Limited modification system derived from pseudomonas aeruginosa and application thereof

Abstract

The invention discloses a limited modification system derived from pseudomonas aeruginosa and application of the limited modification system. The transferase LYSZa7hsdMS gene is composed of a methyltransferase LYSZa7hsdMS and a restriction enzyme LYSZa7hsdR. According to the invention, a 5 '-ACAYNNNNNCCT-3' site of a bacterial genome is subjected to 6mA methylation modification by using methyltransferase M.PaeZa7HsdMS, the bacterial genome is protected by combining a restriction enzyme compound PaeZa7HsdMSR, and the existence of PrrC protein can prevent a restriction-modification system from being inhibited by phage Stp protein. The novel limited modification system provided by the invention has a very wide application prospect in bacteriophage engineering and enhancement of engineering bacterium genome stability.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and specifically relates to a restriction modification system derived from Pseudomonas aeruginosa and an application thereof. Background Art

[0002] The restriction-modification system of bacteria consists of two parts with different enzyme activities: a DNA methyltransferase part that binds to and modifies DNA at a specific recognition sequence and a restriction endonuclease part that specifically cuts modified or unmodified DNA at or near the same site. These two enzyme activities work together to cut foreign DNA with or without specific modifications and protect the host genome from foreign DNA such as phage DNA. In addition, several methyltransferases in the RM system have been shown to regulate phenotypic changes in bacteria at the transcriptional level. Therefore, studying the restriction-modification system is of great significance for optimizing the gene structure of phages and exploring the epigenetic inheritance of bacteria.

[0003] Pseudomonas aeruginosa is a Gram-negative conditional pathogen that often colonizes the lungs of patients and exacerbates symptoms in patients with cystic fibrosis and other lung diseases. As an opportunistic pathogen, P. aeruginosa can cause acute and chronic infections. Its pathogenic characteristics stem from the diverse antibiotic resistance genes and a large number of variable virulence factors in the P. aeruginosa genome, which endows P. aeruginosa with great metabolic flexibility and the ability to adapt to a variety of diseases. P. aeruginosa has natural drug resistance and can further acquire drug resistance through various mechanisms (such as production of β-lactamases, changes in drug targets, and enhanced drug excretion). Drug resistance makes commonly used antibiotics (such as penicillins and cephalosporins) ineffective in treating P. aeruginosa infections. Sometimes, even if the test results show that a certain antibiotic is effective, the actual treatment may not be effective due to insufficient drug penetration or other factors. In addition, how to balance effective treatment with the management of side effects is also an important issue.

[0004] In recent years, in order to deal with multidrug-resistant Pseudomonas aeruginosa strains, phage therapy has been proposed as an alternative to traditional antibiotic treatment after failure. Compared with broad-spectrum antibiotics, phages have less impact on the normal human flora, and can reduce the occurrence of bacterial resistance by constantly adapting to changes in bacteria through natural selection and mutation mechanisms. However, the use of phages may trigger an immune response within the bacteria, reducing the therapeutic effect. Therefore, it is urgent to amplify the phage gene library so that the engineered phages can circumvent the immune response within the bacteria.

[0005] Pseudomonas and other strains are often used to produce industrial enzymes, biopolymers (such as polyhydroxyalkanoates PHAs), etc., but phage contamination is a common problem in microbial fermentation. Bacteriophages have extremely strong infection ability and rapid transmission speed. Once infection occurs, it may cause abnormal fermentation process, thus causing serious impact on industrial fermentation process. Therefore, it is crucial to implement effective prevention and control measures to ensure the stability of production strains for fermentation production of engineered bacteria. Summary of the invention

[0006] The invention discloses a restriction modification (RM) system derived from Pseudomonas aeruginosa and application thereof.

[0007] The RM system is derived from Pseudomonas aeruginosa: the modification part is the N6Adenosine DNA (m6A) methyltransferase M.PaeZa7HsdMS (LYSZa7hsdS, LTSZa7hsdM); the restriction part is the restriction endonuclease R.PaeZa7HsdR (LYSZa7hsdR) that recognizes the same sequence. Plasmid transformation experiments showed that the RM system knockout strains showed different natural transformation rates. In addition, DefenseFinder was used to predict and find a prrC gene in the middle of the RM system ( Figure 4 ). In Escherichia coli, this gene encodes an anticodon nuclease of tRNALys (Blanga-Kanfi, S., et al., PrrC-anticodonnuclease: functional organization of a prototypical bacterial restrictionRNase. Nucleic Acids Res, 2006. 34 (11): p. 3209-19.), which can bind to the restriction endonuclease in the RM system where it is located, and the anticodon nuclease activity of PrrC bound to the restriction endonuclease is silenced. When the phage invades the bacterial cell and the phage expresses proteins such as Stp to inhibit the restriction endonuclease activity in the RM, PrrC will release and activate its ribonuclease function, thereby cutting lysine-related tRNA, inhibiting translation and phage replication. Therefore, we speculate that PrrC in this system can work synergistically with RM to enhance the ability to resist infection by exogenous phages.

[0008] This study identified a restriction modification system derived from a clinical strain of Pseudomonas aeruginosa, which consists of 6mA DNA methyltransferase M.PaeZa7HsdMS and restriction endonuclease R.PaeZa7HsdR, which restricts the entry of exogenous DNA fragments or plasmids containing non-6mA methylated recognition sequences into cells. The cleavage activity of the restriction endonuclease complex PaeZa7HsdMSR is restricted by 6mA DNA methyltransferase M.PaeZa7HsdMS, and specifically cuts double-stranded DNA substrates near a pair of opposite unmethylated 5'-ACAYNNNNNCCT-3' (Y and N are degenerate bases, Y refers to C / T, N refers to A / T / C / G) sequences.

[0009] The restriction modification system derived from the clinical strain of Pseudomonas aeruginosa of the present invention comprises a DNA methyltransferase and a restriction endonuclease LYSZa7hsdR, wherein the DNA methyltransferase comprises a LYSZa7hsdS subunit and a LTSZa7hsdM subunit;

[0010] The LYSZa7hsdS subunit is

[0011] (a) consisting of the amino acid sequence shown in SEQ ID NO.1;

[0012] (b) an enzyme derived from (a) having sequence-specific recognition activity of a methyltransferase by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in (a);

[0013] The LTSZa7hsdM subunit is:

[0014] (a) consisting of the amino acid sequence shown in SEQ ID NO.2;

[0015] (b) an enzyme derived from (a) having methyltransferase activity by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in (a);

[0016] The restriction endonuclease LYSZa7hsdR is:

[0017] (a) consisting of the amino acid sequence shown in SEQ ID NO.5;

[0018] (b) An enzyme derived from (a) having restriction endonuclease activity by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in (a).

