Restriction modification system derived from pseudomonas aeruginosa and application thereof

Abstract

The application discloses a restriction modification system derived from pseudomonas aeruginosa and application thereof. The restriction modification system is composed of a methyltransferase LYZSa7hsdMS and a restriction endonuclease LYZSa7hsdR. The methyltransferase M.PaeZa7HsdMS is used for carrying out 6mA methylation modification on a 5'-ACAYNNNNNCCT-3' site of a bacterial genome, a restriction endonuclease complex PaeZa7HsdMSR is combined to protect the bacterial genome, and the presence of a PrrC protein can prevent the restriction-modification system from being inhibited by a bacteriophage Stp protein. The application has a very broad application prospect in bacteriophage engineering and enhancement of the stability of an engineering bacterial genome.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a restriction modification system derived from Pseudomonas aeruginosa and its applications. Background Technology

[0002] The bacterial restriction-modification (RM) system consists of two parts with different enzymatic activities: a DNA methyltransferase moiety that binds to and modifies DNA based on a specific recognition sequence, and a restriction endonuclease moiety that specifically cleaves modified or unmodified DNA at or near the same site. These two enzymatic activities work together to cleave exogenous DNA with or without specific modifications, protecting the host genome from foreign DNA such as bacteriophage DNA. Furthermore, several methyltransferases in the RM system have been shown to regulate bacterial phenotypic changes at the transcriptional level. Therefore, studying the restriction-modification system is of great significance for optimizing bacteriophage gene structure and exploring bacterial epigenetics.

[0003] *Pseudomonas aeruginosa* is a Gram-negative opportunistic pathogen that commonly colonizes the lungs of patients, exacerbating symptoms in those with cystic fibrosis and other lung diseases. As an opportunistic pathogen, *Pseudomonas aeruginosa* can cause both acute and chronic infections. Its pathogenic characteristics stem from the diverse antibiotic resistance genes and numerous variable virulence factors in its genome, giving it exceptional metabolic flexibility and the ability to adapt to various diseases. *Pseudomonas aeruginosa* possesses natural drug resistance and can further acquire resistance through various mechanisms, such as the production of β-lactamases, alteration of drug targets, and enhanced drug excretion. Drug resistance limits the effectiveness of commonly used antibiotics (such as penicillin and cephalosporins) in treating *Pseudomonas aeruginosa* infections. Even when test results indicate the effectiveness of a particular antibiotic, poor treatment outcomes may occur due to insufficient drug penetration or other factors. Furthermore, balancing effective treatment with the management of side effects is a crucial issue.

[0004] In recent years, phage therapy has been proposed as an alternative to traditional antibiotic treatment after the failure of multidrug-resistant Pseudomonas aeruginosa strains. Compared to broad-spectrum antibiotics, phages have less impact on the normal human flora and can continuously adapt to bacterial changes through natural selection and mutation mechanisms, thus reducing the occurrence of bacterial resistance. However, the use of phages may trigger an internal bacterial immune response, reducing the therapeutic effect. Therefore, there is an urgent need to expand phage gene libraries so that engineered phages can circumvent the internal bacterial immune response.

[0005] Pseudomonas strains are commonly used in the production of industrial enzymes and biopolymers (such as polyhydroxyalkanoates, PHAs), but bacteriophage contamination is a common problem during microbial fermentation. Bacteriophages possess extremely strong infectivity and rapid spread; once infection occurs, it can lead to abnormal fermentation processes, severely impacting industrial fermentation. Therefore, implementing effective prevention and control measures to ensure the stability of production strains is crucial for the fermentation production of engineered microorganisms. Summary of the Invention

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

[0007] The RM system was derived from *Pseudomonas aeruginosa*: the modification portion was an N6Adenosine DNA (m6A) methyltransferase M.PaeZa7HsdMS (LYSZa7hsdS, LTSZa7hsdM); the restriction portion was a restriction endonuclease R.PaeZa7HsdR (LYSZa7hsdR) that recognizes the same sequence. Plasmid transformation experiments showed that the RM system knockout strain exhibited differential natural transformation rates. Furthermore, using DefenseFinder, a prrC gene was predicted to be present in the middle of this RM system. Figure 4 In *E. coli*, this gene encodes an anticodon nuclease of tRNALys (Blanga-Kanfi, S., et al., PrrC-anticodonnuclease: functional organization of a prototypical bacterial restriction RNase. Nucleic Acids Res, 2006. 34(11): p. 3209-19.). This gene can bind to the restriction endonuclease in its RM system, silencing the anticodon nuclease activity of PrrC bound to the restriction endonuclease. When bacteriophages invade bacterial cells and express proteins such as Stp to inhibit the activity of restriction endonucleases in the RM, PrrC is released and its ribonuclease function is activated, thereby cleaving lysine-associated tRNA and inhibiting translation and phage replication. Therefore, we hypothesize that PrrC in this system can synergistically work with the RM to enhance the ability to resist exogenous bacteriophage infection.

