DNA phosphorothioation modification enzyme and application thereof

Abstract

The application discloses a DNA phosphor-thioacylation modification system and application thereof, and belongs to the technical field of bioengineering. The phosphor-thioacylation modification protein comprises a nucleotide sequence of a pptA gene, as shown in SEQ ID NO. 1, and an amino acid sequence encoded by the nucleotide sequence, as shown in SEQ ID NO. 2. The PptA protein encoded by the pptA gene can perform phosphor-thioacylation modification on bacterial and bacteriophage genomes. After the pptA gene is integrated into a bacteriophage genome, the pptA gene can serve as an antagonistic system of a bacterial anti-bacteriophage Ssp defense system, endow the bacteriophage with the ability of resisting the Ssp defense system, or form an anti-bacteriophage defense system together with SspFGH, and help the bacteria resist the infection of the bacteriophage. The application provides a new way for the development of biotechnology, in-vivo and in-vitro DNA phosphor-thioacylation modification, bacteriophage engineering, bacteriophage treatment and application in anti-bacteriophage engineering.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a DNA phosphorus thioylation modification system, an antagonistic system for bacterial anti-phage mechanisms, and the application of this system in biotechnology development, in vivo and in vitro DNA phosphorus thioylation modification, phage engineering, phage therapy, and anti-phage engineered bacteria. Background Technology

[0002] Bacteria constantly face the threat of bacteriophages and have evolved a variety of defense systems. For example, the well-known restriction-modification (RM) system uses methyltransferases to methylate DNA bases to distinguish between the bacteria's own DNA and foreign DNA, and then uses restriction endonucleases to cut and degrade the foreign DNA that has not been methylated (Ren, J. et al., 2022, Biotechnol Lett 44, 33-44). The CRISPR system (clustered regularly interspaced short palindromic repeats) is an acquired immune system of bacteria. When a bacteriophage first infects a bacterium, short DNA fragments in the bacteriophage genome are captured by the bacteria and stored in the bacteria's CRISPR array. When the bacteriophage invades the bacteria again, the bacteria use the CRISPR system to express spacer sequences that bind to the Cas protein, specifically recognizing and cutting the bacteriophage DNA [Horvath, P. et al., 2010, Science 327(5962):167-70]. In addition, abortive infection systems, including the CBASS system and the Gabija system, involve infected cells initiating active suicide or growth arrest programs to prevent viral spread and thus protect surrounding cells from bacteriophage infection [Wang, JG et al., 2025, Cell 188(14):3744-3756; Li, J. et al., 2024, Nature 629, 467-473]. The phosphorothioate modification (PT) system, discovered in recent years, is also an important component of bacterial defense systems.

[0003] Phosphothioylation is an epigenetic modification in DNA where non-bridging oxygen atoms on phosphodiester bonds are replaced by sulfur atoms. Currently, three phosphothioylation systems—Dnd, Ssp, and Tdp—have been discovered in bacteria and archaea. These modification modules can be combined with different restriction modules to achieve phage defense. Taking the Ssp system as an example, the modification module SspABCD specifically recognizes the 5'-CCA-3' / 3'-TGG-5' sequence in bacterial DNA, generating 5'-C... PS CA-3' / 3'-TGG-5' single-strand modification. When bacteriophages infect bacteria, the restriction modules SspE or SspFGH can specifically recognize and cleave the unmodified exogenous bacteriophage DNA, thereby limiting phage proliferation in bacteria. The modification sequences of the Dnd and Tdp systems are significantly different from those of the Ssp system, producing 5'-G... PS AAC-3' / 3'-CTT PS G-5' and 5'-G PS ATC-3' / 3'-CTA PS Double-strand modification of G-5'. Similar to the Ssp modification system, the Dnd modification system is also performed by the multi-protein complex DndABCDE. The Tdp modification system can complete the phosphorylation modification process by a single TdpC protein, but it requires a high temperature environment (above 50 °C) to have significant modification activity (Xiong, XL et al., 2020, Nature Microbiology 5, 917-928; Wu, D. et al., 2022, Nature Catalysis 5, 1131-1144; An, TC et al., 2025, Nature Chemical Biology 21, 1160-1170).

