhoR two-component system at a concentration of 50 µM Pi. Under low phosphorus conditions, PhoB phosphorylation regulates the O-antigen gene cluster to cope with the extreme environmental pressure of low phosphorus by participating in the control of the distribution of the number of short and long O-antigen chains.

[0063] Example 2

[0064] Example 1 shows that in Δ ​ middle ​ Given the increased transcription levels, what is the relationship between PhoB and RfaH, and do PhoB and RfaH jointly regulate the O-antigen gene cluster? This study investigates the relationship between RfaH and O-antigen synthesis, using bacterial two-hybrid and EMSA assays to explore whether PhoB and RfaH have a co-regulatory effect on the O-antigen gene cluster.

[0065] I. Materials and Methods 1.1 Strains and Culture Conditions Plasmids: pKT18 and pKT25 were preserved in the laboratory. Culture conditions: pKT18: 30°C, ampicillin resistant; pKT25: 37°C, chloramphenicol resistant.

[0066] 1.2 Construction of Deletion and Replacement Strains ​ For missing replacement strains, refer to the experimental steps 1.11 and 1.12 in Example 1.

[0067] 1.3 Extraction of total bacterial RNA The RNA extraction test follows the experimental steps in Example 1, Section 1.6.

[0068] 1.4 Reverse Transcription PCR For reverse transcription PCR, refer to the experimental steps in 1.7 of Example 1.

[0069] 1.5 Real-time quantitative PCR For real-time quantitative PCR, refer to the experimental steps in 1.8 of Example 1.

[0070] 1.6 Western blotting detection of target protein The detection procedure for the target protein is as described in Example 1.

[0071] 1.7 Bacterial Two-Hybrid Assay (1) Bacterial two-hybrid plasmid vectors pKT25 and pKT18 were revived from a -80°C freezer and cultured overnight at 37°C. (2) Pick a single colony and inoculate it into 10 mL of LB liquid medium, and incubate at 37℃ and 200 rpm for 10 h; (3) Perform plasmid extraction; (4) Construct pKT25- ​ pKT18- ​ plasmids; (5) PKT25- ​ pKT18- ​ Thermal conversion ​ The competent cells were plated on selection plates containing different concentrations of dual antibiotics, and then verified by PCR and identified by sequencing. (6) Inoculate the positive strain into LB liquid medium containing double antibiotics and culture at 37°C and 200 rpm until the logarithmic growth phase; (7) Take 10 μL of bacterial culture and spot it onto a double antibiotic medium containing X-gal. Let it stand at 37℃ for 10 h and observe whether there is a colorimetric interaction.

[0072] 1.8 Gel Retention Test Take the PhoB protein and O-antigen promoter EMSA assay as an example.

[0073] (1) The O-antigen promoter fragment was amplified by PCR and then purified and recovered by PCR. (2) Biotinylated probe labeling of the O-antigen promoter fragment was performed using the Beyotime EMSA / Gel-Shift kit; (3) EMSA test was performed using Beyotime chemiluminescence EMSA kit; (4) Use a chemiluminescence imager to perform gel migration imaging.

[0074] 1.9 Data Processing and Analysis Refer to Example 1.

