Application of pseudomonas aeruginosa tsrF gene and preparation method of engineering bacteria of pseudomonas aeruginosa tsrF gene

Abstract

The invention relates to application of a pseudomonas aeruginosa tsrF gene and a preparation method of engineering bacteria of the pseudomonas aeruginosa tsrF gene. A novel tsrF gene is identified in a pseudomonas aeruginosa genome, and the expression of the tsrF gene is regulated in the pseudomonas aeruginosa, so that the'biofilm yield ', the'cyclic diguanylate (c-di-GMP) yield', the'type 6 secretion system (T6SS) expression ', the'FleQ receptor yield of c-di-GMP', the'PelD receptor yield of c-di-GMP 'and the like in the pseudomonas aeruginosa can be regulated; further, the TsrF protein can be used as a new target for research and development of drugs for preventing and treating pseudomonas aeruginosa, and the TsrF protein has important significance on prevention and control of pseudomonas aeruginosa infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a Pseudomonas aeruginosa tsrF gene, TsrF protein, engineering bacteria and preparation and application thereof. BACKGROUND

[0002] Pseudomonas aeruginosa Pseudomonas aeruginosa (also known as "green pyocyanin", belonging to gram-negative bacilli), is one of the most toxic respiratory pathogens. Existing research shows that Pseudomonas aeruginosa has high pathogenicity because the bacteria can release a variety of virulence factors such as pyocyanin and protease after invading the host, and can also release a variety of toxic proteins to the host through T6SS (also known as "type VI secretion system"). Studies have shown that the expression of T6SS of Pseudomonas aeruginosa isolated from chronic infected patients is significantly increased, indicating that T6SS promotes chronic infection. T6SS can inject a variety of types of effector proteins (toxin proteins) to adjacent prokaryotic and eukaryotic cells, destroy their cell membranes, cell walls and genetic materials, etc., so as to be in a favorable position in intraspecific or interspecific competition.

[0003] In order to treat Pseudomonas aeruginosa infection, antibiotics are often used in clinic, and it has been found through research that Pseudomonas aeruginosa has strong biofilm formation ability. In the clinic, the bacteria in the biofilm are wrapped by a large amount of extracellular polymers, making it difficult for antibiotics to enter and kill the bacteria. However, after repeated treatment with drugs, drug-resistant strains are easily evolved, and then biofilm of drug-resistant strains is formed, resulting in reduced or even completely ineffective antibiotic treatment.

[0004] Therefore, it is necessary to find a new target for treating Pseudomonas aeruginosa infection and provide a train of thought for analyzing the drug resistance mechanism of Pseudomonas aeruginosa. SUMMARY

[0005] In order to solve the above technical problems, it is necessary to find a new target for treating Pseudomonas aeruginosa infection and affect the biofilm yield and T6SS expression of Pseudomonas aeruginosa.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is as follows: In a first aspect, the present application provides a Pseudomonas aeruginosa tsrF gene, wherein the tsrF gene is a nucleotide sequence as shown in SEQ ID No. 1.

[0007] In a second aspect, the present application also provides an application of knocking out the Pseudomonas aeruginosa tsrF gene, wherein the application is any one of F11) to F15): F11) application in reducing the biofilm yield of Pseudomonas aeruginosa; F12) use in reducing expression of P. aeruginosa T6SS; F13) use in reducing production of P. aeruginosa cyclic di-GMP (may also be referred to as "c-di-GMP"); F14) use in reducing production of P. aeruginosa FleQ receptor of cyclic di-GMP (may also be referred to as "c-di-GMP receptor FleQ"); F15) use in reducing production of P. aeruginosa PelD receptor of cyclic di-GMP (may also be referred to as "c-di-GMP receptor PelD"); The tsrF gene is a nucleotide sequence as shown in SEQ ID No. 1.

[0008] In a third aspect, the present application further provides use of a P. aeruginosa tsrF gene, the use is use in reducing expression of P. aeruginosa T6SS, the T6SS is at least one of H1-T6SS, H2-T6SS, H3-T6SS and H4-T6SS, and the tsrF gene is a nucleotide sequence as shown in SEQ ID No. 1.

[0009] In a fourth aspect, the present application further provides use of a P. aeruginosa tsrF gene, the use is any one of F1) to F6): F1) use in improving production of P. aeruginosa biofilm; F2) use in improving expression of P. aeruginosa T6SS; F3) use in improving production of P. aeruginosa cyclic di-GMP; F4) use in improving production of P. aeruginosa FleQ receptor of cyclic di-GMP; F5) use in improving production of P. aeruginosa PelD receptor of cyclic di-GMP; F6) use in preparing a drug for inhibiting reproduction of E. coli; The tsrF gene is a nucleotide sequence as shown in SEQ ID No. 1.

[0010] In a fifth aspect, the present application further provides use of a P. aeruginosa tsrF gene, the use is use in improving expression of P. aeruginosa T6SS, the T6SS is at least one of H1-T6SS, H2-T6SS, H3-T6SS and H4-T6SS, and the tsrF gene is a nucleotide sequence as shown in SEQ ID No. 1.

[0011] In a sixth aspect, the present application further provides a TsrF protein, wherein the TsrF protein is an amino acid sequence as shown in SEQ ID No. 2, and further wherein the protein is composed of 524 amino acids, has a molecular weight of 57.17 kDa, and has a theoretical isoelectric point of 8.77. tsrF In a seventh aspect, the present application further provides a drug target protein, wherein the drug target protein is the TsrF protein as described above, and further wherein the drug is a drug for treating P. aeruginosa infection.

[0012] In a seventh aspect, the present application further provides a drug target protein, wherein the drug target protein is the TsrF protein as described above, and further wherein the drug is a drug for treating P. aeruginosa infection.

[0013] In an eighth aspect, the present application further provides a P. aeruginosa engineering bacterium, wherein the engineering bacterium is a P. aeruginosa with a tsrF gene knocked out or a P. aeruginosa with the tsrF gene overexpressed. tsrF In an eighth aspect, the present application further provides a P. aeruginosa engineering bacterium, wherein the engineering bacterium is a P. aeruginosa with a gene knocked out or a P. aeruginosa with the

[0014] gene overexpressed. tsrF In a ninth aspect, the present application further provides a preparation method of the P. aeruginosa engineering bacterium, wherein the P. aeruginosa with the tsrF gene knocked out is prepared by a method M1, or the P. aeruginosa with the gene overexpressed is prepared by a method M2. tsrF The method M1 comprises the following steps: knocking out the tsrF gene in the P. aeruginosa by using a gene knockout technology to obtain the P. aeruginosa with the gene knocked out. tsrF The method M2 comprises the following steps: transforming an expression vector containing the tsrF gene into the P. aeruginosa to obtain the P. aeruginosa with the gene overexpressed.