[0019] The second object of the present invention is to provide a gene encoding the above restriction modification system.

[0020] Preferably, the nucleotide sequence of the gene encoding the LYSZa7hsdS subunit is

[0021] (a) consisting of the nucleotide sequence shown in SEQ ID NO.3;

[0022] (b) a DNA molecule that hybridizes to the DNA sequence defined in (a) under stringent conditions and encodes a protein having sequence-specific recognition activity of a methyltransferase;

[0023] The nucleotide sequence of the gene encoding the LTSZa7hsdM subunit is:

[0024] (a) consisting of the nucleotide sequence shown in SEQ ID NO.4;

[0025] (b) a DNA molecule that hybridizes to the DNA sequence defined in (a) under stringent conditions and encodes a protein having methyltransferase activity;

[0026] The nucleotide sequence of the gene encoding the restriction endonuclease LYSZa7hsdR is:

[0027] (a) consisting of the nucleotide sequence shown in SEQ ID NO.6;

[0028] (b) A DNA molecule that hybridizes to the DNA sequence defined in (a) under stringent conditions and encodes a protein having restriction endonuclease activity.

[0029] The present invention also provides the application of the restriction modification system in restricting the entry of exogenous DNA fragments or plasmids containing non-6mA methylation recognition sequences into cells, wherein the restriction endonuclease specifically cuts a double-stranded DNA substrate containing an unmethylated 5'-ACAYNNNNNCCT-3' sequence, and the 5'-ACAYNNNNNCCT-3' site (Y and N are degenerate bases, Y refers to C / T, and N refers to A / T / C / G) of the bacterial genome is subjected to 6mA methylation modification by using a methyltransferase, so that the cutting activity of the restriction endonuclease is restricted by the methyltransferase.

[0030] Preferably, the bacteria may be various bacteria, such as Pseudomonas aeruginosa.

[0031] The innovative features of the present invention are as follows:

[0032] The present invention utilizes methyltransferase M.PaeZa7HsdMS to perform 6mA methylation modification on the 5'-ACAYNNNNNCCT-3' site (Y and N are degenerate bases, Y refers to C / T, N refers to A / T / C / G) of the bacterial genome, combines the restriction endonuclease complex PaeZa7HsdMSR to protect the bacterial genome, and the presence of PrrC protein can prevent the restriction-modification system from being inhibited by the phage Stp protein. The present invention proposes a novel restriction-modification system with very broad application prospects in phage engineering and enhancing the stability of the engineering bacterial genome.

[0033] Pseudomonas aeruginosa LYSZa7 was deposited on October 25, 2024 in the Guangdong Microbiological Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, and the deposit number is: GDMCC No: 65350. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the result of mutant PCR identification by agarose gel electrophoresis; WT, Pseudomonas aeruginosa LYSZa7 wild-type strain; ΔRM1 is the LYSZa7ΔRMI strain in which the PaeZa7hsdMSR gene is knocked out; M, DNA molecular weight marker. Figure 2 This is a methylation motif analysis diagram based on SMRT-seq sequencing results; left: LYSZa7 strain; right: LYSZa7ΔRM1 strain.

[0035] Figure 3 These are the results of the plasmid transformation efficiency experiment and the SMRT-seq results of LYSZa7 and LYSZa7ΔRMI.

[0036] Figure 4 This is a diagram showing the positional relationship between the RM system PaeZa7HsdMSR and PrrC encoding genes. DETAILED DESCRIPTION

[0037] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0038] Embodiment 1:

[0039] In response to the diverse demands for restriction systems in phage engineering and stable genetically engineered strains, the present invention deeply explores the new M.PaeZa7HsdMSR gene system and provides a restriction modification system derived from Pseudomonas aeruginosa and its application.

[0040] A restriction modification system derived from Pseudomonas aeruginosa, composed of 6mA DNA methyltransferase M.PaeZa7HsdMS (LYSZa7hsdS, LTSZa7hsdM) and restriction endonuclease R.PaeZa7HsdR (LYSZa7hsdR), restricting the entry of exogenous DNA fragments or plasmids containing non-6mA methylation recognition sequences into cells; the gene of the enzyme is derived from Pseudomonas aeruginosa LYSZa7, isolated from a patient with secondary pneumonia caused by the new coronavirus.

[0041] In the application of the restriction modification system, the cutting activity of the restriction endonuclease complex R.PaeZa7HsdMSR is restricted by the 6mA DNA methyltransferase M.PaeZa7HsdMS, and specifically cuts the double-stranded DNA substrate containing the unmethylated 5'-ACAYNNNNNCCT-3' sequence (Y and N are degenerate bases, Y refers to C / T, and N refers to A / T / C / G).

[0042] 1. Sequence

[0043] 1. Amino acid sequence of DNA methyltransferase:

[0044] >LYSZa7hsdS (SEQ ID NO.1)

[0045] MSGYDKNPLVPRLRFPEFRDAGEWSAIELGRFSRLVTERVGNTTCTPYTITSGVGLISQEEKL

[0046] GRTIAGNSLKNYIVLQRNDFAYNKSATKAYPQGFIALYVGDDRAAVNSIFTCFRVDQSQVV

[0047] PAFLDKLFSVNLHGRWLRKRISIGARAHGSLQVSDDDLMATPVPLPRGSRSLSEQQKIADCL

[0048] SSLDELITSGIQKFDAINTFKKGLIQELFPRKGGTLPKRRFPEFRAAAEWEIRTLAELAENLD

[0049] NRRIPVAEKDRVKGGTPYYGASGVVDYIHGHIFDEELLCISEDGANLLARNTPIAAFSISGKSW

[0050] VNNHAHVLRFESRYVQRIVEDYLNSISLEDYLTGMAQPKLNRAKLDTIPVPIPQDVDEQKAVADYLACLDSMIVAQSRKVDLLRTHKMGLMQQLFPKGNGGV*

[0051] >LTSZa7hsdM(SEQ ID NO.2)

