Genes prdR and the proteins encoded by the genes prdR for regulating myxobacteria predation and application thereof

Abstract

The application discloses a gene prdR for regulating myxobacteria predation and a protein coded by the gene, and belongs to the technical field of biotechnology. The mutant DK10 with significantly improved predation capacity is obtained by using the strategy of adaptive evolution; genetic variation sites of the mutant DK10 are analyzed by using the bacterial genome resequencing technology, and functions of the genetic variation sites are researched by using the gene knockout technology, and it is found that the gene deletion of MXAN_2902 (prdR, the nucleotide sequence is shown as SEQ ID NO. 1) significantly enhances the predation capacity of Myxococcus xanthus. The nucleic acid sequence and the amino acid sequence of the transcriptional activator PrdR for regulating the predation of Myxococcus xanthus are disclosed, and the functional strain of Myxococcus xanthus capable of efficiently predating Salmonella enteritidis can be developed by using the prdR gene, and a new strategy is provided for the prevention and control of drug-resistant HVPG pathogenic bacteria.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. It relates to a gene prdR that regulates the predation of myxobacteria, its encoded protein, and its applications, particularly a σ... 54 Application of transcription activator PrdR in regulating the predation of drug-resistant Salmonella by Myxococcus faecalis. Background Technology

[0002] Salmonella enteritidis is a drug-resistant, highly virulent / persistent genotype (HVPG) pathogen that can cause enteric fever diseases such as sepsis and typhoid fever, and is one of the leading causes of bacterial gastroenteritis in humans worldwide. In recent years, the detection rate of Salmonella enteritidis has been increasing year by year, and its drug resistance level has continued to rise, posing a serious challenge to clinical treatment and public health control, necessitating the development of novel control strategies. Myxobacteria, as a type of predatory bacteria, can prey on almost all microbial groups, including Gram-negative and Gram-positive bacteria and fungi. Their predatory characteristics and broad environmental adaptability make them a novel type of biocontrol microorganism. Long-term interaction with prey bacteria can drive the evolution of myxobacteria at the adaptive, genomic, and phenotypic levels, and can also accelerate the evolution of predatory traits in myxobacteria, enhancing their predatory abilities. Therefore, identifying the key regulatory factors that control the predation of drug-resistant bacteria by myxobacteria during adaptive evolution will lay the foundation for the prevention and control of drug-resistant HVPG pathogens. Summary of the Invention

[0003] The purpose of this invention is to identify a gene regulating the predatory ability of *Myxococcus faecalis* and its encoded protein PrdR. Loss of function of this gene significantly enhances the ability of *Myxococcus faecalis* to prey on drug-resistant *Salmonella enteritidis*. This invention has important reference value and potential application value for studying how to control drug-resistant *HVPG* pathogens by improving the predatory ability of *Myxococcus faecalis*. Specifically, the technical solution adopted in this invention is as follows:

[0004] This invention employs a central inoculation model to obtain an adaptive evolution mutant, DK10, with significantly enhanced predation ability against drug-resistant Salmonella enteritidis through long-term co-culture experiments. Genetic variation sites on the genome of mutant DK10 were identified using bacterial genome resequencing technology. The effects of genetic variation sites on predation of drug-resistant Salmonella enteritidis by knocking out genes encoding specific sites were analyzed.

[0005] This invention provides adaptive evolutionary mutants of *Myxococcus faecalis* with different co-culture cycles.

[0006] This invention provides genetic variation sites on the genomes of individually cultured and co-cultured *Myxococcus faecalis* in the 9th and 10th generations.

[0007] This invention provides a protein encoding the predatory gene prdR of *Myxococcus faecalis*, the amino acid sequence of which is shown in SEQ ID No. 2.

[0008] This invention provides a gene prdR that regulates the predation of *Myxococcus faecalis*, encoding the protein described above.

[0009] Preferably, the gene prdR that regulates the predation of *Myxococcus faecalis* has the nucleotide sequence shown in SEQ ID No. 1.

[0010] This invention also provides the application of the above-mentioned gene prdR in regulating the predation of drug-resistant Salmonella by Myxococcus faecalis.

[0011] The present invention also provides a protein encoded by a gene that can enhance the predation of Salmonella mutants by Myxococcus faecalis, the amino acid sequence of which is shown in SEQ ID NO.4.

[0012] The present invention also provides the gene encoding the above-mentioned protein that can enhance the predation of drug-resistant Salmonella enteritidis mutants by Myxococcus faecalis, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0013] This invention also provides a method for constructing the *Myxococcus faecalis* mutant DK10, which involves mutating the *Myxococcus faecalis* genome by changing the base at position 3393990 of Genbank accession number MXAN_2902 from C to G, thereby changing alanine to glycine.