[0004] Due to the complexity of Dnd and Ssp modification modules in bacteria, establishing in vitro thiosulfonation modification systems is costly. Furthermore, the Tdp system requires high-temperature catalysis and cannot catalyze thiosulfonation modification reactions at 37 °C or room temperature. Moreover, no effective phage antagonistic system or feasible engineered antagonistic phage modification scheme has been discovered for bacterial thiosulfonation modification defense systems, especially the unique single-chain modified Ssp system. In this invention, we discovered the pptA single gene in the phage genome as a simplified thiosulfonation modification system that can complete thiosulfonation modification at 37 °C or room temperature. After phage engineering, it can endow phages with the ability to antagonize the Ssp system, potentially serving as a solution in phage therapy. Simultaneously, the PptA protein and the SspFGH module can form a novel phage defense system. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of existing technologies and provide a DNA phosphorylation modification enzyme and its application as an antagonistic system for genome modification and bacterial anti-phage mechanisms. This system comprises the pptA gene or its encoded protein. The PptA protein encoded by pptA can confer the ability of phages to successfully penetrate Ssp defense systems, including SspABCD-SspE and SspABCD-SspFGH. This invention provides the gene sequence and protein information of pptA, laying the foundation for genetic modification and applicable to many fields such as biotechnology development, in vitro and in vivo DNA phosphorylation modification, phage engineering, and phage therapy.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention discovers a novel function of the phage PptA protein. The PptA protein, derived from Psychrobacter phage PVHp5 (GeneBank accession number: MN497414), is a novel single-component DNA phosphorothioylation modification system. This system comprises the pptA gene and its encoded amino acid sequence, as shown in SEQ ID NO.1 and SEQ ID NO.2; or a homologous protein encoded by the pptA gene that performs the same function, i.e., a protein whose sequence is significantly similar to that shown in SEQ ID 2 and performs the same or similar function in an organism; or a derivative protein whose pptA gene encodes a sequence as shown in SEQ ID NO.2, i.e., a protein whose sequence is substituted, deleted, or added to one or more amino acids of the sequence shown in SEQ ID 2 encoded by the pptA gene and performs the same or similar function in an organism. The N-terminus of the PptA protein contains a PP-loop domain with the sequence “SXGXXS” (this domain has ATP pyrophosphatase activity, where X represents any amino acid), and its C-terminus contains an iron-sulfur cluster and a key cysteine ​​residue responsible for sulfur transport.

[0007] The present invention also provides an antagonistic system for the Ssp defense system against bacterial phages, the system comprising the pptA gene or the protein encoded thereon, which can provide Psychrobacter phage PVHp5 (GeneBank accession number: MN497414) with resistance against host bacteria containing the SspABCD-SspE or SspABCD-SspFGH system.

[0008] Based on the function of the PptA protein, this invention provides applications for the pptA gene or its encoded protein to exert DNA phosphorylation modification activity in vitro and in vivo, or as an engineered phage antagonistic host Ssp defense system, including SspBCD-SspFGH and SspBCD-SspE defense systems. Furthermore, the PptA protein can work with the SspFGH module to form a novel phage defense system.

[0009] This invention also provides applications of the above-mentioned pptA gene or its encoded protein or the above-mentioned bacterial anti-phage mechanism antagonistic system, the applications of which include the fields of medicine and food, specifically including applications in biotechnology development, in vivo and in vitro DNA phosphorylation modification, phage engineering, or preparation of phage therapeutic products.

[0010] The present invention also provides a vector comprising nucleotides 1 to 753 of the sequence shown in SEQ ID NO.2 above, wherein expression of the protein in bacteria enables phosphorylation modification of the bacterial genome, the modification type being d(C PS C) Modification: Under in vitro reaction conditions, single / double-stranded DNA can be phosphorylated and modified.

[0011] This invention provides two vectors for engineering bacteriophages, which can modify the bacteriophage genome by phosphorylation and thioylation, enabling the bacteriophage to acquire the ability to resist the host Ssp system.

[0012] This invention provides two vectors for expressing the PptA-SspFGH system in bacteria as a bacterial anti-phage defense system.

[0013] The advantages of this invention are as follows: This invention is the first to discover an antagonistic system of bacterial Ssp defense system, composed of PptA protein encoded by the pptA gene, the structure and function of which have not been described in the literature.

[0014] Unique antagonistic function: PptA protein can phosphorylate the phage genome during the phage proliferation cycle, thereby enabling the phage to overcome the cleavage action of the Ssp defense system in the bacteria, including SspBCD-SspFGH and SspBCD-SspE, and ultimately allowing the phage to proliferate successfully.

[0015] Engineering application value: Phage engineering can enable phages that were originally restricted by the Ssp system to gain the ability to escape the Ssp system, providing a solution to the problem of pathogens containing Ssp defense systems in phage therapy.

[0016] Extensive modification activity: PptA protein can perform phosphorylation on DNA in vivo or in vitro, with the modification type being d(C) PS C), and the reaction can be completed at 37 °C or room temperature, overcoming the limitation of the Tdp system requiring high-temperature catalysis.

[0017] Construction of novel defense systems: The combination of PptA and SspFGH systems can form a new bacterial defense system, expanding the application of anti-phages to bacteria.