[0015] Further, the knocking out of the tsrF gene in the P. aeruginosa by using the gene knockout technology can be implemented by the following technical solution: amplifying the upstream homologous arm of the tsrF gene by using a primer pair UF and UR, amplifying the downstream homologous arm of the Primer pair gene by using a primer pair DF and DR; fusing the upstream and downstream homologous arm fragments by using overlap PCR, and introducing the fragments into a host bacterium by using electroporation, and then screening by using sucrose sensitivity and verifying by using PCR to obtain a tsrF gene scarless knockout mutant (one kind of “P. aeruginosa engineering bacterium”). tsrF Further, the transforming of the expression vector containing the gene into the P. aeruginosa can be implemented by the following technical solution: amplifying the tsrF gene by using a primer pair OEF and OER.tsrF The full-length sequence of the gene is cloned into an expression vector to transform a host bacterium to obtain tsrF The overexpression strain (or a kind of "pseudomonas aeruginosa engineering bacteria"); Further, the primers UF, UR, DF, DR, OEF and OER can be selected as the nucleotide sequences shown in SEQ ID No. 3-8, specifically as follows: Primer UF: gagctcggtacccggggatccGCTGACGGCGAAGGCCGT (SEQ ID No. 3); Primer UR: agcttcatgcgatggaCATCGCCTCCATCTCGTCC (SEQ ID No. 4); Primer DF: gatgTCCATCGCATGAAGCTATTGAG (SEQ ID No. 5); Primer DR: acgacggccagtgccaagcttCCAGGGACGGGTGGGCGA (SEQ ID No. 6); Primer OEF: gtcgacggtatcgataagcttTCCCGTATCGCTACCTGGACG (SEQ ID No. 7); Primer OER: cgctctagaactagtggatccCGCAGCCAATCAGGGCCG (SEQ ID No. 8).

[0016] In the tenth aspect, the application further provides a method for reducing the yield of biofilm and / or the expression of T6SS in the pseudomonas aeruginosa engineering bacteria, which comprises knocking out the tsrF gene in the genome of the pseudomonas aeruginosa by using genetic engineering method, wherein the tsrF gene is the nucleotide sequence shown in SEQ ID No. 1.

[0017] The application / method of the above-mentioned second to fifth aspects, ninth aspect and tenth aspect of the application are all non-disease diagnosis and treatment purposes.

[0018] Technical effects of the application: The application first identifies a brand new tsrFThe gene was identified and its application in regulating key bacterial physiological functions has been clarified, specifically in regulating "biofilm production," "c-di-GMP production," "T6SS expression," "FleQ receptor production of c-di-GMP," and "PelD receptor production of c-di-GMP" in *Pseudomonas aeruginosa*. This fills a gap in the research on the regulatory mechanism of class IV T6SS (H4-T6SS) in *Pseudomonas aeruginosa*, providing new molecular targets and theoretical foundations for technological breakthroughs in this field. Furthermore, by constructing [a specific gene] within *Pseudomonas aeruginosa*... tsrF Gene knockout mutants significantly altered T6SS activity, biofilm production, and c-di-GMP synthesis levels. These three factors are key regulatory links in *Pseudomonas aeruginosa*'s competitive survival ability, virulence expression, and drug resistance formation, indicating that this invention can intervene in the core pathogenic pathways of bacteria. This invention provides both an efficient and specific biotechnological means for regulating the pathogenicity of *Pseudomonas aeruginosa* and a new technical direction for solving the infection control challenges caused by the high drug resistance and virulent virulence of *Pseudomonas aeruginosa* in clinical practice (such as...). tsrF As a target protein for drugs used to treat Pseudomonas aeruginosa infection, this protein has significant practical implications and application prospects for improving the treatment efficacy of Pseudomonas aeruginosa infection.

[0019] Other features and aspects of this disclosure will become clear from the following detailed description of exemplary embodiments with reference to the accompanying drawings. Attached Figure Description

[0020] The accompanying drawings, which are included in and form part of this specification, illustrate exemplary embodiments, features, and aspects of this disclosure together with the specification and serve to explain the principles of this disclosure.

[0021] tsrF This image shows the gene cluster "H4-T6SS" and its functional activity test data of the *Pseudomonas aeruginosa* strain LYSZa7 in Example 1. Part A shows the structure of H4-T6SS in *Pseudomonas aeruginosa* LYSZa7; Part B shows schematic diagrams of the four types of T6SS in strain LYSZa7; Part C shows the determination of the number of viable *E. coli*; and Part D shows the movement of H4-T6SS through mobile genetic elements. The blue lines represent GC content, and each data point represents the percentage of GC content in the corresponding 2500 bp upstream and downstream sequences. (Based on pairing...) t Tests showed P < 0.05; P < 0.01; P < 0.001. ns, no significant difference. EV, control used empty vector (same for subsequent figures).

[0022] Figure 1 As in Example 2 Figure 2The graph shows the data from the validation experiment of gene regulation of H4-T6SS. Part A shows the Δ function detected by real-time quantitative PCR (RT-qPCR). tsrF (EV), Δ retS Δ retS (EV) and Δ tsrF Δ retS ( tsrF Part B shows the expression levels of four T6SS strains in the strain; Part B is for RT-qPCR detection of wild-type LYSZa7(EV), Δ tsrF (EV) and Δ tsrF ( tsrF The expression levels of four T6SS strains were analyzed in part C by Western blot. tsrF (EV), Δ retS Δ retS (EV) and Δ tsrF Δ retS ( tsrF The yield of TssB4-Flag protein in strain A was measured, with the RNA polymerase α subunit (α-RNAP) serving as an internal control; Part D was the determination of the number of viable Escherichia coli.