[0014] The present invention also provides a yellow myxococcus mutant DK10 obtained by the above construction method.

[0015] Preferably, the Salmonella is multidrug-resistant Salmonella enteritidis.

[0016] The beneficial effects of this invention are:

[0017] This invention utilizes an adaptive evolution strategy to evolve an adaptive mutant, DK10, from *Myxococcus faecalis* DK1622, exhibiting significantly enhanced predation capabilities. Bacterial genome resequencing identified three specific genetic variation sites on the genome of the adaptive mutant DK10. Experiments revealed that the deletion of the MXAN_2902 gene significantly enhances the predation ability of *Myxococcus faecalis*, which we named prdR. This gene may provide a novel strategy for the control of drug-resistant HVPG pathogens. Attached Figure Description

[0018] Figure 1 It is an adaptive evolutionary mutant that evolved from wild-type yellow myxococcus, with a significantly enhanced predatory ability.

[0019] Figure 2 This study observes the colony morphology of *Myxococcus faecalis* colonies when 9th and 10th generation adaptive evolution mutants prey on 9th and 10th generation *Salmonella enteritidis* Sal188.

[0020] Figure 3 This is the effect of different co-culture cycles on the ability of *Myxococcus faecalis* to prey on Salmonella Sal188.

[0021] Figure 4 The effects of deletions of the MXAN_2902, MXAN_6413, and MXAN_6735 genes on the ability of *Myxococcus faecalis* to prey on *Salmonella Sal188*. Detailed Implementation

[0022] The following are specific implementation examples of the present invention. It should be noted that these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Modifications and substitutions to the details and form of the implementation schemes made within the scope and spirit of the present invention all fall within the protection scope of the present invention.

[0023] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents.

[0024] The drug-resistant HVPG pathogen involved in the following examples is Salmonella enteritidis Sal188.

[0025] Example 1

[0026] This embodiment provides a novel gene prdR that regulates the predation of *Myxococcus faecalis*, its encoded protein, and its applications, including the following steps:

[0027] (1) After culturing *Myxococcus faecalis* DK1622 in CTT liquid medium for 24 h, centrifuge at 8000 rpm for 5 min to collect the cells. Wash once with TPM liquid medium (formulation: 1.98 g MgSO4·7H2O; 10 mM Tris·HCl pH 7.6; 1 mM KH2PO4 / K2HPO4 pH 7.6; adjust the pH of the medium to 7.6 with KOH, and bring the volume to 1 L), then resuspend in TPM liquid medium and adjust the cell concentration to OD. 550 =10. Salmonella enteritidis Sal188 was cultured in LB liquid medium until it reached the logarithmic growth phase, at which point the initial bacterial concentration was adjusted to OD0. 600=10. Then, inoculate 50 μL of Sal188 onto TPM medium. After the colonies have dried, inoculate 10 μL of DK1622 into the center of the Salmonella colony. After culturing for 3 days, proceed as follows: Figure 1 As shown in Figure A, DK1622 and Sal188 were transferred to CTT and LB plates, respectively; after growth, the co-culture experiment was repeated. First, on TPM medium, after 5 transfers, 0.05% glucose was added to the TPM medium, and then 5 more transfers were performed; by gradually increasing the glucose concentration, the adaptive evolution of *Myxococcus faecalis* and *Salmonella* was accelerated.

[0028] (2) Further, we conducted cross-predation experiments on *Myxococcus faecalis* DK1622 and *Salmonella Sal188* during the 9th and 10th co-culture cycles, using the long-term co-culture method. *Myxococcus faecalis* (DK9) during the 9th co-culture cycle preyed on *Salmonella Sal188* during the 9th (S9) and 10th (S10) co-culture cycles, respectively; simultaneously, *Myxococcus faecalis* (DK10) during the 10th co-culture cycle preyed on *Salmonella Sal188* during the 9th (S9) and 10th (S10) co-culture cycles, with 6 replicates for each combination. The study found that two *Myxococcus faecalis* colonies on the DK9 / S9 predation plates exhibited abnormal morphology; and four *Myxococcus faecalis* colonies on the DK10 / S9 predation plates exhibited abnormal morphology. Figure 2 ).

[0029] (3) We used a competition experiment to analyze the differences in predation of wild-type Salmonella Salmonella Sal188 by *Myxococcus faecalis* at different co-culture cycles. The study found that on TPM+0.05% Glucose plates, there was no significant difference in predation ability between *Myxococcus faecalis* DK3 (co-cultured for the third cycle) and wild-type DK1622; the predation ability of *Myxococcus faecalis* after the fifth co-culture cycle was significantly enhanced; the predation ability of DK9 was significantly weakened, while the predation ability of DK10 was significantly stronger than that of the wild-type. Figure 3 ).