[0008] This study identified a restriction modification system derived from a clinical strain of *Pseudomonas aeruginosa*, consisting of a 6mA DNA methyltransferase M.PaeZa7HsdMS and a 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 the 6mA DNA methyltransferase M.PaeZa7HsdMS, specifically cleaving double-stranded DNA substrates near a pair of opposing unmethylated 5'-ACAYNNNNNCCT-3' (Y and N are degenerate bases, Y refers to C / T, and N refers to A / T / C / G) sequences.

[0009] The present invention relates to a restriction modification system derived from clinical strains of Pseudomonas aeruginosa, comprising a DNA methyltransferase and a restriction endonuclease LYSZa7hsdR, wherein the DNA methyltransferase comprises a LYSZa7hsdS subunit and an LTSZa7hsdM subunit.

[0010] The LYSZa7hsdS subunit is

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

[0012] (b) An enzyme derived from (a) that has methyltransferase sequence-specific recognition activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence defined in (a).

[0013] The LTSZa7hsdM subunit is:

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

[0015] (b) An enzyme derived from (a) that has methyltransferase activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence defined in (a).

[0016] The restriction endonuclease LYSZa7hsdR is:

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

[0018] (b) An enzyme derived from (a) that has restriction endonuclease activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence defined in (a).

[0019] A second objective of this invention is to provide a coding gene that encodes the aforementioned restricted modification system.

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

[0021] (a) Consists 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 strict conditions and encodes a protein with methyltransferase sequence-specific recognition activity;

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

[0024] (a) Consists 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 strict conditions and encodes a protein with methyltransferase activity;

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

[0027] (a) Consists 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 strict conditions and encodes a protein with restriction endonuclease activity.

[0029] This invention also provides the application of the above-mentioned restriction modification system in restricting the entry of exogenous DNA fragments or plasmids containing non-6mA methylated recognition sequences into cells. The restriction endonuclease specifically cleaves double-stranded DNA substrates containing unmethylated 5'-ACAYNNNNNCCT-3' sequences. The methyltransferase is used to modify the 5'-ACAYNNNNNCCT-3' site of the bacterial genome with 6mA methylation (Y and N are degenerate bases, Y refers to C / T, and N refers to A / T / C / G), so that the cleavage activity of the restriction endonuclease is limited by the methyltransferase.

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

[0031] The innovative aspects of this invention:

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

[0033] Pseudomonas aeruginosa LYSZa7 was deposited on October 25, 2024 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China. The accession number is GDMCC No. 65350. Attached image description:

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

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

[0036] Figure 4 This is a diagram showing the positional relationship between the PaeZa7HsdMSR and PrrC coding genes in the RM system. Detailed Implementation

[0037] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0038] Example 1:

[0039] In response to the diverse needs of phage engineering and stable genetically engineered strains for restriction systems, this invention delves into a novel M.PaeZa7HsdMSR gene system, providing a restriction modification system derived from Pseudomonas aeruginosa and its applications.

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

[0041] The application of the restriction modification system, wherein the cleavage activity of the restriction endonuclease complex R.PaeZa7HsdMSR is restricted by the 6mA DNA methyltransferase M.PaeZa7HsdMS, specifically cleaves double-stranded DNA substrates containing the unmethylated 5'-ACAYNNNNNCCT-3' sequence (Y and N are degenerate bases, Y refers to C / T, 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] II. Experimental Methods and Results

[0212] 1. Reagent preparation

[0213] (1) LB solid medium: Dissolve 16g LB Agar Powder (Sangon) in 400mL of deionized water, sterilize in an automatic high-pressure steam sterilizer at 121℃ for 20min, remove and let the medium cool to about 50℃, pour 15mL into 9cm round petri dishes in a biosafety cabinet, open the lid and cool for 15min, then seal with sealing film and store.

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

[0215] 2. Activation and culture conditions of the strain

[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 introduced from a -80℃ bacterial library using an inoculation loop. This application also holds and guarantees to make it available to the public for 20 years from the date of application, and it was deposited on October 25, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, Guangdong Province, China, accession number: GDMCC. (No. 65350) was inoculated onto the surface of antibiotic-free LB solid medium, streaked to isolate single colonies, and then incubated statically at 37°C. Activation of Pseudomonas aeruginosa strain LYSZa7 requires 18-20 hours. The newly grown single colonies were inoculated into 2 mL of LB liquid medium and incubated overnight on a shaker at 37°C and 220 rpm. An uninoculated LB liquid medium was placed as a control.

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

[0218] (1) Construct a shuttle plasmid that mutates LYSZa7 RMⅠ deletion.