[0023] tsrF This is a phylogenetic analysis diagram of the comprehensive regulatory role of TsrF and its homologous proteins in Example 3, based on RNA-seq data ( ), LYSZa7 and Δ Figure 3 GO enrichment analysis (as shown in Part A) and KEGG enrichment analysis (as shown in Part B) of differentially expressed genes; as shown in Part C, BLAST analysis of TsrF was performed to screen out the top 26 representative homologous proteins. The right panel shows the operon structures (containing these homologous proteins) in the corresponding species, and the conserved T6SS protein in each operon is marked with a different color.

[0024] tsrF This is a graph showing experimental data related to the direct regulation of H4-T6SS and biofilm formation by TsrF in Example 4. Part A shows the predicted TsrF domains; Part B shows the electrophoretic mobility detection of TsrF and... Figure 4 and tssB4 Direct binding effect of promoters; tssH4 Gene promoters serve as negative controls; section C shows wild-type LYSZa7 and Δ algB Δ tsrF Δ retS Δ retS And the biofilm yield data of their complementary strains after 12 hours of culture in LB medium; Part D shows the RT-qPCR detection of Δ tsrF (EV), ΔretS Δ retS (EV) and Δ tsrF Δ retS ( tsrF ) strains tsrF - D expression levels of the genes.

[0025] pelA Figures showing experimental data related to the role of c-di-GMP in TsrF-mediated H4-T6SS expression and biofilm formation in Example 5, wherein Part A shows determination of intracellular c-di-GMP levels by detecting mRNA levels of the genes Figure 5 cdrA (EV), Δ retS Δ retS (EV) and Δ tsrF Δ retS ( tsrF ) strains tsrF and gcbA expression levels of the genes; Part C shows Western Blot detection of TssB4-Flag protein expression levels in Δ roeA (EV), Δ retS Δ retS (EV), Δ tsrF Δ retS ( tsrF ) and Δ bifA Δ retS ( tsrF ) strains, with a-RNAP as the internal control; Part D shows E. coli survival number determination; Part E shows biofilm production detection of Δ sadC (EV), Δ retS Δ retS (EV), Δ tsrF Δ retS ( tsrF ) and Δ bifA Δ retS ( tsrF ) strains.

[0026] sadC Figures showing experimental data related to the role of TsrF in mediating AmrZ regulation of c-di-GMP production in Example 6, wherein Part A shows RT-qPCR detection of Δ Figure 6 (EV), Δ retS Δ retS (EV) and Δ tsrF Δ retS ( tsrF ) strains tsrF ​mRNA levels of the genes shown in part B, determined by detecting amrZ mRNA levels of the genes shown in part B, determined by detecting cdrA (EV), Δ retS (EV), and Δ retS (EV), and Δ tsrF (EV), and Δ retS (EV), and Δ tsrF (EV), and Δ amrZ (EV), and Δ tssH4 mRNA levels of the genes shown in part D, determined by detecting tssB4 (EV), Δ retS (EV), and Δ retS (EV), and Δ tsrF (EV), and Δ retS (EV), and Δ tsrF (EV), and Δ amrZ (EV), and Δ

[0027] amrZ FIG. 7 is a graph of experimental data related to the response of FleQ to TsrF regulation of c-di-GMP in Example 7, where part A shows mRNA levels of the genes shown in part A, determined by detecting Figure 7 (EV), Δ retS (EV), and Δ retS (EV), and Δ tsrF (EV), and Δ fleQ (EV), and Δ pelD (EV), and Δ retS (EV), and Δ tsrF (EV), and Δ retS (EV), and Δ tsrF (EV), and Δ sadC (EV), and Δ retS (EV), and Δ tsrF (EV), and Δ fleQ (EV), and Δ sadC (EV), and Δ retS (EV), and Δ tsrF (EV), and Δ pelD (EV), and Δ sadC (EV), and Δ tssH4 mRNA levels of the genes shown in part C, determined by detecting tssB4 (EV), Δ retS (EV), and Δ tsrF (EV), and Δ retS (EV), and Δ tsrF (EV), and Δ sadC (EV), and Δ retS (EV), and Δ tsrF (EV), and Δ fleQ (EV), and Δ sadC (EV), and ΔretS Δ tsrF ( pelD ) Biofilm production of the strain after 12 hours of culture in LB medium. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present application will now be described in detail, with reference to the figures. The detailed description is not to be regarded as limiting the application, but rather as a description of certain aspects, features and embodiments of the application.

[0029] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, for any numerical ranges recited herein, the endpoints are included and the intervals formed between the endpoints are also included. The endpoints are included in the ranges and the intervals are included in the endpoints, unless specifically stated otherwise. Any smaller range or interval that falls within the recited ranges are also specifically included.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials which are related to the present application. In case of conflict, the content of the specification will prevail.

[0031] Many modifications and variations of this application of the application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is to be understood that the application is not limited in scope by the specific embodiments described herein. Rather, the specific embodiments are intended to illustrate the application and the following claims are intended to cover all equivalents thereof.

[0032] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended terms that are intended to mean "including but not limited to".

[0033] In a first aspect, the present application provides a Pseudomonas aeruginosa sadC The gene is a nucleotide sequence as shown in SEQ ID No. 1. tsrF

[0034] ​

[0035] The nucleotide sequence shown in SEQ ID No. 1 is a transcription factor TsrF first identified in the genome of a clinically isolated Pseudomonas aeruginosa, which can regulate the production of a biofilm, the production of c-di-GMP, the expression of T6SS, the production of a FleQ receptor of c-di-GMP, the production of a PelD receptor of c-di-GMP and the like in Pseudomonas aeruginosa, has great potential as a target for the development of anti-Pseudomonas aeruginosa drugs, can provide an efficient and specific biological technology for regulating the pathogenicity of Pseudomonas aeruginosa, can provide a new technical direction for solving the infection prevention and control problem of Pseudomonas aeruginosa caused by high drug resistance and strong virulence in the clinic, and has important practical significance and application prospect for improving the treatment effect of Pseudomonas aeruginosa infection.

[0036] In a second aspect, the present application further provides an application of knocking out a Pseudomonas aeruginosa tsrF gene, and the application is any one of F11) to F15): F11) the application in reducing the production of a biofilm of Pseudomonas aeruginosa; F12) the application in reducing the expression of T6SS of Pseudomonas aeruginosa; F13) the application in reducing the production of c-di-GMP of Pseudomonas aeruginosa; F14) the application in reducing the production of a FleQ receptor of c-di-GMP of Pseudomonas aeruginosa; F15) the application in reducing the production of a PelD receptor of c-di-GMP of Pseudomonas aeruginosa; The tsrF gene is a nucleotide sequence shown in SEQ ID No. 1.