[0075] II. Experimental Results 2.1 Successful expression and purification of RfaH recombinant protein and preparation of RfaH polyclonal antibody Since no studies in DE17 have shown that RfaH directly regulates the O2 antigen gene cluster, for the sake of experimental rigor, a recombinant RfaH protein was constructed to express the regulation of the O2 antigen gene cluster by EMSA assay. A prokaryotic expression plasmid pET30a-RfaH using pET30a as a vector was successfully constructed using *E. coli* Rosetta, named pSL2185. Identification using T7 primers showed that the pSL2185 band size was 754 bp.​ (Part A) Sequencing results were consistent with expectations, and pSL2185 was successfully constructed. The RfaH recombinant protein was expressed using an E. coli protein expression system. The induction conditions for the RfaH recombinant protein were 1 mM IPTG, 120 rpm, and 30℃ for 4 h. The protein was purified by washing with gradient concentrations of imidazole. SDS-PAGE and His antibody Western blotting were used to verify the purified target protein. The results showed that the size of the RfaH recombinant protein, as detected by SDS-PAGE, was 20.7 kDa (…). ​ Part B); Western blotting detection clearly revealed a single band at the target band location: RfaH recombinant protein was 20.7 kDa ( ​ (Part C), consistent with expectations. Freund's adjuvant was emulsified with the recombinant protein, and mice were repeatedly injected subcutaneously at multiple sites. Blood was collected from the heart, and serum was separated to prepare a polyclonal antibody against RfaH. Antibody purity was verified by Western blotting. The results showed that the RfaH protein exhibited a single band at 18.4 kDa. ​ (Part D) RfaH polyclonal antibody was successfully prepared, and the negative control showed no obvious band, which was consistent with the expected protein size.

[0076] 2.2 RfaH binds to the 5'UTR of the O-antigen synthesis gene cluster EMSA assays were used to verify that RfaH regulates the DE17 O-antigen gene cluster. The EMSA assays showed that as the concentration of RfaH protein increased, the probe bands exhibited significant banding, indicating that RfaH directly binds to the 5'UTR region of the O-antigen synthesis gene cluster. However, no slowly migrating bands were observed in the mixture of RfaH negative control 16S rRNA promoters. ​ This indicates that RfaH directly regulates the O-antigen synthesis gene cluster.

[0077] 2.3 Successfully built ​ Missing and Replaced Plants In order to study gene function and conduct in-depth research ​ Genes in regulating O-antigen and ​ We constructed the Δ relationship. ​ Gene deletion strains and complemented strains. Δ gene deletion was constructed using Red homologous recombination technology. ​ Gene deletion strains, using ​ -UF and ​ -DR primers amplified a band of 1009 bp, and DE17 a band of 1400 bp, successfully constructing the Δ... ​ ( ​Part A of the study involved PCR amplification of the promoter region using the DE17 genome as a template. ​ The fragment was 655 bp. The PCR-purified fragment was ligated to the pSTV28 plasmid by enzyme digestion. The successfully constructed complement plasmid was named pSL2187, and then electroporated to Δ ​ The competent cells, identified as 743 bp using primer M13, were confirmed to be of expected size by sequencing, and CΔ was successfully constructed. ​ ( ​ Part B). The protein levels of the deleted and complemented strains were verified using the RfaH polyclonal antibody constructed in Part 2.1 of this example. The results are shown in Δ. ​ No obvious band was observed at the RfaH protein site, while DE17 and CΔ ​ The expected bands were observed at the RfaH protein location. ​ Part C of the middle section), proof ​ Deleted and supplemented strains were successfully constructed. This was confirmed by silver nitrate staining. ​ (Middle D part left) and Western blotting ( ​ (Right side of section D) Verification analysis of the LPS spectrum, the results are shown below. ​ Gene deletion strains did not form complete O-antigen ladder bands, CΔ ​ The presence of a distinct O-antigen band compared to the wild-type strain proves... ​ The missing and supplemented strains were successfully constructed.

[0078] 2.4 ​ The absence of [something] does not affect in vitro growth capacity. To detect Δ ​ To investigate whether it would affect the growth capacity of bacteria in vitro, the growth curves of each strain were measured over 12 hours. The results showed... ​ Gene deletion does not affect the growth of DE17 in LB medium. ​ ).