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

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

[0221] The *E. coli* DH5α (containing 60 μg / mL gentamicin), *E. coli* HBI01 (containing 6 μg / mL chloramphenicol), and corresponding *Pseudomonas aeruginosa* strains from step (1) were cultured overnight in LB broth with shaking. The next day, the *E. coli* DH5α containing the shuttle plasmid and the *E. coli* HBI01 containing the pRK600 plasmid were transferred at a volume ratio of 1:100 to LB broth containing the corresponding antibiotics and cultured on a shaker at 37°C and 220 rpm until OD (out of control). 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℃ and 220rpm until OD. 600 =0.8. Take 1 mL each of *E. coli* DH5α carrying the shuttle plasmid (requiring 60 μg / mL gentamicin), *E. coli* HBI01 carrying the pRK600 plasmid (requiring 6 μg / mL chloramphenicol), and the corresponding *Pseudomonas aeruginosa*, and place them in 1.5 mL centrifuge tubes. Centrifuge at 5000 rpm for 10 min to collect the bacterial cells, then resuspend them in sterile deionized water. Repeat this process three times to remove residual antibiotics. Resuspend the three strains in 0.05 mL of deionized water and incubate them in antibiotic-free LB agar for 8 h. After incubation, scrape off the bacterial cells and resuspend them in 1 mL of deionized water. Take 0.1 mL of this solution and spread it on LB agar containing gentamicin (60 μg / mL) and chloramphenicol (6 μg / mL). Once colonies have grown, pick single colonies and streak them onto LB agar containing 20% ​​sucrose (by volume). The following day, 16 positive clones were selected from the monoclonal colonies grown on sucrose solid medium and PCR amplified using primers UF / DR (see Table 6). After identification of the target band by agarose gel electrophoresis, the bacterial strain was preserved. Figure 1 Thus, the ΔRM1 with the PaeZa7hsdMSR gene knocked out was obtained, namely the RM system deletion mutant LYSZa7ΔRMI.

[0222] 4. SMRT sequencing for motif identification

[0223] SMRT-seq was performed on Pseudomonas aeruginosa LYSZa7 and LYSZa7ΔRMI, respectively. The specific method was as follows: the bacterial cultures were cultured to OD... 600=1.5 After aliquoting, 1 mL was transferred to 1.5 mL centrifuge tubes and centrifuged at 13000 rpm for 5 min in a pre-chilled centrifuge at 4°C. Excess culture medium was removed, and the cells were resuspended in sterile PBS buffer. The centrifugation process was repeated twice. The cells were then rapidly frozen in liquid nitrogen and stored at -80°C or transported on dry ice. Genomic DNA was extracted by Guangdong Meggene Biotechnology Co., Ltd., and DNA integrity and purity were tested on a 1% agarose gel. Subsequently, DNA concentration and purity were measured using a Qubit 3.0 and Nanodrop One. Finally, genome sequencing was performed using a PacBio RSII sequencer (PacBio, Menlo Park, USA) according to standard protocols. Sequencing data was downloaded and transmitted via Huawei Cloud platform. The raw data from the sequencing was stored on a local server using Huawei Cloud obsutil software. SequelTools was used for quality control of the sequencing data. The pb_basemods_hifi module in SMRTLink v11.1 developed by PacBio was used to process the HiFi data from the sequencing 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 are visualized from left to right as the bases with modifications in the motifs of LYSZa7 and LYSZa7ΔRMI SMRT-seq data relative to the Motification QV and Friction Motif Sites. Sequencing results show that LYSZa7ΔRMI exhibits the disappearance of the methylation motif 5'-ACAYNNNNNCCT-3' (Y and N are degenerate bases, Y refers to C / T, and N refers to A / T / C / G), further demonstrating the methylation function of the RM system containing PaeZa7HsdMS and PaeZa7HsdMSR in Pseudomonas aeruginosa LYSZa7 and its targeted motifs.

[0225] 5. Plasmid transformation efficiency determination:

[0226] (1) Construct plasmids with corresponding methyltransferase recognition motifs.