[0018] Multi-field application prospects: This invention provides new approaches and applications for the development of biotechnology, in vivo and in vitro DNA phosphorylation and thioylation modification, phage engineering and / or preparation of phage therapeutic products, and prevention and control of phage contamination. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the PptA protein domain and its location on the PVHp5 phage genome; Figure 2 This is a schematic diagram of plasmid pPT1069, which uses pET28a(+) as its backbone and contains nucleotide sequences from position 1 to 753 of the pptA gene from PVHp5 phage. Figure 3 Figure a shows the phosphorus-thioylated modified dinucleotide d(C). PS Figure C) shows the structural formula; Figure b shows the mass spectrometry detection signal after the bacterial genome is digested by enzymes in BL21(DE3) strain when pPT1069 plasmid or pET28a(+) empty plasmid is induced to be expressed. Figure 4 These are schematic diagrams of plasmids pPT1090 and pPT1091. pPT1090 uses pCas9 plasmid as a backbone and contains a spacer sequence targeting the ninC gene in λ phage. pPT1091 uses pBluescript II SK(+) plasmid as a backbone and integrates the pptA gene sequence and the left and right homologous arm sequences required for homologous recombination. Figure 5 It is an engineered λ phage consisting of a wild-type λ phage and an integrated pptA gene induced by the T7 promoter. pptAFigure 1. Results of a patchy assay in E. coli to resist the SspBCD-SspE and SspBCD-SspFGH defense systems. Figure 6 These are schematic diagrams of plasmids pPT1112 and pPT1086. pPT1112 uses pACYC184 plasmid as a backbone and integrates the pptA gene expressed under the BBa_J23100 promoter; pPT1086 uses pBAD24 plasmid as a backbone and integrates the sspFGH gene cluster from E. coli swine43 (GeneBank accession number: SAMN04376781). Figure 7 This is a diagram showing the experimental results of resistance to T4 phage in Escherichia coli with heterologous expression of the PptA-SspFGH defense system.

[0021] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention in any way. Unless otherwise stated, the present invention is implemented using conventional molecular biology, biochemistry, and microbiology techniques in the art.

[0023] Materials and general methods used in the examples: Strains and plasmids: Escherichia coli BL21(DE3), JM109(DE3), and MG1655(DE3) were preserved in our laboratory; strains MG1655(DE3)-sspBCDE and JM109(DE3)-sspBCDFGH containing the Ssp defense system were constructed according to the literature method (Zou, X. et al., 2022, Nature Communications 13(1):4491); plasmids pET28a(+), pCas9 (Addgene #42876), pBluescript II SK(+), pACYC184, and pBAD24 were preserved in our laboratory; λ phage and T4 phage were preserved in our laboratory; Psychrobacter phage PVHp5 (GenBank accession number: MN497414) was isolated and preserved in our laboratory; E. coli swine43 strain (GenBank accession number: SAMN04376781) was preserved in our laboratory.

[0024] Enzymes and reagents: Restriction endonucleases, Gibson splicing cloning kit, high-fidelity DNA polymerase, and T4 DNA ligase were purchased from Takara Bio Engineering (Dalian) Co., Ltd.; IPTG and L-arabinose were purchased from Sigma-Aldrich; nuclease NP1 and alkaline phosphatase CIP were purchased from NEB; bacterial genome extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; HPLC-grade methanol and acetonitrile were purchased from Merck; other routine reagents were domestically produced analytical grade.

[0025] Culture conditions: Escherichia coli was cultured in LB medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride), and appropriate antibiotics were added as needed (kanamycin 50 μg / mL, ampicillin 100 μg / mL, chloramphenicol 34 μg / mL).

[0026] Mass spectrometry detection method: TSQ mass spectrometer was used for detection in negative ion mode with electrospray ionization (ESI) source and selected ion monitoring (SIM) mode for detection of target products.

[0027] Example 1: Identification and Sequence Analysis of the pptA Gene 1.1 Discovery of the pptA gene This invention utilizes bioinformatics analysis of the genome of Psychrobacter phage PVHp5 (GenBank accession number: MN497414) to identify an open reading frame of 753 bp, named pptA. The nucleotide sequence of this gene is shown in SEQ ID NO.1, encoding a 250-amino acid protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0028] 1.2 PptA protein domain analysis Conserved domain analysis revealed that the PptA protein exhibits typical dual-domain characteristics. Figure 1 ): The N-terminal domain (approximately amino acids 1-120) contains a PP-loop domain with the sequence “SXGXXS”. This domain has ATP pyrophosphatase activity and is responsible for ATP binding and pyrophosphate hydrolysis.

[0029] The C-terminal domain (approximately amino acids 121-250) contains iron-sulfur cluster binding sites and key cysteine ​​residues responsible for sulfur transport, participating in the activation and transfer of sulfur.

[0030] This structural feature suggests that PptA may be a single-component phosphorylation modifying enzyme with dual functions of ATP activation and sulfur transfer.

[0031] 1.3 Location of the pptA gene in the phage genome like Figure 1 As shown, the pptA gene is located in an early expression region of the PVHp5 phage genome, with genes related to DNA metabolism both upstream and downstream, suggesting that this gene may play a role in phage DNA replication or modification.

[0032] Example 2: Construction of plasmid pPT1069 encoding PptA protein 2.1 Primer Design Primers for Gibson splicing were designed based on the pptA gene sequence and the multiple cloning site sequence of the pET28a(+) vector: Primers for amplifying the pptA gene: Upstream primer P1: 5'-CTGGTGCCGCGCGGCAGCCATATGTCTACAAATAGTCCAAGAGTGT-3' Downstream primer P2: 5'-GTGGTGGTGGTGGTGCTCGAGTTAATTATTCAGTGTTTCTAAGGTGCTACTG-3' The underlined part represents a sequence homologous to the vector.