[0037] In a third aspect, the present application further provides an application of knocking out a Pseudomonas aeruginosa tsrF gene, and the application is the application in reducing the expression of T6SS of Pseudomonas aeruginosa, and the tsrF gene is a nucleotide sequence shown in SEQ ID No. 1; and further, the T6SS is at least one of H1-T6SS, H2-T6SS, H3-T6SS and H4-T6SS.

[0038] In a fourth aspect, the present application further provides an application of overexpressing a Pseudomonas aeruginosa tsrF gene, and the application is any one of F1) to F6): F1) the application in improving the production of a biofilm of Pseudomonas aeruginosa; F2) the application in improving the expression of T6SS of Pseudomonas aeruginosa; F3) application in improving production of c-di-GMP of Pseudomonas aeruginosa; F4) application in improving production of FleQ receptor of c-di-GMP of Pseudomonas aeruginosa; F5) application in improving production of PelD receptor of c-di-GMP of Pseudomonas aeruginosa; F6) application in preparing a drug for inhibiting reproduction of Escherichia coli; The tsrF The gene is a nucleotide sequence as shown in SEQ ID No. 1 sequence.

[0039] In a fifth aspect, the present application further provides an application of overexpressing a Pseudomonas aeruginosa tsrF The application is application in improving expression of T6SS of Pseudomonas aeruginosa, the tsrF The gene is a nucleotide sequence as shown in SEQ ID No. 1 sequence; and further, the T6SS is at least one of H1-T6SS, H2-T6SS, H3-T6SS and H4-T6SS.

[0040] In a sixth aspect, the present application further provides a TsrF protein, the TsrF protein is expressed by the tsrF The gene of claim 1, the TsrF protein is an amino acid sequence as shown in SEQ ID No. 2 sequence; and further, the protein is composed of 524 amino acids, the molecular weight is 57.17 kDa, and the theoretical isoelectric point is 8.77.

[0041] In the present application, the amino acid sequence represented by SEQ ID No. 2 is: MEAMDVATLIRELLQAADLEAAAQQFLTRLCRSPGIDQGYCYWREIGWDRLLPVAGHGGPDSTLPVISLGELDNPLVYGLMSNRPCHVERLSRLVDVGAGFDALRERLAGAEAMLVLPIAGESRNAALGVAAIIGSDAALRSWRADRTWQEVFRCHELIIGRLHAQHGETAQAQRQRAADQRREVEQGRARASRLLAGGFIGVGATAKKLRAEMLRLADSSLSLLITGETGSGKDHAAWLIHQASARQGNFVPVNCAAIPKDLIEAELFGAVRGAYTGALQSRKGLVAEADGGTLFLDEIGDMPLGLQGTLLRLLNEKKYRPVGATREQASDFRLICATHRSLVDLVKEGAFREDLYFRIRQHTLHIPALRDRAEDIPALVAHVLLQHNRERQGCVAGISANALARLQAYGFPGNVREMRSLVLAAAERTEPGRRIRSSLLGELADSQPQRGSVRASMAGVLQELVHTDNLPKALLMIERLIVTERLRKVAGSRRSAALSLGIAKRTLARKCLEWNLDGEDSIA.

[0042] In a seventh aspect, the present application further provides a drug target protein, which is the TsrF protein as described above, and a drug for treating P. aeruginosa infection.

[0043] In an eighth aspect, the present application further provides a P. aeruginosa engineering bacterium, which is a P. aeruginosa with a knocked-out tsrF gene or a P. aeruginosa with over-expressed tsrF gene. tsrF The gene is a nucleotide sequence represented by SEQ ID No. 1.

[0044] In a ninth aspect, the present application further provides a preparation method of the P. aeruginosa engineering bacterium as described above, which comprises the following steps: tsrF M1) knocking out the tsrF gene in the P. aeruginosa by using a gene knockout technology to obtain the P. aeruginosa with the knocked-out gene, or M2) over-expressing the tsrF gene in the P. aeruginosa by using a gene over-expression technology to obtain the P. aeruginosa with the over-expressed tsrF gene. The M2 method includes the following steps: [The text abruptly ends here, likely due to an incomplete sentence or a missing section tsrF The gene expression vector was transformed into Pseudomonas aeruginosa, resulting in overexpression. tsrF Genetically modified Pseudomonas aeruginosa.

[0045] Furthermore, gene knockout technology was used to remove Pseudomonas aeruginosa. tsrF Gene knockout can be achieved using the following technical approach: amplification with primer pairs UF and UR. tsrF Upstream homologous arm of gene, using Primer pair DF and DR amplification tsrF Downstream homologous arms of the gene; the upstream and downstream homologous arm fragments were fused by overlap PCR, and after electroporation and introduction into the host bacteria, sucrose sensitivity screening combined with PCR verification was used to obtain... tsrF Gene knockout mutant strain (a type of "engineered Pseudomonas aeruginosa"); Furthermore, it will contain tsrF The gene expression vector can be transformed into Pseudomonas aeruginosa using the following technical methods: amplification using primer pairs OEF and OER. tsrF The full-length gene sequence was cloned into an expression vector and then transformed into a host bacterium to obtain... tsrF Overexpression strain (or a type of "engineered Pseudomonas aeruginosa"); Furthermore, the primers UF, UR, DF, DR, OEF, and OER can be selected as nucleotide sequences as shown in SEQ ID NO. 3-8, specifically as follows: Primer UF: gagctcggtacccggggatccGCTGACGGCGAAGGCCGT (SEQ ID No. 3); Primer UR: agcttcatgcgatggaCATCGCCTCCATCTCGTCC (SEQ ID No. 4); Primer DF: gatgTCCATCGCATGAAGCTATTGAG (SEQ ID No. 5); Primer DR: acgacggccagtgccaagcttCCAGGGACGGGTGGGCGA (SEQ ID No. 6); Primer OEF: gtcgacggtatcgataagcttTCCCGTATCGCTACCTGGACG (SEQ ID No. 7); Primer OER: cgctctagaactagtggatccCGCAGCCAATCAGGGCCG (SEQ ID No. 8).