[0079] 2.5 ​ The transcriptional level of the O-antigen gene cluster decreases after deletion. In this embodiment 2.3, the present invention found that DE17 is missing. ​ Following gene sequencing, bacteria are unable to synthesize O-antigen. What impact would this have on the transcriptional level of the O-antigen gene cluster? Therefore, the transcriptional levels of genes related to O-antigen synthesis were examined under low phosphorus conditions and normal LB culture. The results show that Δ ​ Compared to 13 genes in the DE17 O-antigen gene cluster under low phosphorus conditions ​ - ​ - ​ - ​ - ​ - ​ -​ - rmlC - wzy - wegQ - wegR - wegS The transcription level decreased. fepE , phoB There was no significant difference in genes. Δ rfaH Under LB culture, compared to the 13 genes in the DE17 O-antigen gene cluster... rmlB - rmlD - rmlA - fdtA - fdtB - wzx - wegP - rmlC - wzy - wegQ - wegR - wegS The transcriptional level decreased significantly (Log2 fold change < -4), and the downregulation was more significant compared with the 50 μM Pi concentration. Figure 13 ).

[0080] 2.6 RfaH expression levels increase under low phosphorus conditions In section 2.5 of this embodiment, it was discovered that RfaH participates in the biological process of O-antigen synthesis in bacteria under low phosphorus conditions. Therefore, does RfaH participate in the regulation of O-antigen under low phosphorus conditions? Thus, the expression level of RfaH in wild-type strains under different Pi concentrations was detected. The results showed that under low phosphorus conditions, the expression level of RfaH in DE17 increased compared to high phosphorus conditions and LB. Figure 14 Part A). Gray-scale analysis results show a significant increase in phosphorus levels under low-phosphorus conditions compared to high-phosphorus conditions ( P <0.01, a significant increase compared to LB ( P <0.001)( Figure 14 Part B of the middle section.

[0081] 2.7 Δ under low phosphorus conditions phoB Increased expression level of RfaH protein In sections 2.8 and 2.9 of Example 1, the present invention discovered Δ phoB middle rfaH If the transcriptional level increases, then at the protein level Δ phoB What changes occur in RfaH under low phosphorus conditions? Therefore, ΔH was measured. phoB The expression level of RfaH in the middle molecule. The results showed that under low phosphorus conditions, Δ phoB Compared to DE17 RfaH, the protein expression level in RfaH increased ( Figure 15 Part A). Gray-scale analysis results show that Δ under low phosphorus conditions phoBThe protein expression level in the middle was significantly increased compared to that in DE17 RfaH. Figure 15 (Part B) P <0.01).

[0082] 2.8 PhoB does not interact with RfaH protein. In sections 2.8 and 2.9 of Example 1 and section 2.7 of this Example, the present invention discovered Δ phoB The transcriptional and protein levels of RfaH show differential changes. What is the relationship between PhoB and RfaH proteins, and are there any interactions? Therefore, bacterial two-hybrid technology was used to detect the interaction between PhoB and RfaH proteins in vitro. PCR amplification was performed using the DE17 genome as a template. phoB, rfaH CDS sequence, PCR purified fragment ligated with pKT25 and pKT18 plasmids by restriction enzyme digestion, pKT25- phoB The plasmids were named pSL2188 and pKT25- phoB The plasmids were named pSL2188 and pKT18- rfaH The plasmid was named pSL2189. Results showed that pSL2188 and pSL2189 were co-transfected. E.coli BTH101 No blue product was formed by enzymatic digestion of X-gal on the double antibiotic medium containing X-gal, showing no significant difference from the negative control pT25 / pT18. In contrast, the positive control pT25-zip / pT18-zip formed a blue product by enzymatic digestion of X-gal, resulting in blue-green colonies. Figure 16 This indicates that PhoB does not interact with the RfaH protein.