[0033] 2.2 PCR amplification Using genomic DNA from Psychrobacter phage PVHp5 as a template, PCR amplification was performed using primers P1 / P2. The reaction mixture (50 μL) consisted of 100 ng template DNA, 0.4 μM each of forward and reverse primers, 0.2 mM dNTPs, 1 U high-fidelity DNA polymerase, 5 μL 10× buffer, and sterile water to a final volume of 50 μL. The amplification program was as follows: 94 °C pre-denaturation for 3 min; 94 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C extension for 45 s, for a total of 30 cycles; and a final extension at 72 °C for 10 min.

[0034] The PCR product was detected by 1% agarose gel electrophoresis, yielding a pptA gene fragment of approximately 750 bp. The fragment was then purified using a gel extraction kit.

[0035] 2.3 Vector linearization The pET28a(+) plasmid was digested overnight at 37 °C with NdeI and XhoI restriction endonucleases. The digestion products were subjected to 1% agarose gel electrophoresis, and the approximately 5.3 kb linearized vector fragment was recovered.

[0036] 2.4 Gibson splicing connection Prepare the Gibson splicing reaction (20 μL) according to the following system: pptA gene DNA fragment: 100 ng Linearized pET28a(+) vector fragment: 200 ng 2×Gibson splice premix: 10 μL Sterile water: Add to a final volume of 20 μL After gently mixing, the mixture was placed at 50 °C for 60 min. After the reaction was complete, the product was placed on ice to cool and store for later use.

[0037] 2.5 Transformation and Identification Add 10 μL of the recombinant product to 100 μL of *E. coli* DH5α competent cells, incubate on ice for 30 min, heat shock at 42 ℃ for 90 s, and immediately incubate on ice for 2 min. Add 900 μL of LB medium and incubate at 37 ℃ for 1 h. Spread 100 μL of the bacterial culture onto an LB agar plate containing kanamycin (50 μg / mL) and incubate overnight at 37 ℃.

[0038] Single clones were picked and inoculated into LB liquid medium (containing kanamycin) and cultured overnight at 37 °C with shaking. The plasmid was then extracted and sequenced for verification. Sequencing results showed that the pptA gene was correctly inserted into the vector without mutation, indicating that the recombinant plasmid pPT1069 was successfully constructed. Its plasmid map is shown below. Figure 2 As shown in the figure. In this plasmid, the pptA gene is fused with a 6×His tag at the N-terminus, expressed under the control of the T7 promoter, and has a kanamycin resistance marker, making it suitable for inducing recombinant PptA protein expression in Escherichia coli BL21(DE3).

[0039] Example 3: Verification of DNA phosphorus thioylation modification of PptA protein 3.1 Heterologous expression in vivo The pET28a(+) empty plasmid (negative control) and pPT1069 plasmid were transformed into Escherichia coli BL21(DE3) competent cells, plated on LB agar plates containing kanamycin, and incubated overnight at 37 °C.

[0040] Single colonies were picked and inoculated into 5 mL of LB medium (containing kanamycin) and cultured overnight at 37 °C with shaking at 220 rpm. The overnight culture was then transferred to 50 mL of fresh LB medium (containing kanamycin) at a ratio of 1:100 and cultured at 37 °C until OD (occurrence limit) was reached. 600 Approximately 0.6. Add IPTG to a final concentration of 1 mM and continue induction culture at 37 °C overnight (approximately 16 h).

[0041] 3.2 Bacterial genome extraction Genomic DNA was extracted from each bacterial strain using a bacterial genomic DNA extraction kit, following the kit's instructions. The concentration of the extracted genomic DNA was determined using a nucleic acid quantification instrument and adjusted to 200 ng / μL.

[0042] 3.3 DNA enzymatic digestion treatment Take 20 μg of bacterial genomic DNA and treat it at 95 °C for 10 min to denature the DNA into a single-stranded state.

[0043] Add the following to the denatured DNA sample: Sodium acetate buffer (pH 5.4): Final concentration 50 mM ZnCl2: Final concentration 1 mM nuclease NP1: 5 U The DNA was completely hydrolyzed into single nucleotides by treating it in a 50 ℃ water bath for 2 h.

[0044] Subsequently, 5 U of alkaline phosphatase CIP was added, and the mixture was treated in a 37 °C water bath for 2 h to dephosphorylate the nucleotides into nucleosides.

[0045] 3.4 Sample purification and concentration The enzymatically hydrolyzed reaction solution was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa and centrifuged at 4 °C and 12000 × g for 20 min to remove the enzyme protein in the reaction system and collect the solution from the bottom tube.

[0046] The collected solution was freeze-dried using a lyophilizer, and the sample was reconstituted with 10 μL of double-distilled water.

[0047] 3.5 TSQ mass spectrometry detection The reconstituted sample was analyzed by TSQ mass spectrometry, and the target product d(C) was detected in ion monitoring (SIM) mode. PS Characteristic ions of C).

[0048] Phosphorylated modified dinucleotide d(C PS The structural formula of C) is as follows Figure 3 As shown in Figure a. The mass spectrometry detection results are as follows: Figure 3 As shown in b: In the control group sample expressing the pET28a(+) empty plasmid, d(C) was not detected. PS C) characteristic signals.