[0052] MTEFEKQKLGKTLWAIADQLRGAMNADDFRDYMLAFLFLRYLSDNYEAAAQKELGTDYP

[0053] QQTNDSVSTPLQLWYESNLDDVQEFEKQMRRKVHYVIEPQYLWGNIAEMARTQDLLLLNT

[0054] LQSGFSYIENESFASTFRGLFSEINLASDKLGKTYPERNARLCKIIAEIAKGLGQFSTDSDTLG

[0055] DAYEYLIGQFAAGSGKKAGEFYTPQPISSILSAIVTLDSQEPATGKRSHLDGVFDFACGSGSL

[0056] LLNVRRLMGTHGIGKIYGQEKNITTYNLARMNMLLHGVKDSEFEIFHGDTLLNEWDMLRE

[0057] TNPAKMPRFDAVVANPPFSYRWEPGEALGEDARFKNYGLAPKSAADFAFLLHGFHFLKQD

[0058] GVMAIILPHGVLFRGGAEARIRTKLLKDGHIDTVIGLPANLFFSTSIPVCILVLKKCKKPDDV

[0059] LFINAAEYFEKGKRQNRLLKTDEMPNGEIGHIEKIIDTYQYRKEEPRYSRRVDMEEIEKNDFNLNISRYVSTAEAEEIIDLKAIHEELVSIDQSIKKVTKKHNEFLSALGLPLLP*

[0060] 2. Nucleic acid sequence of DNA methyltransferase:

[0061] >LYSZa7hsdS(SEQ ID NO.3)

[0062] ATGAGTGGGTATGACAAAAATCCGTTGGTTCCTAGATTACGATTTCCGGAGTTTCGTGAT

[0063] GCGGGAGAATGGAGCGCTATCGAGTTAGGCAGATTTTCTCGACTTGTGACCGAGAGAGT

[0064] CGGGAACACTACTTGCACACCTTACACGATTACATCTGGAGTCGGTCTTATTAGCCAAGA

[0065] GGAGAAGCTCGGTCGTACCATCGCGGGGAACTCTCTGAAGAATTATATTGTCCTGCAGA

[0066] GAAATGACTTTGCATATAATAAGAGTGCAACGAAAGCGTATCCGCAAGGTTTTATTGCTC

[0067] TCTATGTTGGGGATGATCGAGCCGCTGTTCCAAATAGCATTTTTACTTGCTTTCGTGTTGA

[0068] TCAGAGTCAGGTGGTTCCAGCTTTTTTGGATAAGCTTTTCTCCGTTAACTTGCATGGGCG

[0069] GTGGTTAAGAAAGCGCATTTCTATTGGCGCGCGTGCGCACGGCTCGCTGCAAGTAAGTG

[0070] ATGATGACCTAATGGCAACGCCTGTGCCACTACCTCGCGGCTCTCGCTCACTTTCCGAAC

[0071] AACAGAAAATTGCCGATTGCCTGTCATCCCTTGATGAGCTGATTACGTCGGGCATCCAGA

[0072] AGTTTGACGCCATCAATACCTTCAAAAAGGGGTTGATCCAAGAGCTTTTTCCTCGTAAA

[0073] GGTGGAACTTTGCCGAAGCGACGTTTTCCAGAGTTTCGGGCTGCGGCAGAGTGGGAGA

[0074] TTAGAACGCTGGCCGAGCTGGCAGAGAATTTGGATAATCGTCGAATTCCAGTGGCTGAA

[0075] AAGGATCGGGTCAAGGGAGGGACTCCTTATTATGGAGCATCTGGGGTCGTTGATTACATT

[0076] CACGGCCATATCTTTGACGAAGAATTACTTTGCATTTCCGAAGATGGTGCGAATCTATTG

[0077] GCGAGAAACACACCAATCGCCTTCTCAATTTCAGGAAAGTCATGGGTTAACAACCATGC

[0078] TCATGTTTTGAGGTTCGAGAGCAGATACGTCCAAAGAATTGTTGAAGACTATTTAAACTC

[0079] TATTTCTCTTGAAGATTATTTGACAGGTATGGCCCAGCCCAAGCTGAACCGCGCAAAGCT

[0080] AGACACTATCCCCGTACCCATTCCACAGGATGTGGACGAGCAAAAGGCAGTCGCCGATT

[0081] ACTTAGCTTGTTTGGATAGTATGATTGTCGCTCAAAGTCGGAAGGTTGATTTGCTCAGGA

[0082] CTCATAAAATGGGCTTGATGCAGCAACTTTTTCCCAAGGGGAATGGTGGGGTATAG

[0083] >LTSZa7hsdM(SEQ ID NO.4)