[0046] In a tenth aspect, the present invention also provides a method for reducing biofilm production and / or T6SS expression in engineered Pseudomonas aeruginosa, the method comprising reducing biofilm production and / or T6SS expression in the Pseudomonas aeruginosa genome. tsrF The gene was knocked out using genetic engineering methods. tsrF The gene is the nucleotide sequence shown in SEQ ID No. 1.

[0047] The applications / methods of the second to fifth aspects, the ninth aspect and the tenth aspect of the present invention are all applications / methods for purposes other than disease diagnosis and treatment.

[0048] The nucleotide sequence shown in SEQ ID No. 1 of this invention (i.e., Pseudomonas aeruginosa) tsrF The TsrF gene has been shown to positively regulate biofilm production in Pseudomonas aeruginosa. By knocking out this gene, biofilm production in Pseudomonas aeruginosa can be reduced, thereby reducing the protective effect of extracellular polymers on Pseudomonas aeruginosa and making it easier for antibiotics to enter the bacteria and kill them. Furthermore, the TsrF protein can also be used as a target for Pseudomonas aeruginosa infection, providing a new direction and idea for the development of drugs against Pseudomonas aeruginosa infection.

[0049] The nucleotide sequence shown in SEQ ID No. 1 of this invention (i.e., Pseudomonas aeruginosa) tsrF Studies have shown that T6SS expression in *Pseudomonas aeruginosa* isolated from patients with chronic infection is significantly elevated, indicating that T6SS promotes chronic infection. By knocking out this gene, the corresponding T6SS expression is inhibited, which can reduce the release of virulence factors, such as effector proteins, from *P. aeruginosa* into patients, thereby reducing its virulence. Furthermore, TsrF protein can also be used as a target for *P. aeruginosa* infection, providing new directions and ideas for the development of anti-*P. aeruginosa* drugs.

[0050] The nucleotide sequence shown in SEQ ID No. 1 of this invention (i.e., Pseudomonas aeruginosa) tsrF The gene has been shown to regulate c-di-GMP production, c-di-GMP receptor FleQ production, and c-di-GMP receptor PelD production in Pseudomonas aeruginosa. These substances are positively correlated with the virulence of Pseudomonas aeruginosa (including biofilm production and T6SS expression). Knocking out this gene can reduce the production / expression of these factors, thereby reducing the virulence of Pseudomonas aeruginosa. Furthermore, the TsrF protein can also be used as a target for Pseudomonas aeruginosa infection, providing a new direction and idea for the development of drugs against Pseudomonas aeruginosa infection.

[0051] In addition, by analyzing the nucleotide sequence shown in SEQ ID No. 1 (i.e., Pseudomonas aeruginosa) tsrF Overexpression of genes can also play a role in the development of related drugs, such as in the application of anti-E. coli drugs.

[0052] The present invention will be further illustrated by the following embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0053] Example 1: Functional verification of H4-T6SS in Pseudomonas aeruginosa strain LYSZa7 A novel T6SS gene cluster was first identified in a *Pseudomonas aeruginosa* strain LYSZa7 isolated from a patient (see [link to relevant documentation]). Figure 1 (As shown in Parts A and B), this invention names it H4-T6SS. Using the genome of wild-type LYSZa7 strain as a template, the coding genes of the core components of H1-T6SS, H2-T6SS, H3-T6SS, and H4-T6SS were knocked out respectively through homologous recombination technology. tssH1 , tssH2 , tssH3 , tssH4 Single mutants ΔT6SS-1, ΔT6SS-2, ΔT6SS-3, and ΔT6SS-4, and four T6SS complete deletion mutants ΔT6SS-1, 2, 3, and 4 were obtained; simultaneously, a variant of ΔT6SS-4 was constructed. tssH4 Gene-complemented strain ΔT6SS-4 (T6SS-4).

[0054] The specific process of constructing the complement in this embodiment is as follows: [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] tssH4 The gene was cloned into the pUCP20 vector (PCR amplification primers PPF and PPR are shown in SEQ ID No. 9-10), and the complement plasmid was transformed into the ΔT6SS-4 mutant strain via triparental conjugation. The successful construction of the complement was confirmed by resistance screening (clobenzylpenicillin, 150 μg / mL) and DNA sequencing.

[0055] The sequences of the PPF and PPR mentioned above are shown in SEQ ID No. 9-10, as follows: Primer PPF: gagctcggtacccggggatccTCCCGTATCGCTACCTGGAC (SEQ ID No. 9) Primer PPR: acgacggccagtgccaagcttCGCAGCCAATCAGGGCCG (SEQ ID No. 10) Using *Escherichia coli* DH5α as the recipient bacterium, a bacterial competition experiment was conducted to detect the killing ability of various mutant strains. The specific procedure for the bacterial competition experiment in this embodiment was as follows: *Escherichia coli* containing pBBR1-MCS5 was mixed with *Pseudomonas aeruginosa* in equal proportions. A sterile 0.45 μM nylon membrane was placed on LB agar plates, and 20 μl of the mixed bacterial solution was placed on the nylon membrane. The plates were then incubated at 37°C for 16 h. The bacteria on the membrane were washed off with sterile water, the bacterial solution was serially diluted, and then spread onto LB agar plates containing 50 μg / ml gentamicin. The plates were incubated at 37°C, and the colony counts were recorded.

[0056] The results showed that the ΔT6SS-4 mutant strain had a significantly reduced killing ability against Escherichia coli DH5α, and its defective phenotype was similar to that of the ΔT6SS-1 and ΔT6SS-3 mutant strains; while the complement strain ΔT6SS-4 (T6SS-4) could completely restore the killing efficiency against DH5α. Figure 1 (As shown in section C); the killing ability of the four T6SS complete deletion mutants ΔT6SS-1,2,3,4 was significantly reduced compared to each single mutant. Figure 1 As shown in section C). The GC content of the upstream and downstream sequences of the H4-T6SS gene cluster is lower than the average GC content of 66.2% for strain LYSZa7 ( Figure 1 As shown in section D), integrase-related genes and functional genes involved in bacterial virulence and drug resistance were detected in the upstream and downstream regions of this gene cluster. Figure 1 (As shown in section D).

[0057] The above results indicate that H4-T6SS in Pseudomonas aeruginosa strain LYSZa7 has functional activity and can significantly affect the bacteria's ability to kill other microorganisms. At the same time, H4-T6SS is located in an integrin, which enables the movement and spread of gene clusters, providing a molecular basis for Pseudomonas aeruginosa to adapt to the environment and enhance its competitive ability.