[0049] In the experimental group samples expressing the pPT1069 plasmid, significant d(C) was detected. PS C) Characteristic signals, whose retention time and mass-to-charge ratio are consistent with those of the standard.

[0050] 3.6 Conclusion The experimental results indicate that the PptA protein possesses DNA phosphorothioylation activity, enabling it to perform phosphorothioylation on genomic DNA in bacteria, with the modification type being d(C). PS C). This is the first time that a single-component PptA protein has been demonstrated to have phosphorylation modification function.

[0051] Example 4: Functional confirmation of the antagonistic system of the bacterial Ssp defense system based on the pptA gene To verify whether modifying the phage genome with the pptA gene can provide phages with the ability to resist the Ssp system, we knocked the pptA gene into λ phage and verified that the pptA gene can act as an antagonistic system for the bacterial Ssp defense system through phage plaque assays.

[0052] 4.1 Construction of plasmids pPT1090 and pPT1091 4.1.1 Construction of pPT1090 plasmid Primers P3 (5'-AAACGTTGATATCAACCCGGAGCT-3') and P4 (5'-AAAAAGCTCCGGGTTGATATCAAC-3') were mixed, heated at 95 °C for 5 min, and then naturally cooled to room temperature to anneal the double-stranded DNA fragment containing sticky ends.

[0053] The pCas9 plasmid was digested with BsaI restriction endonuclease overnight at 37 °C, and the linearized vector fragment was recovered by gel extraction.

[0054] The annealed double-stranded DNA fragment was ligated to the linearized pCas9 vector fragment using T4 DNA ligase and incubated overnight at 16 °C. The ligation product was transformed into *E. coli* DH5α, plated on LB agar plates containing kanamycin, and incubated overnight at 37 °C. Single colonies were picked, cultured, and the plasmid was extracted and sequenced for verification, yielding the correct pPT1090 plasmid. Figure 4 This plasmid contains a spacer sequence that targets the ninC gene in λ phage for subsequent gene editing.

[0055] 4.1.2 Construction of pPT1091 plasmid Amplification of homologous arm fragment 1: Using the λ phage genome as a template, PCR amplification was performed using primers P5 (5'-TCGACGGTATCGATAAGCTTAGAGCTTATACAGGCATTCGG-3') and P6 (5'-TAAACAAAATTATTTCTAGAGGCCTATAGTGAGTCGTATTAGCAGCCCTGTCTCCCCAT-3') to obtain homologous arm fragment 1 (including the left homologous arm).

[0056] Amplification of homologous arm fragment 2: Using the λ phage genome as a template, PCR amplification was performed using primers P7 (5'-TAGAAACACTGAATAATTAATGCGATGTTATCGGTGCG-3') and P8 (5'-GCTCTAGAACTAGTGGATCCTTGGTGTTCTTTCACACCGG-3') to obtain homologous arm fragment 2 (containing the right homologous arm).

[0057] Amplification of the pptA gene fragment: Using the PVHp5 phage genome as a template, PCR amplification was performed using primers P9 (5'-AAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGTCTACAAATAGTCCAAGAGTG-3') and P10 (5'-GCACCGATAACATCGCATTAATTATTCAGTGTTTCTAAG-3') to obtain the pptA gene fragment 3.

[0058] Fusion PCR: Using fragments 1, 2, and 3 as templates, fusion PCR was performed using primers P5 and P8 to obtain fusion fragment 4 containing the left homologous arm-pptA gene-right homologous arm.

[0059] Amplification of the linearized vector: Using pBluescript II SK(+) plasmid as a template, PCR amplification was performed using primers P11 (5'-AAGCTTATCGATACCGTCGAC-3') and P12 (5'-GGATCCACTAGTTCTAGAGCG-3') to obtain the linearized vector fragment 5.

[0060] Gibson assembly: Fragments 4 and 5 were ligated using the Gibson assembly and cloning kit, transformed into *E. coli* DH5α, plated on LB agar plates containing ampicillin, and incubated overnight at 37 °C. Single colonies were picked, cultured, and then the plasmid was extracted and sequenced for verification, yielding the correct pPT1091 plasmid. Figure 4 ).

[0061] 4.2 Engineering modification of λ phage The pPT1090 and pPT1091 plasmids were co-transformed into E. coli JM109(DE3) strain to obtain JM109(DE3) (pPT1090, pPT1091) strain.

[0062] A double-layer plate method was used to prepare plates containing JM109(DE3) (pPT1090, pPT1091) in the upper layer. Wild-type λ phage solution was serially diluted, with 10 μL of each dilution spotted onto the plates. After drying, the plates were incubated overnight at 37 °C.

[0063] Pick a single phage plaque and resuspend it in SM buffer. Mix an appropriate amount of phage solution with JM109(DE3) bacterial culture, replate, and incubate overnight at 37 °C. Pick a single phage plaque again and repeat the above plating steps 3 times to purify the recombinant phage.

[0064] The purified phage genomic DNA was extracted, the pptA gene region was amplified by PCR and sequenced for verification, and finally an engineered phage λ with the pptA gene knocked in was obtained. pptA .