[0084] ATGACCGAGTTCGAGAAGCAAAAGCTAGGCAAGACCCTTTGGGCCATCGCTGACCAGC

[0085] TACGTGGTGCGATGAATGCGGATGACTTCCGCGACTACATGCTGGCTTTCCTGTTTCTGC

[0086] GCTATTTGTCGGACAACTACGAAGCTGCCGCACAAAAAGAATTGGGGACGGACTATCCG

[0087] CAGCAGACTAACGATTCGGTTTCCACACCTCTGCAGCTTTGGTACGAAAGCAATCTAGA

[0088] CGATGTGCAGGAGTTCGAGAAGCAGATGCGCCGGAAGGTGCATTACGTGATCGAGCCTC

[0089] AATACCTGTGGGGCAACATCGCCGAGATGGCGCGCACGCAAGATTTGTTACTGCTAAAC

[0090] ACGCTACAAAGTGGTTTCAGCTATATCGAGAATGAGTCCTTTGCCAGCACCTTCCGGGG

[0091] ACTGTTCTCGGAAATTAACCTAGCCTCCGATAAACTGGGCAAAACTTACCCGGAACGAA

[0092] ACGCGCGCCTGTGTAAGATAATTGCCGAAATCGCCAAGGGCCTGGGTCAGTTCTCCACC

[0093] GACAGCGACACGCTGGGCGATGCCTATGAATACTTAATCGGCCAGTTTGCGGCTGGCTC

[0094] GGGCAAGAAAGCTGGTGAGTTCTACACACCGCAGCCTATTTCCAGCATTCTGTCTGCTAT

[0095] CGTCACGCTGGATAGTCAAGAGCCCGCTACGGGCAAGCGCTCGCACTTGGACGGCGTTT

[0096] TCGATTTTGCCTGTGGCTCTGGCTCACTGCTGCTCAATGTGCGCCGCCTTATGGGGACGC

[0097] ATGGCATTGGCAAGATCTACGGACAGGAAAAGAACATTACCACTTACAACCTGGCGCGC

[0098] ATGAACATGTTGCTGCATGGGGTGAAGGATTCAGAGTTTGAAATTTTCCATGGCGATACC

[0099] TTGCTCAATGAGTGGGACATGCTACGCGAGACCAATCCTGCCAAGATGCCCAGATTCGA

[0100] TGCCGTTGTGGCCAATCCGCCGTTTAGCTACCGCTGGGAACCGGGCGAGGCATTGGGCG

[0101] AGGACGCCCGCTTCAAGAACTATGGTCTGGCACCGAAGTCAGCAGCGGACTTCGCCTTT

[0102] CTGCTGCATGGCTTCCACTTCCTTAAGCAAGACGGCGTGATGGCCATCATCCTGCCTCAT

[0103] GGTGTGTTGTTCCGTGGTGGTGCCGAGGCGCGCATCCGCACTAAGTTGCTGAAAGACGG

[0104] CCACATCGACACTGTGATCGGCCTGCCGGCCAACCTGTTTTTCTCTACCAGCATCCCGGT

[0105] GTGCATCCTGGTGCTGAAGAAGTGCAAGAAACCTGACGACGTGTTGTTCATCAACGCC

[0106] GCCGAGTATTTCGAGAAGGGCAAGCGGCAGAACCGATTGCTTAAAACAGATGAGATGC

[0107] CTAACGGTGAAATCGGGCACATTGAAAAGATTATCGATACCTACCAGTACCGTAAGGAG

[0108] GAGCCTCGTTATTCGCGTAGGGTGGACATGGAGGAAATCGAGAAGAACGATTTCAACCT

[0109] AAATATCTCGCGTTATGTGAGTACTGCAGAGGCGGAGGAGATAATAGATCTCAAGGCGAT

[0110] CCATGAAGAGCTAGTTTCGATAGACCAAAGTATCAAGAAGGTAACGAAGAAGCACAAC

[0111] GAGTTCTTGAGTGCTTTGGGTTTGCCGCTGCTGCCATAG

[0112] 3. Amino acid sequence of the restriction endonuclease:

[0113] >LYSZa7hsdR (SEQ ID NO.5)

[0114] MTTTERQIELDLIAKLGDLKYTYRPDISDRAALEANFRAKFEALNRVHLTDSEFARLLDSIIT

[0115] PDVYNAAQTLRNINSFERDDDTPLNYTLVNIRDWCKNDFEVIHQLRINTDNSHHRYDVMLL

[0116] INGVPVVQIELKTLAVSPRRAMQQIVDYKTDPGNGYGKTLLCFLQLFIVSNRTDTWYFANN

[0117] NARHFSFNADERFLPVYQFASEDNKKITQLDGFAEKFLAKCTLGQMISRYMVLVASEQRLL

[0118] MMRPYQIYAVKAIVECIHQNCGNGYIWHTTGSGKTLTSFKASTLLKDNPDIDKCLFVVDRK

[0119] DLDRQTREEFNRFQEGCVEENTNTETLVRRLLSDDYADKVIVTTIQKLGLALDGANKRNYK

[0120] ERLEPLRNQRMVFIFDECHRSQFGDNHKAIKEFFPNAQLFGFTGTPIFEKNASYQQIEGQQA

[0121] SYRTTDDLFQRCLHQYTITHAIEDRNVLRFHVDYFKPEGKNPPKPGEGVAKPKVIETILAKH

[0122] DSATNGRKFNALLATASITDAIEYFELFASIQNSKAEQDPEFRPLNVACVFSPPAEGNKDVQQ

[0123] IQEDLPQEKEDNQQDPEGKKAALTRIVADYNARFGTNHRISEFDLYYQDVQKRIKDQQYPN

[0124] ADLPSTQKIDITIVVDMLLTGFDSKYLNTLYVDKNLKHHGLIQAFSRTNRVLNDSKPYGNIL

[0125] DFRQQQNPVEEAIALFSGEKIDNPREIWLVDPAPKVIDSLQAATRKLADFMQSQGVANAPEE

[0126] VVNLKGDAARAQFVNLFKEVQRLKTQLDQYTDLSEEQKAQIIQIAPPDQLQGFKGVYLETA

[0127] KRLKEQQDRDEAPPEVQQLDFEFVLFASAVIDYDYIMGLIAKLTQQKPGKLTMNREQLIGLI

[0128] QSDAKFIDEREDIAEYIRGLPVNEALDEKQIRIGFDRFKSEKKVRELTDIANRHGLEPNALQG

[0129] FVDEILRRRIFDGERLSELMAPLELGWKARTKAELALMEELAPLLHKLAQGREIAGLAAYEDGR*

[0130] 4. Nucleic acid sequence of the restriction endonuclease:

[0131] >LYSZa7hsdR(SEQ ID NO.6)