[0083] 2.9 PhoB does not regulate the RfaH promoter and co-regulates the O antigen with RfaH. In section 2.8 of this embodiment, it was found that PhoB and RfaH proteins do not interact. Therefore, as an upstream regulator of phosphokinases, does PhoB regulate...? rfaH Does the O-antigen mediate biological function through genes? Therefore, the EMSA assay is used to detect whether PhoB regulates it. rfaH The gene was amplified by PCR, and the resulting DNA fragment was 349 bp in size and labeled with fluorescent biotin. The results showed that no significant banding of the probe band occurred with increasing PhoB protein concentration. PhoB is similar to previously reported... waaH The promoter, as a positive control, exhibited similar banding, while no slowly migrating bands were observed in the mixture of PhoB and the negative control 16S rRNA promoter. Figure 17 Part A of the text indicates that PhoB does not regulate... rfaHGenes. Furthermore, this experiment used competitive EMSA to analyze whether PhoB and RfaH had a synergistic effect on the O-antigen. The results showed that as the concentration of PhoB protein increased, the probe band with the addition of RfaH protein exhibited a more pronounced banding phenomenon compared to the addition of PhoB protein alone. [[ID= Part B of the text explains that RfaH and PhoB synergistically regulate the O-antigen.

[0084] This invention expresses and purifies recombinant RfaH protein. EMSA assays demonstrate that RfaH regulates the APEC O2O-antigen gene cluster. This result confirms that the O-antigen gene cluster is dually regulated by PhoB and RfaH, and also indicates that RfaH regulates the O-antigen in various bacteria by binding to the conserved OPS sequence. RfaH's binding to the OPS sequence ensures complete transcription of longer operons. OPS elements are also present in other operons, including the core polysaccharide operon and the T6SS secretion system operon. Therefore, RfaH also regulates the core polysaccharide and T6SS. However, the degree of RfaH's regulation varies among different operons; for example, the dependence of the core polysaccharide on RfaH is lower than that of the O-antigen. The results of this embodiment show that Δ in the LPS map... ​ The lipid A core polysaccharide was still present, while the O-antigen principle was completely lost. Simultaneously, the transcriptional level of the O-antigen gene cluster was detected, indicating Δ... ​ The transcription level of O-antigen was significantly decreased. Furthermore, compared to low phosphorus conditions, Δ ​ The downregulation of O-antigen transcription was more significant in LB culture, indicating that PhoB affects the transcription level of O-antigen under low phosphorus conditions. However, compared with PhoB, RfaH has a more stringent regulatory hierarchy on the O-antigen gene cluster, resulting in the complete absence of the O-antigen band in the LPS map. This result further consolidates the status of RfaH as the core executor of O-antigen synthesis.

[0085] The experimental studies in conjunction with Example 1 showed that PhoB and RfaH both directly bind to the O-antigen promoter and are functionally related. Furthermore, competitive EMSA indicated that they synergistically regulate the O-antigen. However, bacterial two-hybrid and EMSA experiments ruled out direct interaction between the two or PhoB-mediated interaction with the O-antigen promoter. ​ Direct regulation of genes. This invention hypothesizes that the PhoB-RfaH regulatory axis possesses unique regulatory properties: the two work synergistically in O-antigen synthesis through independent mechanisms (PhoB senses environmental signals and regulates transcription initiation, while RfaH mediates transcriptional elongation), rather than through traditional cascade regulation. Furthermore, RfaH protein expression levels significantly increase under low phosphorus conditions compared to high phosphorus and normal LB culture conditions. P <0.001), indicating that the RfaH protein also participates in resisting low phosphorus environmental stress. Example 1 showed that Δ ​ middle​ The transcription level was elevated, therefore, in this embodiment, the RfaH polyclonal antibody was used to detect Δ. ​ The expression level of RfaH protein was analyzed, and the results showed that the expression level of RfaH protein increased significantly under low phosphorus conditions. P <0.001). This indicates Δ ​ This leads to compensatory expression of the RfaH protein.