[0065] 4.3 Phage Spot Test Wild-type λ phage and engineered λ phage pptA Perform 10-fold serial dilutions, and spot 10 μL of each dilution onto plates containing different bacterial strains: Control strain 1: MG1655(DE3) (without Ssp defense system) Experimental strain 1: MG1655(DE3)-sspBCDE (containing the SspBCD-SspE defense system) Control strain 2: JM109(DE3) (without Ssp defense system) Experimental strain 2: JM109(DE3)-sspBCDFGH (containing the SspBCD-SspFGH defense system) All plates were incubated overnight at 37 °C to observe plaque formation.

[0066] 4.4 Experimental Results Experimental results are as follows Figure 5 As shown: On control strains MG1655(DE3) and JM109(DE3), wild-type λ phage and engineered λ phage... pptA Both phages were able to form clear plaques, indicating that both phages could normally infect host bacteria that lack a defense system.

[0067] On the MG1655(DE3)-sspBCDE strain containing the SspBCD-SspE defense system, wild-type λ phage could not form plaques, indicating that it was effectively restricted by the Ssp system; while engineered λ phage... pptA The ability to form plaques indicates that it has acquired the ability to resist the SspBCD-SspE system.

[0068] On the JM109(DE3)-sspBCDFGH strain containing the SspBCD-SspFGH defense system, wild-type λ phage could not form plaques; while engineered λ phage... pptAThe ability to form plaques indicates that it has acquired the ability to resist the SspBCD-SspFGH system.

[0069] 4.5 Conclusion The above experimental results demonstrate that the expression of the pptA gene in the phage genome can perform phosphorylation modification of the phage genome, thereby enabling the phage to acquire the ability to resist the host's Ssp defense system (including SspBCD-SspE and SspBCD-SspFGH). Therefore, the PptA protein can serve as an effective antagonistic system against the bacterial Ssp defense system.

[0070] Example 5: PptA-SspFGH system confers host phage resistance By constructing a PptA-SspFGH heterologous expression system and then heterologously expressing the PptA-SspFGH system, the PptA and SspFGH proteins were used to verify the formation of a new bacterial anti-phage system through phage plaque assays.

[0071] 5.1 Construction of plasmids pPT1112 and pPT1086 5.1.1 Construction of pPT1112 plasmid Preparation of promoter fragment: Primers P13 (5'-TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCGAATTCTCACACAGGAAACAGACC-3') and P14 (5'-GGTCTGTTTCCTGTGTGAGAATTCGCTAGCACTGTACCTAGGACTGAGCTAGCCGTCAA-3') were mixed, heated at 95 °C for 5 min, and then naturally cooled to room temperature to anneal into double-stranded DNA fragment 1.

[0072] Using fragment 1 as a template, PCR amplification was performed using primers P15 (5'-TTGACAGCTTATCATCGATATTGACGGCTAGCTCAGTCC-3') and P16 (5'-CTTGGACTATTTGTAGACATGGTCTGTTTCCTGTGTGAG-3') to obtain fragment 2 containing the constitutive promoter BBa_J23100.

[0073] Amplification of the pptA gene fragment: Using the PVHp5 phage genome as a template, PCR amplification was performed using primers P17 (5'-ATGTCTACAAATAGTCCAAGAGTG-3') and P18 (5'-TAAACTACCGCATTAAAGCTTTAATTATTCAGTGTTTCTAAGGTGCTACTGC-3') to obtain the pptA gene fragment 3.

[0074] Fusion PCR: Using fragments 2 and 3 as templates, fusion PCR was performed using primers P15 and P18 to obtain fusion fragment 4 containing the pptA gene promoter.

[0075] Amplification of the linearized vector: Using pACYC184 plasmid as a template, PCR amplification was performed using primers P19 (5'-TATCGATGATAAGCTGTCAAACATG-3') and P20 (5'-AGCTTTAATGCGGTAGTTTATCAC-3') to obtain the linearized vector fragment 5.

[0076] Gibson assembly: Fragments 4 and 5 were ligated using the Gibson assembly and cloning kit, transformed into *E. coli* DH5α, plated on LB agar plates containing chloramphenicol, and incubated overnight at 37 °C. Single colonies were picked, cultured, and then the plasmid was extracted and sequenced for verification, yielding the correct pPT1112 plasmid. Figure 6 This plasmid contains the pptA gene expressed by a constitutive promoter.

[0077] 5.1.2 Construction of pPT1086 plasmid Amplification of the sspFGH gene cluster: Using the genome of E. coli swine43 strain as a template, PCR amplification was performed using primers P21 (5'-GCTAGCAGGAGGAATTCACCATGGAGGAGCCAAGCAGTTTG-3') and P22 (5'-GCGTTCTGATTTAATCTGTACTACTCCCATGCCTTTTGAATAATG-3') to obtain fragment 6 of the sspFGH gene cluster.

[0078] Amplification of the linearized vector: Using pBAD24 plasmid as a template, PCR amplification was performed using primers P23 (5'-TACAGATTAAATCAGAACGCAGAAGC-3') and P24 (5'-GGTGAATTCCTCCTGCTAGC-3') to obtain the linearized vector fragment 7.