[0132] ATGACCACCACAGAACGCCAGATCGAGCTGGATCTGATCGCAAAGCTCGGCGACCTTAA

[0133] GTACACCTACCGCCCTGACATTAGTGACCGCGCAGCGCTGGAAGCTAACTTCCGTGCCA

[0134] AGTTCGAGGCGCTCAATCGGGTGCACCTGACCGACAGCGAATTTGCGCGTCTGCTCGAC

[0135] AGCATCATCACGCCTGATGTCTATAACGCTGCCCAGACGCTGCGCAACATCAACAGCTTC

[0136] GAGCGCGATGATGACACGCCGCTGAACTACACCCTGGTGAACATCAGGGATTGGTGCAA

[0137] GAACGACTTCGAGGTCATCCACCAGCTACGCATCAATACCGACAACAGCCACCATCGCT

[0138] ACGACGTGATGCTGCTCATCAACGGCGTCCCGGTAGTGCAAATTGAGTTGAAGACCCTC

[0139] GCCGTCAGCCCGCGCCGAGCTATGCAGCAGATCGTCGATTACAAGACCGATCCGGGCAA

[0140] CGGATACGGCAAGACACTGTTGTGCTTCCTGCAACTCTTCATTGTCAGCAACCGTACCG

[0141] ATACTTGGTACTTCGCCAACAACAACGCACGGCACTTCAGCTTCAACGCTGATGAACGC

[0142] TTCTTGCCGGTCTACCAGTTCGCCAGCGAGGACAACAAGAAGATTACGCAGCTTGATGG

[0143] CTTTGCTGAGAAGTTTCTGGCCAAGTGCACCTTGGGCCAGATGATCAGCCGCTACATGG

[0144] TGCTGGTGGCCAGCGAACAGAGGCTGCTGATGATGCGGCCGTACCAAATCTATGCCGTC

[0145] AAAGCCATCGTGGAGTGCATCCACCAGAACTGCGGCAACGGCTACATCTGGCACACCA

[0146] CAGGCTCAGGTAAGACGCTGACCTCATTCAAGGCATCTACCTTACTGAAGGACAACCCG

[0147] GACATCGACAAGTGTCTGTTCGTTGTGGATCGCAAAGACCTGGATCGCCAGACCCGCGA

[0148] GGAATTCAACCGCTTTCAGGAGGGCTGCGTCGAAGAAAATACCAACACCGAAACCCTG

[0149] GTGCGCCGCCTGCTATCGGACGACTACGCCGATAAGGTCATCGTCACGACAATTCAGAA

[0150] GCTGGGTCTTGCTCTAGATGGGGCCAACAAGCGCAACTACAAAGAGCGTCTGGAACCG

[0151] CTACGCAATCAGCGTATGGTATTTATCTTTGATGAATGCCACCGCTCGCAGTTTGGCGAC

[0152] AACCACAAAGCCATCAAGGAGTTCTTCCCTAACGCCCAGCTCTTTGGCTTCACCGGCAC

[0153] GCCCATCTTCGAGAAGAACGCCAGCTATCAGCAAATCGAAGGTCAGCAGGCCAGCTAC

[0154] CGTACTACCGACGATCTGTTCCAGCGCTGCCTACACCAGTACACCATCACCCACGCCATT

[0155] GAGGATCGTAACGTCCTGCGTTTCCACGTGGATTACTTCAAGCCCGAGGGCAAGAACCC

[0156] GCCCAAGCCCGGCGAAGGCGTGGCCAAACCGAAGGTCATCGAGACCATCCTCGCCAAG

[0157] CATGACTCTGCGACTAATGGCCGTAAGTTCAACGCGCTGCTGGCAACGGCCAGCATCAC

[0158] CGACGCCATCGAATACTTCGAGCTGTTTGCCAGCATTCAGAACTCCAAAGCGGAACAAG

[0159] ATCCTGAGTTCCGCCCGCTGAATGTCGCCTGCGTGTTCTCCCCGCCAGCCGAAGGCAAC

[0160] AAGGACGTGCAGCAGATTCAGGAAGACCTGCCGCAAGAAAAAGAAGACAACCAGCAA

[0161] GACCCCGAGGGTAAGAAGGCAGCGCTCACCCGCATCGTCGCCGACTACAACGCTCGCT

[0162] TTGGCACCAACCATCGAATCAGTGAGTTCGACCTGTACTACCAGGACGTGCAGAAGCGC

[0163] ATTAAGGATCAGCAGTACCCCAATGCCGATCTGCCTTCTACGCAGAAAATAGACATCACC

[0164] ATCGTGGTGGATATGCTGCTGACCGGCTTCGATTCCAAGTACCTCAACACCTTGTACGTG

[0165] GACAAGAACCTCAAACACCACGGCCTGATCCAAGCATTCTCACGGACCAACCGCGTAC

[0166] TCAACGACAGCAAGCCCTACGGTAACATCCTCGACTTCCGCCAACAGCAGAACCCGGT

[0167] CGAGGAAGCGATTGCACTCTTCTCCGGCGAGAAAATCGACAACCCGCGCGAAATCTGG

[0168] CTGGTAGACCCTGCACCGAAGGTCATCGACAGCCTGCAGGCGGCTACTCGGAAGCTGG

[0169] CTGATTTCATGCAGTCGCAAGGTGTGGCCAACGCACCGGAAGAAGTTGTGAACCTGAA

[0170] AGGCGATGCTGCTCGAGCACAGTTCGTCAACCTGTTCAAGGAAGTGCAGCGCCTAAAG

[0171] ACGCAGCTCGATCAATACACCGACCTTTCCGAAGAACAGAAAGCGCAGATCATCCAGAT

[0172] TGCGCCACCCGATCAGTTGCAAGGCTTCAAAGGGGTGTACCTGGAGACGGCCAAACGG

[0173] TTGAAGGAACAACAGGATCGCGACGAAGCTCCGCCCGAAGTGCAACAGCTTGATTTCG

[0174] AGTTCGTGCTCTTCGCGTCAGCGGTGATCGACTACGACTACATTATGGGCCTCATTGCGA

[0175] AGCTGACGCAGCAGAAGCCCGGCAAGCTCACCATGAATCGAGAGCAACTAATTGGCCT

[0176] GATCCAGTCCGACGCCAAGTTCATCGACGAGCGTGAGGACATTGCCGAGTACATCCGCG

[0177] GCTTGCCGGTCAATGAAGCCTTGGATGAAAAGCAAATTCGCATCGGATTTGATCGCTTC

[0178] AAGTCGGAGAAAAAGGTACGGGAACTCACCGACATAGCCAACCGCCATGGGCTTGAGC

[0179] CCAATGCCTTGCAAGGCTTTGTCGATGAAATCTTGCGTCGTCGCATCTTCGACGGCGAA

[0180] CGTCTTTCCGAATTAATGGCCCCTTTGGAGCTCGGCTGGAAAGCCCGCACCAAGGCCGA

[0181] GTTGGCACTGATGGAAGAGCTGGCACCGCTGCTACACAAGCTTGCACAAGGCCGAGAG

[0182] ATTGCGGGCCTAGCTGCCTATGAGGATGGCCGATGA

[0183] 5. Amino acid sequence of PrrC homologous protein

[0184] >PrrC(SEQ ID NO.7)

[0185] MKIEDIAKQLKELGENIVLIYAFNATGKTRLSVAYKNATKNIENGQHTGVYYNAFSEDLFV

[0186] WDNDENNDGANIRLNVLPSSLNRFHSFLYENPDAVMDKLAMYSPKFKFNLNSHDNPEDGI

[0187] ESVTFFSNDDDAVPIKISRGEERIFVWCFFLALLEVDGWANAQDAHIFIDDPVSSLDEHNIYV

[0188] TAETIFQQIEQHYLKKKIIVTTHHIGLFSILADRLKKGEKSDRYKHLAKLFILKNLGGEYTLG

[0189] TPRNSVFLFHLHLLQLLEEASKEQLYSHHVVMLRQALENIASFFGRGNIGYTLAQIGVEDYE

[0190] NAANVINSLSHKDAYYYQSDLMSPAVEAVFKDVFAKLLDKYKFALYVG

[0191] 6. Nucleic acid sequence of prrC homologous gene

[0192] >prrC(SEQ ID NO.8)