[0086] Studies have shown that the OPS sequence induces RNA polymerase to form a conformation more favorable for interaction with RfaH; the ternary interaction space consisting of RNA polymerase, RfaH, and the exposed non-template strand of the OPS sequence favors the strong binding of RfaH to RNA polymerase. The PhoB protein, upon phosphorylation of aspartate, forms a dimer. This dimer binds to the DNA sequence upstream of the Pho regulator gene in a head-to-tail manner, thereby recruiting RNA polymerase. Specificity and σ of RNA polymerase... 70 Subunit interactions facilitate the entry of RNA polymerase into the Pho promoter region and the initiation of transcription. Both require the recruitment of RNAPs to initiate O-antigen transcription, and bacteria prioritize energy conservation to enhance their adaptability in extreme environments. Therefore, this invention concludes that: in low-phosphorus environments, both PhoB and RfaH proteins perform biological functions, with PhoB playing a dominant role in regulating O-antigen biosynthesis, leading to the disappearance of short-layer O-antigens, thus conserving bacterial energy and helping bacteria resist low-phosphorus extreme environments; when the environment is at a high phosphorus concentration, PhoB is continuously dephosphorylated and does not bind to O-antigens, with RfaH dominating O-antigen transcription to ensure the complete transcription of the long O-antigen chain, helping bacteria better infect and colonize organs; when PhoB is absent... ​ Following gene expression, RfaH expression increases compensatorily to ensure transcriptional stability of the O-antigen.

Claims

1. A pathogenic avian Escherichia coli strain tolerant to low-phosphorus environments, characterized in that, The strain can maintain LPS spectrum integrity at phosphorus concentrations of 20-200 μM, enabling [the study of] the integrity of [specific parameters]. rmlB, rmlD, rmlA, fdtA, fetB, rmlC, wzy, wegR and wegS Gene transcription levels are upregulated, and upregulated rfaH Transcription level; The strain underwent Δ phoB Gene deletion mutation.

2. The avian pathogenic Escherichia coli strain tolerant to low phosphorus environments according to claim 1, characterized in that, The strain was named DE17-Δ phoB The accession number is CGMCC No.36537, and it is deposited at the China General Microbiological Culture Collection Center on November 7, 2025.

3. The Listeria monocytogenes strain according to claim 2, characterized in that, The phoB The nucleotide sequence of the gene is shown in SEQ ID No.

1.

4. A method for preparing a low-phosphorus-tolerant pathogenic Escherichia coli strain in birds, characterized in that, The preparation method includes the following steps: (1) Using the DE17 strain genome and pKD3 plasmid as templates, amplification was performed respectively. phoB The upstream and downstream homologous arms of the gene and the antibiotic resistance gene were fused and amplified using the overlap PCR method. (2) The homologous arm containing the antibiotic resistance fragment was electroporated into DE17 competent cells, positive clones were screened for antibiotic resistance, and then competent cells were prepared. (3) Eliminate the chloramphenicol resistance gene in the positive clone to obtain the result.

5. The method for preparing a low-phosphorus-tolerant pathogenic Escherichia coli strain in birds according to claim 4, characterized in that, The antibiotics include chloramphenicol.

6. The method for preparing a low-phosphorus-tolerant pathogenic Escherichia coli strain in birds according to claim 4, characterized in that, The competent states were prepared using the electro-sensor method.

7. The method for preparing a low-phosphorus-tolerant pathogenic Escherichia coli strain in birds according to claim 6, characterized in that, The electric shock receptive state method includes the following steps: (1) Resuscitate the streaked strain from a -80℃ freezer and incubate at 37℃ overnight; (2) Pick a single colony and inoculate it into 5 mL of LB liquid medium. When the bacteria grow to the OD value... 600 When the concentration is 0.8, transfer it to 100 mL of LB liquid medium at a ratio of 1:100; (3) When bacteria grow to OD 600 When the concentration is 0.6, let it stand on ice for 30 minutes, while pre-cooling sterile 10% glycerol in advance; (4) Centrifuge at 5000 rpm and 4℃ for 8 min to collect the bacterial culture, and wash twice with sterile 10% glycerol. (5) Finally, resuspend the bacterial cells in 1 mL of sterile 10% glycerol, dispense into 100 μL / tube, and store at -80℃.

Images