[0079] Gibson assembly: Fragments 6 and 7 were ligated using the Gibson assembly and cloning kit, transformed into *E. coli* DH5α, plated on LB agar plates containing ampicillin, and incubated overnight at 37 °C. Single colonies were picked, cultured, and then the plasmid was extracted and sequenced for verification, yielding the correct pPT1086 plasmid. Figure 6 This plasmid contains the sspFGH gene cluster induced by arabinose.

[0080] 5.2 Heterologous expression of the PptA-SspFGH system in Escherichia coli The pPT1086 and pPT1112 plasmids were co-transformed into the JM109(DE3) strain to obtain the JM109(DE3) (pPT1086, pPT1112) strain. A control group was also included. Control 1: JM109(DE3) (without plasmid) Comparison 2: JM109(DE3) (pPT1112) (Only PptA is expressed) Control 3: JM109(DE3) (pPT1086) (Expresses only SspFGH, requires arabinose induction) 5.3 Phage Spot Test A plate containing different bacterial strains in the upper layer was prepared using the double-layer plate method: Tablet 1: JM109(DE3) (Control) Tablet 2: JM109(DE3) (pPT1112) Plate 3: JM109(DE3) (pPT1086), with 0.2% arabinose added during plate formation to induce SspFGH expression. Plates 4-7: JM109(DE3) (pPT1086, pPT1112), with 0%, 0.02%, 0.1%, and 0.5% arabinose added respectively to induce SspFGH expression during plate formation. T4 phage was serially diluted 10-fold, and 10 μL of each dilution was spotted onto the above plates. After drying, the plates were incubated overnight at 37 °C, and plaque formation was observed.

[0081] 5.4 Experimental Results Experimental results are as follows Figure 7 As shown: On control plate 1 (JM109(DE3)), T4 phage formed clear plaques, indicating that the strain was sensitive to T4 phage.

[0082] On plate 2 (expressing PptA only), T4 phages were also able to form clear phage plaques, indicating that expressing PptA alone cannot confer phage resistance to the host bacteria.

[0083] On plate 3 (expressing only SspFGH), T4 phages could also form plaques, but the number of plaques was slightly reduced, indicating that expressing SspFGH alone has a certain inhibitory effect on phages, but not a complete one.

[0084] On plate 4 (co-expressing PptA and SspFGH, 0% arabinose), since SspFGH expression was not induced, only PptA was actually expressed, and the plaque formation was similar to that on plate 2.

[0085] On plates 5-7 (co-expressing PptA and SspFGH, with 0.02%, 0.1%, and 0.5% arabinose added, respectively), the number of T4 phage plaques decreased significantly with increasing arabinose concentration (i.e., increased SspFGH expression), and the plaque formation efficiency declined markedly. Under 0.5% arabinose induction conditions, plaque formation was almost unobservable.

[0086] 5.5 Conclusion The experimental results above demonstrate that the synergistic effect of PptA and SspFGH proteins can constitute a novel bacterial anti-phage defense system. When PptA modifies the bacterial genome with phosphorus thioylation, SspFGH, acting as a restriction module, can recognize and cleave unmodified exogenous phage DNA, thereby conferring host bacteria resistance to phages. With increasing SspFGH protein expression, the host bacteria's resistance to T4 phage is significantly enhanced, confirming that the PptA-SspFGH system can serve as a novel bacterial defense system.

[0087] Example 6: In vitro activity verification of PptA protein 6.1 Expression and purification of PptA recombinant protein The pPT1069 plasmid was transformed into E. coli BL21(DE3) competent cells, and expression was induced according to the method in Example 3. After collecting the bacterial cells, they were resuspended in lysis buffer, and purified by high-pressure disruption, nickel column affinity chromatography, and size exclusion chromatography to obtain high-purity PptA recombinant protein. SDS-PAGE verification showed a single protein band with a molecular weight of approximately 28 kDa, consistent with the theoretical value.

[0088] 6.2 In vitro phosphorylation modification reaction Add the following to a 100 μL reaction system: Tris-HCl buffer: 50 mM, pH 8.0 MgCl2: 10 mM ATP: 2 mM Sulfur donor: 1 mM (such as Na2S or L-cysteine) Substrate DNA: 10 μg single-stranded or double-stranded DNA PptA protein: 2 μM The reaction was carried out at 37 °C for 2 h. After the reaction, DNA recovery, enzymatic digestion, and mass spectrometry detection were performed according to the method in Example 3.

[0089] 6.3 Test Results Mass spectrometry results showed that d(C) was detected in the complete reaction system. PSThe characteristic signal in C) indicates that PptA can perform phosphorylation modification of DNA under in vitro conditions. No product signal was detected in the control group (lacking any key component).

[0090] This result indicates that PptA, as a single-component enzyme, can independently complete DNA phosphorylation at 37 °C without the need for other auxiliary proteins, and has modification activity on both single-stranded and double-stranded DNA.