[0193] GTGAAGATTGAAGATATCGCAAAGCAGTTGAAGGAGCTGGGCGAGAACATCGTCCTAAT

[0194] CTATGCCTTCAATGCTACTGGAAAGACTAGGTTATCGGTGGCGTATAAGAATGCGACCAA

[0195] GAATATCGAGAATGGTCAGCACACTGGTGTCTACTATAACGCCTTCAGCGAAGACCTTTT

[0196] TGTCTGGGATAACGATGAAAACAACGATGGGGCGAACATTAGGCTTAATGTTTTGCCGA

[0197] GTAGCTTAAATCGCTTCCATAGCTTCTTGTATGAAAATCCGGATGCTGTCATGGATAAATT

[0198] GGCGATGTATTCGCCAAAATTTAAATTTAATTTAAATTCGCATGACAATCCCGAGGATGGT

[0199] ATCGAGTCGGTCACTTTTTTTTCGAACGACGATGATGCTGTGCCAATTAAAATCTCGCGG

[0200] GGTGAAGAGAGGATCTTCGTCTGGTGCTTTTTCTTGGCACTTCTGGAAGTTGATGGGTG

[0201] GGCCAATGCTCAAGATGCGCATATTTTCATTGATGACCCAGTGTCCAGTCTTGATGAACA

[0202] CAATATTTATGTCACCGCTGAAACAATTTTTCAGCAAATTGAGCAGCATTATCTCAAGAA

[0203] AAAGATAATAGTCACAACCCATCACATAGGGTTGTTTTCAATTTTGGCGGATCGCTTGAA

[0204] GAAAGGCGAAAAAAGCGATAGATACAAGCATCTTGCCAAACTTTTCATTCTCAAGAATC

[0205] TCGGCGGCGAGTATACCTTGGGGACACCAAGAAATAGTGTTTTCCTATTTCACTTACACC

[0206] TTCTTCAGTTGCTCGAAGAAGCGAGCAAGGAGCAGCTCTACTCTCATCATGTCGTCATG

[0207] CTGCGACAAGCGCTTGAGAACATCGCCTCATTTTTTGGGAGAGGGAATATTGGGTACAC

[0208] ACTCGCCCAAATAGGCGTTGAAGATTACGAGAATGCAGCCAACGTCATAAATTCGCTTTC

[0209] TCATAAAGATGCTTATTACTACCAGAGCGACCTCATGTCGCCTGCCGTTGAGGCAGTGTT

[0210] CAAGGATGTTTTCGCCAAATTGCTAGATAAATACAAATTCGCATTGTATGTAGGCTGA

[0211] 2. Experimental Methods and Results

[0212] 1. Reagent preparation

[0213] (1) LB solid culture medium: Dissolve 16 g of LB Agar Powder (Shenggong) in 400 mL of deionized water and sterilize in a fully automatic high-pressure steam sterilizer at 121°C for 20 min. After cooling to about 50°C, pour 15 mL of the culture medium into 9 cm round culture dishes in a biosafety cabinet. After cooling for 15 min with the lid open, seal with sealing film and store.

[0214] (2) LB liquid culture medium: Dissolve 10 g LB Broth Powder (Shenggong) in 400 mL deionized water, sterilize in a fully automatic high-pressure steam sterilizer at 121°C for 20 min, and seal and store.

[0215] 2. Strain activation and culture conditions

[0216] Pseudomonas aeruginosa LYSZa7 (published in Qu J, Cai Z, Liu Y, Duan X, Han S, Liu J, Zhu Y, Jiang Z, Zhang Y, Zhuo C, Liu Y, Liu Y, Liu L, Yang L. 2021. Persistent bacterial coinfection of a COVID-19 patient caused by agenetically adapted Pseudomonas aeruginosa chronic colonizer 11:641920) was inoculated from a -80°C bacterial library using an inoculating loop. This application also holds and guarantees to be provided to the public within 20 years from the date of application, and was also deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on October 25, 2024, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, and the deposit number is: GDMCC No: 65350) was inoculated onto the surface of LB solid medium without resistance, and single colonies were isolated by streaking and cultured in a 37°C incubator. The activation of Pseudomonas aeruginosa LYSZa7 strain required 18-20 hours. The grown fresh monoclonal colonies were inoculated into 2 mL LB liquid medium, cultured overnight at 37°C 220 rpm on a shaker, and LB liquid medium without any strain inoculation was placed as a control.

[0217] 3. Construction of RM system deletion mutant strain LYSZa7ΔRMI

[0218] (1) Construction of a shuttle plasmid that causes the LYSZa7 RMⅠ deletion mutation.

[0219] Two pairs of primers (UF / UR, DF / DR, see Table 6) were designed, and Pseudomonas aeruginosa LYSZa7 was used as a template. The 800 bp sequence centered on the mutation target gene site was amplified by PCR. The pK18mobsacB vector digested with BamHⅠ and HindⅢ was connected to the two DNA fragments using a seamless cloning kit. The ligation product was then transformed into Escherichia coli DH5α and screened with gentamicin (60 μg / mL). After PCR amplification of the target fragment, it was sent to Biotechnology for sequencing, and the positive Escherichia coli clone of the shuttle plasmid carrying the mutation sequence was identified.

[0220] (2) Construct a LYSZa7 RMⅠ deletion mutant strain.