[0058] Example 2: Verification of the regulatory function of TsrF on H4-T6SS A predicted transcription factor, TsrF, was identified in the H4-T6SS gene cluster. It was knocked out in the RetS deletion mutant (H4-T6SS activation background) and the wild-type LYSZa7 strain, respectively. tsrF The double mutant Δ was constructed. retS Δ tsrF and single mutant Δ tsrF RT-qPCR results showed that, retS In deletion mutants, tsrF Mutations can inhibit the activity of H1-, H2, H3, and H4-T6SS (e.g., Figure 2 (As shown in Part A of the middle section).

[0059] Similarly, compared with wild type LYSZa7, the expression of H1-, H2-, H3-, H4-T6SS in Δ tsrF H1-, H2-, H3-, H4-T6SS in mutant strains were inhibited (as shown in part B of Figure 2

[0060] Western Blot experiment showed that the expression of Δ retS Δ tsrF The expression of TssB4 in mutant strains was significantly reduced, and the expression could be restored by exogenous expression of tsrF (as shown in part C of Figure 2

[0061] By knocking out the membrane protein TssM of H1-, H2-, H3-T6SS, mutant strains with only active H4-T6SS were constructed (ΔT6SS-1,2,3). The results showed that, compared with Δ retS ΔT6SS-1,2,3 mutant strains, the survival rate of E. coli DH5α was significantly increased after co-cultured with Δ retS Δ tsrF ΔT6SS-1,2,3 mutant strains, the survival rate of E. coli DH5α was significantly increased after co-cultured with Δ retS Δ tsrF ΔT6SS-1,2,3 mutant strains, the expression of tsrF after, the effect can be restored (as shown in part D of Figure 2 In addition, when the four T6SS full deletion mutant strains were co-cultured with E. coli DH5α, the survival rate of E. coli was further increased, and tsrF The mutation did not affect the killing ability of the mutant strain to E. coli (as shown in part D of Figure 2 D). By comparing ΔT6SS-1,2,3 and ΔT6SS-1,2,3,4 mutant strains, the antibacterial effect of H4-T6SS can be observed (as shown in part D of Figure 2 D). The above results show that TsrF is a global activator of T6SS in P. aeruginosa LYSZa7, which can positively regulate the expression and antibacterial activity of H4-T6SS.

[0062] The process of Western Blot experiment in this example is as follows: (1) Add 5x SDS loading buffer to the protein sample, and boil for 10 min; prepare SDS-PAGE gel according to the protein gel kit; (2) Take 15 μL protein sample and add it to the SDS-PAGE gel loading well, 90V electrophoresis for 30 min, then adjust the voltage to 120V electrophoresis to the distance of SDS loading buffer from the bottom of the protein gel 2-3 cm stop; ​​(3) Adjust the PVDF membrane and filter paper to the size of the SDS-PAGE gel. Before the transfer operation, soak the PVDF membrane in methanol solution for at least 15 seconds. After electrophoresis, soak the PVDF membrane, transfer filter paper, and SDS-PAGE gel in the transfer solution. Stack the filter paper-protein gel-PVDF membrane-filter paper neatly from bottom to top (black side of the transfer clamp down), and place it in the electrophoresis tank. Wet transfer at a constant current of 400mA for 1.5 hours (adjust as needed). (4) After the transfer is completed, the PVDF membrane is removed and placed in the blocking solution (PBST solution containing 5% skim milk powder) for 1 hour; (5) Primary antibody incubation: Dilute the primary antibody with PBST at a ratio of 1:1000, place the blocked PVDF membrane in the primary antibody dilution solution and incubate overnight at 4°C or at room temperature for 2 hours; (6) After the primary antibody incubation is completed, the PVDF membrane is washed with PBST 4 times, 15 min each time; (7) Secondary antibody incubation: Dilute the secondary antibody with PBST at a ratio of 1:20000, transfer the PVDF membrane to the secondary antibody dilution solution, and incubate at room temperature for 1 hour with shaking at 50 rpm. (8) After the secondary antibody incubation is completed, the PVDF membrane is washed with PBST 4 times, 15 min each time; (9) Use ECL chemiluminescence solution for development. Generally, the ECL chemiluminescence solution is prepared by mixing the two solutions in the ECL chemiluminescence detection kit at a volume ratio of 1:1. The luminescence solution should be prepared and used immediately.

[0063] Example 3: TsrF is a globally distributed regulatory factor widely distributed in Gram-negative bacteria. Extracting wild LYSZa7 strains and Δ tsrF Total RNA from mutant strains was sequenced using RNA-Seq; differential expression analysis was then used for screening. Genes with a p-value of ≤0.05 were identified, and functional and pathway enrichment analyses were performed using the GO and KEGG databases. GO enrichment results indicated that differentially expressed genes are involved in multiple biological functions, including T6SS, metal ion binding, extracellular polysaccharides, pyocyanin synthesis, membrane components, and transport. Figure 3 (As shown in Part A of the middle section).

[0064] Differentially expressed genes were mapped to 13 KEGG pathways, covering secondary metabolism, two-component systems, biofilm formation, etc. (e.g.) Figure 3 (As shown in Part B).

[0065] Phylogenetic analysis showed that TsrF is not only present in the genus *Pseudomonas* (… Pseudomonas ), and also exists in the genus Vibrio ( Vibrio Burkholderia ( ) Burkholderia) and Achromobacter Achromobacter ) and in all the above-mentioned bacterial species, tsrF The presence of at least one T6SS structural gene upstream or downstream of the gene indicates that TsrF is mainly a Regulators associated with T6SS (As shown in Part C of Figure 3 ).

[0066] Example 4 Transcriptional factor TsrF directly regulates the expression of H4-T6SS and biofilm related genes Protein structure prediction The structure of TsrF contains an AAA domain (ATPase domain associated with various cellular activities) and an HTH domain (helix-turn-helix DNA binding domain) (as shown in Part A of Figure 4 According to the transcriptional direction (arrow direction) of the genomic annotation, the H4-T6SS gene cluster can be divided into two parts (as shown in Part A of tssB4 - tssL4 and tssH4 - tssM4 (as shown in Part A of Figure 1 ).