[0030] (4) Further, we analyzed the mutant sites on the genomes of *Myxococcus faecalis* DK9 and DK10 from the 9th and 10th co-culture cycles, as well as those from individually cultured *Myxococcus faecalis* D9 and D10, using bacterial genome resequencing technology. In the 9th co-culture cycle, we identified 20 genetic variations in D9, including 18 single nucleotide polymorphisms (SNPs), 1 insertion mutation (INS), and 1 deletion mutation (DEL); and 21 genetic variations in DK9, including 20 single nucleotide polymorphisms (SNPs) and 1 deletion mutation (DEL) (Table 1). During the 10th co-culture cycle, we identified 23 genetic variations in strain D10, including 20 single nucleotide polymorphisms (SNPs), 2 insertion mutations (INS), and 1 deletion mutation (DEL); and 25 genetic variations in strain DK10, including 22 SNPs, 1 multinucleotide polymorphism (MNP), and 2 deletion mutations (Table 1). We further analyzed the genomic locations of these genetic variation sites, identifying four specific mutation sites in DK10: 165757 (MXAN_0141), 3393990 (MXAN_2902), 7899408 (MXAN_6413), and 8283443 (MXAN_6735) (Table 2).

[0031] Table 1

[0032] Table 2

[0033] (4) Since mutation site 165757 (MXAN_0141) is a synonymous mutation and does not change the protein sequence, we constructed deletion mutants of the MXAN_2902, MXAN_6413, and MXAN_6735 genes. First, we constructed the upstream homologous arm 2902-Up-flank of the MXAN_2902 gene. Using primers 2902-1F and 2902-2R (Table 3) with genomic DNA of *Myxococcus faecalis* DK1622 as a template, we amplified the upstream homologous arm. At the same time, using primers 2902-3F and 2902-4R (Table 3) with genomic DNA of *Myxococcus faecalis* DK1622 as a template, we amplified the downstream homologous arm 2902-Down-flank. Using homologous recombination, we constructed 2902-Up-flank and 2902-Down-flank into the HindIII and EcoRI sites of the pBJ113 plasmid to obtain pBJ113-2902 knockout. The pBJ113-2902 knockout plasmid was transformed into *E. coli*, and the pBJ113-2902 knockout plasmid was extracted. *Myxococcus faecalis* DK1622 competent cells were prepared, and the pBJ113-2902 knockout plasmid was transformed into *Myxococcus faecalis* DK1622 competent cells. Single-crossover strains were obtained by screening with kanamycin-containing medium, and double-crossover strains were obtained by screening with galactose-containing medium, resulting in *Myxococcus faecalis* ∆MXAN_2902 with the MXAN_2902 gene knocked out. *Myxococcus faecalis* ∆MXAN_6413 and ∆MXAN_6735 with the MXAN_6413 and MXAN_6735 knocked out were constructed using the same method (primers are shown in Table 3). The differences in the predatory ability of the ∆MXAN_2902, ∆MXAN_6413, and ∆MXAN_6735 mutants were analyzed using a competition experiment. The competitive experiment involved culturing *Myxococcus faecalis* mutants DK1622, ∆MXAN_2902, ∆MXAN_6413, and ∆MXAN_6735 in CTT liquid medium for 24 h, centrifuging at 8000 rpm for 5 min, collecting the cells, washing once with TPM liquid medium, resuspending in TPM liquid medium, and adjusting the cell concentration to OD0.05. 550 =10. Salmonella enteritidis Sal188 was cultured in LB liquid medium until it reached the logarithmic growth phase, at which point the initial bacterial concentration was adjusted to OD0. 600=10. Then, 50 μL of Salmonella enteritidis Sal188 and 50 μL of Myxococcus faecalis were mixed thoroughly and inoculated onto TPM (formula: 1.98 g MgSO4·7H2O; 10 mM Tris·HCl pH 7.6; 1 mM KH2PO4 / K2HPO4 pH 7.6; 15 g agar; adjust the pH of the medium to 7.6 with KOH, and bring the volume to 1 L) and TPM + 0.05% Glucose medium (formula: 1.98 g MgSO4·7H2O; 10 mM Tris·HCl pH 7.6; 1 mM KH2PO4 / K2HPO4 pH 7.6; 0.5 g glucose; 15 g agar; adjust the pH of the medium to 7.6 with KOH, and bring the volume to 1 L). A control group was prepared by inoculating 50 μL of Salmonella enteritidis Sal188 alone. 30 Incubate at ℃ for 18 hours; collect residual bacterial cells on the plates using TPM liquid medium, serially dilute, and spread onto LB+TCR plates (LB medium supplemented with 20 μg / ml tetracycline). Calculate the CFU of *Salmonella flavum* mutants DK1622, ∆MXAN_2902, ∆MXAN_6413, and ∆MXAN_6735 after they prey on *Salmonella* Sal188.