[0091] The above embodiments describe in detail the specific implementation of the present invention, and fully demonstrate that the PptA protein has DNA phosphorus thioylation modification activity, and can serve as an effective antagonistic system against the bacterial Ssp defense system, enabling bacteriophages to acquire the ability to resist the Ssp system; at the same time, PptA and SspFGH can jointly constitute a new bacterial defense system.

[0092] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention's specification and drawings under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A DNA phosphorus thioylation modifying enzyme PptA, characterized in that, Its amino acid sequence is shown in SEQ ID NO.2: MSTNSPRVLC HFSCGAPSAV ATKLAIEKYG KDNVTVFNIQ ITEEHPDNQR FLKECELWFG 60 VPVTTVRNEN FKGSIYEVFK QGFIKSPQGA ACTTQLKRKV RASFQNPDDI HVFGFTTEEE 120 QRAIDFNERN PSLTTDWVLL DAGFNRNDCL GVLAGVGIGI PQMYKLGYNN NNCVGCVKGG 180 MGYWNKIRKD FPHVFARMAM VEREVGHSLL KDKDGAVWLD ELDPDRGRMS KEPDIECSLV 240 CSSTLETLNN 250.

2. The DNA phosphorus thioylation modifying enzyme PptA according to claim 1, characterized in that, The DNA phosphorus thioylation modifying enzyme PptA is a derived protein with DNA phosphorus thioylation modification activity obtained by substituting, deleting or adding one or more amino acids of the amino acid sequence shown in SEQ ID NO.

2.

3. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the DNA phosphorothioylation modifying enzyme PptA as described in claim 1 or 2, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 1: atgtctacaa atagtccaag agtgttgtgc cacttctcat gtggtgcacc tagcgcagta 60 gccacaaaac tagcaataga gaagtatggt aaagataatg tcactgtatt taatattcaa 120 attacagaag aacatccaga taaccagagg tttttgaaag agtgtgagtt gtggtttggt 180 gtccctgtca cgacggtacg taacgagaat tttaaaggtt ctatatatga agtatttaaa 240 caaggtttta ttaaaagccc tcaaggtgca gcttgcacca ctcagttgaa acgtaaagta 300 cgcgccagct tccaaaaccc agatgatata cacgtcttcg ggtttactac cgaggaagaa 360 cagcgagcta tagattttaa tgagcgtaac ccttcgctca ctacggattg ggtactactg 420 gacgcaggtt ttaaccgtaa tgattgttta ggtgtattgg cgggtgtcgg tatcggtata 480 ccacagatgt ataagttagg ttataacaat aacaactgtg tgggttgtgt gaaaggtggt 540 atgggttat ggaataaaat acgtaaagat tttccacacg tctttgcgcg tatggctatg 600 gttgagcgtg aagtaggaca cagtctattg aaggataagg acggtgcagt atggttagat 660 gaacttgacc ctgaccgtgg gcgcatgtct aaagagcctg atatagagtg cagtttagtt 720 tgcagtagca ccttagaaac actgaataat taa 753.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule as described in claim 3.

5. An engineered cell, characterized in that, The engineered cell comprises the recombinant expression vector of claim 4, and the host cell of the engineered cell is a prokaryotic cell or a eukaryotic cell; the prokaryotic cell is preferably Escherichia coli, Streptomyces, Bacillus subtilis or Pseudomonas aeruginosa; the eukaryotic cell is preferably yeast, insect cell, plant cell, mammalian cell or human cell.

6. A method for DNA phosphorus thioylation modification, characterized in that, Using DNA as a substrate, a reaction is carried out in the presence of the DNA phosphorus thioylation modifying enzyme PptA as described in claim 1 or 2, ATP, a sulfur donor, and divalent metal ions to generate phosphorus thioylated modified DNA; wherein the phosphorus thioylation modification is d(C PS C) Modification.

7. A method for acclimating bacteriophages to possess the ability to resist bacterial Ssp defense systems, characterized in that, The nucleic acid molecule of claim 3 or the recombinant expression vector of claim 4 is introduced into the phage genome to express the PptA protein in the phage; The method for constructing a bacterial antiphage defense system is as follows: the DNA phosphorothioylation modifying enzyme PptA and SspFGH protein as described in claim 1 or 2 are co-expressed in bacteria, so that PptA modifies the bacterial genome with phosphorothioylation, and SspFGH acts as a restriction module to cut unmodified foreign DNA, thereby enabling the bacteria to acquire antiphage ability.

8. A reaction system for DNA phosphorus thioylation modification, characterized in that, It contains the DNA phosphorothioylation modifying enzyme PptA as described in claim 1 or 2, ATP, a sulfur donor, and a divalent metal ion.

9. An engineered bacteriophage, characterized in that, The genome of the engineered bacteriophage contains the nucleic acid molecule of claim 3 or the recombinant expression vector of claim 4, and is capable of expressing the PptA protein.

10. The application of the DNA phosphorothioylation modifying enzyme PptA according to claim 1 or 2, or the recombinant expression vector according to claim 4, or the engineered cell according to claim 5, or the reaction system according to claim 8, or the engineered phage according to claim 9 in the preparation of phosphorothioylation modified DNA products, phage therapy products, anti-phage engineered bacteria, or in biotechnology development.

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