[0067] Electrophoretic mobility shift assay (EMSA) experiments showed that after incubation of TsrF with tssB4 or tssH4 promoter probe, a retarded band was observed, indicating that TsrF can bind to the promoter of the H4-T6SS gene cluster (as shown in Part B of Figure 4 The incubation reaction of TsrF with algB gene promoter probe was used as a negative control (as shown in Part B of Figure 4 ).

[0068] The process of the EMSA experiment in this example is as follows: (1) PCR to obtain a 300-500 bp fragment before the start codon of the target gene; (2) preparation of the EMSA electrophoresis gel: the formula is 1 ml 5xTBE buffer, 1 ml acrylamide / bisacrylamide, 313 μL 80% glycerol, 75 μL 10% ammonium persulfate (APS), 5 μL tetramethyl ethylenediamine (TEMED), 7.607 ml sterile water; (3) EMSA binding reaction: 1 μL binding buffer, 100 ng probe, 100 μg TsrF, and make up to 10 μL with water. After 30 min of reaction at room temperature, 5 μL 5xLoading Buffer was added, and the reaction system was gently pipetted; (4) electrophoresis: 0.5xTBE was used as the buffer, and pre-electrophoresis was performed at 90 V for 30 min to remove factors that might damage the interaction between proteins and nucleic acids in the gel. After pre-electrophoresis, 20 μL of the reaction system was loaded, and electrophoresis was performed at 90 V until the bromophenol blue dye reached 2 / 3-3 / 4 of the gel, and then the electrophoresis was stopped; (5) the migration band was observed on the DNA gel photographing system.

[0069] As shown in Figure 4As shown in section C, the experimental results show that Δ retS Δ tsrF The biofilm formation ability of the double mutant strain was significantly lower than that of the Δ strain. retS Mutant strain, replacement tsrF It can be partially recovered later; Δ retS Δ tsrF Double mutant biofilm synthesis gene pelA - D Gene expression levels were significantly lower than Δ retS mutant strain ( Figure 4 (As shown in section D).

[0070] The process for determining the biofilm yield in this embodiment is as follows: (1) Pick a single colony from an LB plate and place it into 3 ml of LB medium. Incubate overnight at 37°C with shaking. Re-inoculate the overnight culture onto fresh medium and incubate at 37°C with shaking until the logarithmic phase. (2) Adjust the OD600 value of the test strain to 0.002, add it to a 96-well plate, add 120µL of bacterial solution to each well, make 5 replicates for each test strain, and incubate at 37℃. (3) After the appropriate time has been incubated, remove the 96-well plate, use a pipette to extract the bacterial culture, and wash it three times with sterile water to remove the bacteria from the bottom of the well. (4) Add 150µL of 0.1% (m / v) crystal violet solution to each well and let stand for 20min; (5) Use a pipette to extract the crystal violet and wash off any unadsorbed crystal violet with sterile water; (6) Blow dry the water on the 96-well plate, add 200µL of 95% (v / v) ethanol to each well, and let stand for 20 min; (7) Use a multi-functional microplate reader to read the absorbance at 570 nm. The experiment should be repeated at least 3 times.

[0071] Example 5: TsrF regulates H4-T6SS and biofilm formation in a c-di-GMP dependent manner. RT-qPCR detection Δ retS With Δ retS Δ tsrF c-di-GMP indicator gene in mutant strains cdrA The expression reveals Δ retS Δ tsrF middle cdrA Expression was significantly lower than Δ retS exogenous expression tsrF The expression can be restored ( Figure 5 (As shown in Part A of the diagram); In addition, RT-qPCR also confirmed that TsrF can regulate the c-di-GMP synthesis gene. gcbA ,roeA The expression ( Figure 5 (As shown in Part B). Subsequently, in Δ retS Δ tsrF Exogenous expression in mutant strains sadC (Improving c-di-GMP) and bifA (Degradation of c-di-GMP), discovered sadC It can upregulate H4-T6SS expression and bifA No impact ( Figure 5 (as shown in Part C), and sadC Can eliminate Δ retS Δ tsrF The phenomenon of increased E. coli survival rate when ΔT6SS-1,2,3 is co-cultured with E. coli ( Figure 5 (As shown in section D). Finally, through biofilm detection experiments, it was found that increasing Δ retS Δ tsrF The mutant strain's c-di-GMP level can restore its biofilm formation defect, while in Δ retS Δ tsrF Decreased c-di-GMP levels in mutant strains had no effect on biofilm formation. Figure 5 (As shown in section E).

[0072] The above experimental results indicate that TsrF can regulate the expression activity and biofilm formation of H4-T6SS in a c-di-GMP-dependent manner by activating c-di-GMP synthesis, providing experimental evidence for clarifying the regulatory mechanism of TsrF and subsequent drug target development.

[0073] Example 6: TsrF regulates c-di-GMP synthesis by enhancing AmrZ activity. The expression of c-di-GMP metabolic regulatory genes in each strain was detected by RNA-seq and RT-qPCR, and it was found that only amrZ In Δ tsrF Significantly downregulated in mutant strains, and exogenous expression tsrF Its expression can be restored ( Figure 6 (as shown in Part A); then pBBR1-MCS5- amrZ Import Δ retS Δ tsrF The mutant strain was detected by RT-qPCR, which showed the presence of the c-di-GMP indicator gene. cdrA The expression of was significantly increased ( Figure 6 (As described in Part B). Next, the H4-T6SS gene was detected by RT-qPCR ( tssB4 , tssH4 The expression of AmrZ was found to upregulate Δ. retS Δ tsrF Expression of H4-T6SS in mutant strains (Figure 6 Competition experiments further showed that Δ retS Δ tsrF After Δ Figure 6 T6SS-1,2,3 expressed AmrZ, the antibacterial activity against E. coli was significantly improved (as described in section D of the detailed description). retS Δ tsrF After the mutant strain expressed AmrZ, the total amount of biofilm could be restored to the level of Δ retS Δ Figure 6 (as described in section E of the detailed description). Finally, EMSA experiments confirmed that the purified TsrF could directly bind to the promoter region of amrZ (as described in section F of the detailed description). Figure 6

[0074] The above experimental results show that TsrF can directly bind to the promoter of amrZ and positively regulate its expression, increase the synthesis of intracellular c-di-GMP by improving the activity of AmrZ, and further enhance the activity of H4-T6SS and biofilm formation, thereby clarifying the key molecular link of the TsrF regulatory pathway, and providing experimental basis for taking the TsrF-AmrZ-c-di-GMP axis as a target for prevention and control of P. aeruginosa infection.