[0034] (5) The study found that the deletion of the MXAN_2902 gene on TPM and TPM+0.05% Glucose plates significantly enhanced the ability of *Myxococcus faecalis* to prey on Salmonella. Figure 4 ).

[0035] Table 3

[0036] Nucleic acid and amino acid sequences of the prdR gene

[0037] The nucleotide sequence of the original prdR gene is SEQ ID No. 1:

[0038] The amino acid sequence encoded by the original prdR gene SEQ ID No.2: MTRPVSAYAPRSERKPLYVKVVTQPALHGCWAVNISETGIGLIATPHTPSEGPREGEDVELSFSLPDSGEHIRVRGVVRWRHDASGAVAAMGVSFRAFEEADGVKLARYLASSHLQVVVAFATEQESRAVTAALEGVAQLHFAASPSDAHALVTRGDAACLVVCGQDEEQALALVESLAARRADADPSGAGPPSDLASRIVYCAPAAPERLVALFNDGRIFRALGPSPTREALHQAVLQAGREHGVRTEQWRMALELERSLLRERALSQKLPPAPGGRGGDEVGFRSAAMQRVMELVRLVAPHRVAVLLQGETGTGKEVLARILHRLSGRGDVPLVVQDCGALSETLLESELFGHVKGSFTGAVADHPGLFVLANGGTIFLDEIENTTANLQAKLLRVLETGDVRPVGGTQVRHVDVRVVAASNKDMGEEVRAGRFRADLFYRLNSFTVDIPPLRDRPEDVPELALYFLELFNRTLRRSASGIAPDAVDALRAYAWPGNVRELRNVMERSVLLSRPGEVVSRRLLPPVLGSTLALRNEPGGDGSLRARLDRVEREFIREALERHGGVLRRAAVALGMDPVTLGRRVRRHGLWKGDS

[0039] The nucleotide sequence of the mutant prdR gene (mutant DK10) SEQ ID No.3

[0040] The amino acid sequence encoded by the mutant prdR gene (mutant DK10) is SEQ ID No. 4: MTRPVSAYAPRSERKPLYVKVVTQPALHGCWAVNISETGIGLIATPHTPSEGPREGEDVELSFSLPDSGEHIRVRGVVRWRHDASGAVAAMGVSFRAFEEADGVKLARYLASSHLQVVVAFATEQESRAVTAALEGVAQLHFAASPSDA HALVTRGDAACLVVCGQDEEQALALVESLAARRADADPSGAGPPSDLASRIVYCAPAAPERLVALFNDGRIFRALGPSPTREALHQAVLQAGREHGVRTEQWRMALELERSLLRERALSQKLPPAPGGRGGDEVGFRSAAMQRVMELVR LVAPHRVAVLLQGETGTGKEVLARILHRLSGRGDVPLVVQDCGALSETLLESELFGHVKGSFTGAVADHPGLFVLANGGTIFLDEIENTTANLQAKLLRVLETGDVRPVGGTQVRHVDVRVVAGSNKDMGEEVRAGRFRADLFYRLNSF TVDIPPLRDRPEDVPELALYFLELFNRTLRRSASGIAPDAVDALRAYAWPGNVRELRNVMERSVLLSRPGEVVSRRLLPPVLGSTLALRNEPGGDGSLRARLDRVEREFIREALERHGGVLRRAAVALGMDPVTLGRRVRRHGLWKGDS

[0041] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A protein encoding the gene prdR that regulates the predation of *Myxococcus faecalis*, characterized in that, The amino acid sequence is shown in SEQ ID No.

2.

2. The gene prdR encoding the predatory gene of *Myxococcus faecalis* as described in claim 1.

3. The gene prdR according to claim 2, characterized in that, The nucleotide sequence is shown in SEQ ID No.

1.

4. A protein encoded by a gene that enhances the predation of Salmonella by *Myxococcus faecalis* mutants, characterized in that, The amino acid sequence is shown in SEQ ID NO.

4.

5. A mutant gene encoding the protein of claim 4.

6. The mutant gene according to claim 5, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

3.

7. A method for constructing a *Myxococcus faecalis* mutant DK10, characterized in that, The mutation involves altering the genome of *Myxococcus faecalis* by changing the base at position 3393990 of Genbank accession number MXAN_2902 from C to G, resulting in alanine being replaced by glycine.

8. A yellow myxococcus mutant DK10 obtained according to the construction method of claim 7.

9. The use of the gene described in claim 2, 3, 5 or 6 in regulating the predation of drug-resistant Salmonella by Myxococcus faecalis.

10. The application according to claim 9, characterized in that, The Salmonella mentioned is multidrug-resistant Salmonella enteritidis.

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