[0221] The E. coli DH5α with the shuttle plasmid constructed in step (1) (need to add gentamicin 60 μg / mL), the E. coli HBI01 with the pRK600 plasmid (need to add chloramphenicol 6 μg / mL) and the corresponding Pseudomonas aeruginosa were shaken and cultured overnight in LB liquid culture medium. The next day, the E. coli DH5α with the shuttle plasmid and the E. coli HBI01 with the pRK600 plasmid were transferred to LB liquid culture medium with corresponding antibiotics at a volume ratio of 1:100 and placed in a shaker at 37°C and 220 rpm to OD 0. 600 =0.8, then the corresponding Pseudomonas aeruginosa was transferred to fresh LB liquid medium at a volume ratio of 1:10, and cultured at 42°C and 220 rpm until OD 600 =0.8. Take 1 mL of each of E. coli DH5α with shuttle plasmid (need to add gentamicin 60 μg / mL), E. coli HBI01 with pRK600 plasmid (need to add chloramphenicol 6 μg / mL) and corresponding Pseudomonas aeruginosa and place them in a 1.5 mL centrifuge tube. Collect the bacteria by centrifugation at 5000 rpm for 10 min in a centrifuge, resuspend with sterile deionized water, repeat 3 times to wash away the residual antibiotics, and then resuspend the three strains with 0.05 mL deionized water and drop them in LB solid medium without resistance for 8 hours. After the culture is completed, scrape the bacteria and resuspend them in 1 mL deionized water, take 0.1 mL and apply it on LB solid medium with gentamicin (60 μg / mL) and chloramphenicol (6 μg / mL), and pick the monoclonal colony after the colony grows and streak it on LB solid medium containing 20% ​​sucrose (mass volume ratio). The next day, 16 positive clones were selected from the monoclonal colonies grown in the sucrose solid medium, and PCR amplification was performed using primers UF / DR (see Table 6). After the target bands were identified by agarose gel electrophoresis, the strains were preserved ( Figure 1 ), thereby obtaining ΔRM1 with the PaeZa7hsdMSR gene knocked out, namely the RM system deletion mutant strain LYSZa7ΔRMI.

[0222] 4. SMRT sequencing to identify motifs

[0223] SMRT-seq was performed on Pseudomonas aeruginosa LYSZa7 and LYSZa7ΔRMI. The specific method was as follows: the bacterial solution was cultured to OD 600=1.5, and then dispense 1 mL into a 1.5 mL centrifuge tube, centrifuge at 13000 rpm for 5 min in a precooled centrifuge at 4 ° C, remove excess culture medium, resuspend the bacteria in sterile PBS buffer, repeat the above centrifugation operation twice, and quickly freeze the bacteria in liquid nitrogen, store at -80 ° C or place in dry ice for delivery. Genomic DNA was extracted by Guangdong Meige Gene Biotechnology Co., Ltd., and DNA integrity and purity were tested on 1% agarose gel, and then DNA concentration and purity were measured using Qubit 3.0 and Nanodrop One. Finally, the PacBio RSII sequencer (PacBio, Menlo Park, USA) was used for genome sequencing according to the standard protocol. Sequencing data was downloaded and transmitted through the Huawei Cloud Platform. Use Huawei Cloud obsutil software to store the downloaded raw data to the local server, use SequelTools to perform quality control on the sequencing data, and use the pb_basemods_hifi module in SMRTLink v11.1 developed by PacBio to process the downloaded HiFi data to obtain the modified base information in the genome.

[0224] like Figure 2 As shown, the SMRT-seq results of wild-type Pseudomonas aeruginosa LYSZa7 and LYSZa7ΔRMI, from left to right are the visualization of the bases with modifications in the motifs in the SMRT-seq sequencing data of LYSZa7 and LYSZa7ΔRMI on Motification QV and Fraction Motif Sites. The sequencing results showed that LYSZa7ΔRMI showed the disappearance of the methylation motif 5'-ACAYNNNNNCCT-3' (Y and N are degenerate bases, Y refers to C / T, N refers to A / T / C / G), further proving the methylation function of the RM system where PaeZa7HsdMS and PaeZa7HsdMSR in Pseudomonas aeruginosa LYSZa7 are located and the motifs they target.

[0225] 5. Determination of plasmid transformation efficiency:

[0226] (1) Construct a plasmid with the corresponding methyltransferase recognition motif.

[0227] The 300bp DNA fragment with the corresponding methyltransferase recognition motif in the genome was amplified by polymerase chain reaction (PCR) using two pairs of primers (HsdM-TE-F / HsdM-TE-R, see Table 6) with the LYSZa7 genome as a template, and BamHI and HindⅢ restriction sites and 20bp sequences outside the restriction sites in the pUCP20 plasmid were added to its end. Each fragment contains three RM system recognition motifs. Similarly, the 300bp DNA fragment without the corresponding methyltransferase recognition motif in the genome was amplified by polymerase chain reaction (PCR) using two pairs of primers (HsdM-CV-F / HsdM-CV-R, see Table 6) with the LYSZa7 genome as a template, and BamHI and HindⅢ restriction sites and 20bp sequences outside the restriction sites in the pUCP20 plasmid were added to its end. After the PCR product was purified and recovered, the corresponding fragment was connected to the linearized pUCP20 plasmid using seamless cloning, and transformed into E. coli DH5α by heat shock method for amplification. After the construction of PCR verification plasmid was completed, the positive clone was picked and expanded in LB liquid medium with Amp resistance. The next day, the plasmid was extracted and the E. coli DH5α carrying the corresponding plasmid was placed in 30% glycerol and stored in a -80°C bacterial library. The system (Table 1) and cycle (Table 2) used for PCR detection, the system (Table 3) and cycle (Table 4) for PCR amplification of the target fragment for downstream experiments, and the seamless cloning system (Table 5) are as follows.

[0228] Table 1 PCR system used for detection bands

[0229]

[0230]

[0231] Table 2 PCR cycles used for detection bands

[0232]

[0233] Table 3 PCR system used to amplify target bands

[0234]

[0235] Table 4 PCR cycles used to amplify target bands

[0236]

[0237]

[0238] Table 5 Seamless cloning system

[0239]

[0240] Table 6 Primers used in this patent

[0241]

[0242]

[0243] (2) Preparation of JM110 competent cells.

[0244] In order to remove the extra methylation modification introduced by the dam and dcm genes in E. coli DH5α, the plasmid needs to be re-transformed into the methyltransferase-deficient E. coli JM110 for amplification. Take the E. coli JM110 (Beyotime / Biyuntian, Catalog No. D0361) from the -80℃ bacterial library and streak to resuscitate. This strain lacks the dam and dcm methyltransferase genes to prevent the introduction of additional methylation modification interference. Inoculate the E. coli JM110 monoclonal colony into 10mL LB liquid culture medium and culture it in a shaker at 37℃220rpm until the OD 600 =0.35-0.5 (about 5h) and then put it in ice, and then package it into 1.5mL centrifuge tubes in a biosafety cabinet, and put it into a refrigerated centrifuge precooled at 4°C and centrifuged at 5000rpm for 10min to collect the bacteria. The Competent Cell Preparation Kit was used to prepare Escherichia coli JM110 competent cells, and the prepared competent cells were stored at -80°C.