[0075] Example 7 FleQ acts as a c-di-GMP effector in the process of TsrF regulating H4-T6SS and biofilm formation.

[0076] The expression of retS and retS in Δ tsrF was detected by RT-qPCR, confirming that both were down-regulated in Δ fleQ Δ pelD (as described in section A of the detailed description); then Δ retS Δ tsrF was constructed, and Figure 7 plasmid was introduced to increase the level of c-di-GMP. RT-qPCR detection showed that only Δ retS Δ tsrF Δ fleQ Δ retS Δ tsrF Δ pelD strains could restore the expression of sadC and retS genes, Δ tsrF Δ pelD ( sadC ) strain. tssH4 tssB4 retS Δ tsrF Δ fleQ ( sadC ​​​) strains failed to recover ( Figure 7 Bacterial competition experiments showed that in the presence of Δ retS Δ tsrF Knocking out FleQ (but not PelD) in ΔT6SS-1,2,3 abolished the c-di-GMP-induced increase in antibacterial activity (Fig. 6C); biofilm detection showed that Figure 7 pelA / pelB Gene expression analysis also showed that sadC Overexpression of FleQ in Δ retS Δ tsrF The total amount of biofilm and pelA / pelB expression could not be restored (Fig. 6D). Figure 7

[0077] The above results confirmed that FleQ is a key c-di-GMP effector in the TsrF regulatory pathway, mediating c-di-GMP signaling to regulate the expression of H4-T6SS, antibacterial activity, and biofilm formation, which provides core experimental evidence for further clarifying the molecular mechanism of the TsrF-c-di-GMP regulatory axis and the development of targeted drugs.

[0078] In the description of the present specification, the description referring to the terms "one embodiment", "an example", "a specific example" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0079] Unless otherwise defined, technical terms or scientific terms used in the present application shall be understood as having the meaning commonly understood by one of ordinary skill in the art to which the present application pertains. The terms "one", "a", "the" and the like in the present application do not denote a quantity restriction, but can denote a singular or a plurality. The terms "include", "contain", "have" and any variation thereof in the present application are intended to cover non-exclusive inclusion. The terms "connect", "connect to", "couple" and the like in the present application are not limited to physical or mechanical connection, but include electrical connection, whether direct or indirect. The term "multiple" in the present application means two or more, and "and / or" describes the association relationship of the associated objects, which means that there can be three relationships. The character " / " generally represents an "or" relationship between the associated objects. The terms "first", "second", "third" and the like in the present application are only to distinguish similar objects, and do not represent a specific order of the objects. ​​

[0080] The above embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it cannot be understood as a limitation on the patent scope of the application. It should be noted that for ordinary skilled persons in the art, several modifications or improvements can be made without departing from the concept of the present application, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. An application for knocking out a Pseudomonas aeruginosa tsrF gene, characterized in that, The use is any one of F21) - F26): F21) use in increasing production of P. aeruginosa biofilm; F22) use in increasing expression of P. aeruginosa T6SS; F23) use in increasing production of P. aeruginosa cyclic di-GMP; F24) use in increasing production of P. aeruginosa cyclic di-GMP FleQ receptor; F25) use in increasing production of P. aeruginosa cyclic di-GMP PelD receptor; The F26) use in the manufacture of a medicament for inhibiting E. coli reproduction; The nucleotide sequence of the gene is shown in the sequence of SEQ ID No.

1.

2. Use of a knockout Pseudomonas aeruginosa tsrF gene characterized in that, The application is the use in reducing the expression of P. aeruginosa T6SS, the T6SS is at least one of H1-T6SS, H2-T6SS, H3-T6SS and H4-T6SS, and the tsrF The gene is a nucleotide sequence shown in the sequence of SEQ ID No.

1.

3. Use of overexpression of Pseudomonas aeruginosa tsrF gene characterized in that, tsrF The use is any one of F21) - F26): F21) use in increasing production of P. aeruginosa biofilm; F22) use in increasing expression of P. aeruginosa T6SS; F23) use in increasing production of P. aeruginosa cyclic di-GMP; F24) use in increasing production of P. aeruginosa cyclic di-GMP FleQ receptor; F25) use in increasing production of P. aeruginosa cyclic di-GMP PelD receptor; The F26) use in the manufacture of a medicament for inhibiting E. coli reproduction; The gene is a nucleotide sequence as shown in SEQ ID No. 1 sequence.

4. Use of overexpression of Pseudomonas aeruginosa tsrF gene characterized in that, The application is the use in improving the expression of Pseudomonas aeruginosa T6SS, the T6SS is at least one of H1-T6SS, H2-T6SS, H3-T6SS and H4-T6SS, and the tsrF The gene is a nucleotide sequence shown in the sequence of SEQ ID No.

1.

5. A method for preparing a Pseudomonas aeruginosa engineering bacterium, characterized by, The engineered bacteria is Pseudomonas aeruginosa with the gene knocked out tsrF The gene is a nucleotide sequence as shown in SEQ ID No. 1 sequence tsrF The gene is a nucleotide sequence as shown in SEQ ID No. 1 sequence Knockouts were prepared using the M1 method. tsrF Genes of Pseudomonas aeruginosa, including: M1) The method comprises the following steps: using a gene knockout technique to knockout the gene in Pseudomonas aeruginosa tsrF to obtain Pseudomonas aeruginosa with the knockout tsrF gene.

6. A method for preparing a Pseudomonas aeruginosa engineering bacterium, characterized by, The engineering bacteria is Pseudomonas aeruginosa overexpressing the tsrF gene, the tsrF gene is a nucleotide sequence as shown in SEQ ID No. 1 sequence; Overexpression was prepared using the M2 method. tsrF Genes of Pseudomonas aeruginosa, including: M2) The method comprises the following steps: transforming the expression vector containing tsrF The expression vector of the gene into P. aeruginosa to obtain P. aeruginosa overexpressing tsrF The gene.

7. A method of reducing biofilm production and / or T6SS expression in a P. aeruginosa engineered bacterium, characterized in that, The method comprises knocking out the gene in the genome of Pseudomonas aeruginosa by using a genetic engineering method tsrF The gene is a nucleotide sequence as shown in the sequence of SEQ ID No.

1. tsrF tsrF The gene is a nucleotide sequence as shown in the sequence of SEQ ID No. 1.

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