[0245] (3) Preparation of wild-type Pseudomonas aeruginosa LYSZa7 and LYSZa7ΔRMI Pseudomonas aeruginosa competent cells required for electroporation transformation of plasmids.

[0246] Inoculate the overnight culture into 10 mL of LB liquid medium containing appropriate antibiotics. Incubate the culture at 37°C with shaking at 220 rpm until OD 600 The cell mass was 1.0, and then the cells were collected by centrifugation (5000 rpm, 4°C, 10 min), and washed twice with 0.3 M sucrose solution pre-cooled on ice. Finally, the cells were resuspended in 1 mL of 0.3 M sucrose solution, and 100 μL of competent cells were aliquoted and stored at -80°C.

[0247] (4) During the electrotransformation operation, 100 ng of plasmid DNA (the plasmid in step 5, plasmid transformation efficiency determination (1) constructing a plasmid with the corresponding methyltransferase recognition motif) was added to the Pseudomonas aeruginosa competent cells thawed on ice. After incubation on ice for 10 minutes, electrotransformation was performed in a Bio-Rad electroporation cup using a Bio-Rad GenePulser Xcell electroporation instrument (2500V, 25μF, 200Ω) with an electrode gap of 0.2 cm, and then diluted in a non-resistant LB liquid medium at a ratio of 1:10 and revived at 37°C and 220 rpm for 1 hour. The transformed bacterial solution was diluted 10 times, and 100 μL of the bacterial solution was spread on a LB solid medium with carbenicillin (150 μg / mL) resistance using a coating rod.

[0248] (5) In order to compare the transformation ability of plasmids in different strains, the concentration of purified and extracted plasmids was quantitatively measured three times using a NanoDrop One (Thermo Fisher) spectrophotometer and finally adjusted to 100 ng / μL based on the average value. Plasmid transformation was performed by electroporation as described above. The transformed cells were resuspended in 1 mL LB and stored at 37°C for 1 h. A 10-fold dilution gradient was prepared, and 100 μL of each gradient was applied to LB solid culture medium containing appropriate antibiotics. The number of transformants was calculated as colony forming units (CFU) per μg of plasmid DNA per mL of culture.

[0249] (6) The plasmids carrying the three recognition motifs (all three motifs are recognized by the methyltransferase HsdMS in the RMⅠ system) were electroporated into the wild-type LYSZa7 and the RM system deletion mutant LYSZa7ΔRMI, and the plasmid transformation efficiency was calculated by counting the colony forming units (CFU) formed per microgram of plasmid DNA per milliliter of culture (refer to step (5)).

[0250] Plasmid transformation efficiency experimental results ( Figure 3 ), dark blue (EV) indicates the use of a plasmid without the PaeZa7HsdMSR recognition motif, and light red (RMⅠMotif) indicates a plasmid of the same size with three recognition motifs (5'-ACAYNNNNNCCT-3'). ns P>0.05, **P<0.01. When the artificially constructed plasmid with an unmethylated motif (i.e., the plasmid constructed in 5(1)) was transformed into the LYSZa7 wild-type strain, its transformation efficiency was lower than that of the empty plasmid (i.e., the original pUCP20 plasmid), while when the artificially constructed plasmid with an unmethylated motif was transformed into the RM system-deficient mutant LYSZa7ΔRMI, its plasmid transformation efficiency was ( Figure 3 , RMⅠMotif) and plasmid without methyltransferase recognition motif ( Figure 3The results showed that PaeZa7HsdMSR can recognize and cut exogenous plasmids with (5'-ACAYNNNNNCCT-3') recognition motif without methylation modification in vivo, and has a certain ability to resist exogenous plasmid DNA.

Claims

1. A restriction modification system, characterized in that: It is composed of a DNA methyltransferase and a restriction endonuclease LYSZa7hsdR, wherein the DNA methyltransferase includes a LYSZa7hsdS subunit and a LTSZa7hsdM subunit, and; The LYSZa7hsdS subunit is: (a) consisting of the amino acid sequence shown in SEQ ID NO.1; (b) an enzyme derived from (a) having sequence-specific recognition activity of a methyltransferase by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in (a); The LYSZa7hsdM subunit is: (a) consisting of the amino acid sequence shown in SEQ ID NO.2; (b) an enzyme derived from (a) having methyltransferase activity by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in (a); The restriction endonuclease LYSZa7hsdR is: (a) consisting of the amino acid sequence shown in SEQ ID NO.5; (b) An enzyme derived from (a) having restriction endonuclease activity by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in (a).

2. A gene encoding the restriction modification system according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the gene encoding the LYSZa7hsdS subunit is: (a) consisting of the nucleotide sequence shown in SEQ ID NO.3; (b) a DNA molecule that hybridizes to the DNA sequence defined in (a) under stringent conditions and encodes a protein subunit having sequence-specific recognition activity of a methyltransferase; The nucleotide sequence of the gene encoding the LTSZa7hsdM subunit is: (a) consisting of the nucleotide sequence shown in SEQ ID NO.4; (b) a DNA molecule that hybridizes to the DNA sequence defined in (a) under stringent conditions and encodes a protein subunit having methyltransferase activity; The nucleotide sequence of the gene encoding the restriction endonuclease LYSZa7hsdR is: (a) consisting of the nucleotide sequence shown in SEQ ID NO.6; (b) A DNA molecule that hybridizes to the DNA sequence defined in (a) under stringent conditions and encodes a protein having restriction endonuclease activity.

4. The restriction modification system of claim 1 is used to restrict the entry of exogenous DNA fragments or plasmids containing non-6mA methylation recognition sequences into cells, wherein the restriction endonuclease specifically cuts a double-stranded DNA substrate containing an unmethylated 5'-ACAYNNNNNCCT-3' (Y and N are degenerate bases, Y refers to C / T, and N refers to A / T / C / G) sequence, and the 5'-ACAYNNNNNCCT-3' site of the bacterial genome is subjected to 6mA methylation modification by using a methyltransferase, so that the cutting activity of the restriction endonuclease is restricted by the methyltransferase.

5. The use according to claim 4, characterized in that: The bacteria is Pseudomonas aeruginosa.

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