[0227] Using polymerase chain reaction (PCR) with two pairs of primers (HsdM-TE-F / HsdM-TE-R, see Table 6), a 300bp DNA fragment containing the corresponding methyltransferase recognition motif was amplified from the LYSZa7 genome as a template. BamHI and HindIII restriction enzyme sites and a 20bp sequence outside the restriction enzyme sites in the pUCP20 plasmid were added to the ends of each fragment. Each fragment contained three RM system recognition motifs. Similarly, using polymerase chain reaction (PCR) with two pairs of primers (HsdM-CV-F / HsdM-CV-R, see Table 6), a 300bp DNA fragment without the corresponding methyltransferase recognition motif was amplified from the LYSZa7 genome as a template. BamHI and HindIII restriction enzyme sites and a 20bp sequence outside the restriction enzyme sites in the pUCP20 plasmid were added to the ends of each fragment. After purification and recovery of the PCR product, the corresponding fragment was ligated into the linearized pUCP20 plasmid using seamless cloning, and then transformed into *E. coli* DH5α for amplification via heat shock. After PCR verification plasmid construction, positive clones were picked and cultured in LB liquid medium with Amp resistance. The next day, the plasmid was extracted, and *E. coli* DH5α containing the corresponding plasmid was placed in 30% glycerol and stored in a -80°C cellar. The PCR detection system (Table 1) and cycles (Table 2), the PCR amplification system (Table 3) and cycles (Table 4), and the seamless cloning system (Table 5) are as follows.

[0228] Table 1. PCR system used for band detection.

[0229]

[0230]

[0231] Table 2. PCR cycles used for band detection.

[0232]

[0233] Table 3. PCR systems used to amplify the target band.

[0234]

[0235] Table 4. PCR cycles used to amplify the target band.

[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] To remove the unnecessary methylation modifications introduced by the dam and dcm genes in *E. coli* DH5α, the plasmid needed to be re-transformed into *E. coli* JM110, a strain lacking both methyltransferase genes, for amplification. *E. coli* JM110 (Beyotime, catalog number D0361) was streaked and revived from a -80℃ library. This strain lacks both the dam and dcm methyltransferase genes, preventing the introduction of additional methylation modifications. Single colonies of *E. coli* JM110 were inoculated into 10 mL of LB broth and cultured on a shaker at 37℃ and 220 rpm until OD500 was reached. 600 =0.35~0.5 (about 5h) and then placed on ice. In a biosafety cabinet, the cells were aliquoted into 1.5mL centrifuge tubes and centrifuged at 5000rpm for 10min in a pre-cooled refrigerated centrifuge at 4℃ to collect the cells. Escherichia coli JM110 competent cells were prepared using the Competent Cell Preparation Kit and stored at -80℃.

[0245] (3) Prepare wild-type Pseudomonas aeruginosa LYSZa7 and LYSZa7ΔRMI competent cells for plasmid transformation by electroporation.

[0246] The overnight cultured strain was inoculated into 10 mL of LB liquid medium containing an appropriate antibiotic. The culture was incubated at 37°C with shaking at 220 rpm until OD reached. 600 The concentration was set to 1.0, and the cells were collected by centrifugation (5000 rpm, 4°C, 10 min). The cells were washed twice with 0.3 M sucrose solution pre-chilled 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 electroporation operation, 100 ng of plasmid DNA (the plasmid in step 5, plasmid transformation efficiency determination, (1) constructing the 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, electroporation was performed in a Bio-Rad electroporator (2500V, 25μF, 200Ω) in a Bio-Rad electroporator cuvette with an electrode gap of 0.2 cm. Subsequently, the cells were diluted 1:10 in antibiotic-free LB liquid medium and thawed 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 LB solid medium with carbenicillin (150 μg / mL) resistance using a spreader.

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

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

[0250] Experimental results of plasmid transformation efficiency ( Figure 3 Dark blue (EV) indicates the use of plasmids containing the PaeZa7HsdMSR recognition motif, and light red (RMⅠMotif) indicates plasmids of the same size containing 3 recognition motifs (5'-ACAYNNNNNCCT-3'). ns P>0.05, **P<0.01. When the artificially constructed plasmid with unmethylated motifs (i.e., the plasmid constructed in 5(1)) was transformed into the wild-type LYSZa7 strain, its transformation efficiency was lower than that of the empty vector plasmid (i.e., the original pUCP20 plasmid). However, when the artificially constructed plasmid with unmethylated motifs was transformed into the RM system deletion mutant LYSZa7ΔRMI, its plasmid transformation efficiency ( Figure 3 ,RMⅠMotif) and plasmids without methyltransferase recognition motifs ( Figure 3The transformation efficiency of PaeZa7HsdMSR is comparable to that of EV. This result indicates that PaeZa7HsdMSR can recognize and cleave unmethylated exogenous plasmids with the (5'-ACAYNNNNNCCT-3') recognition motif in vivo, and has a certain ability to resist exogenous plasmid DNA.

Claims

1. The application of a restriction modification system in restricting the entry of exogenous DNA fragments or plasmids containing non-6mA methylated recognition sequences into bacteria, wherein a restriction endonuclease specifically cleaves double-stranded DNA substrates containing an unmethylated 5'-ACAYNNNNNCCT-3' sequence, and a methyltransferase is used to modify the 5'-ACAYNNNNNCCT-3' site of the bacterial genome with 6mA methylation, thereby limiting the cleavage activity of the restriction endonuclease to methyltransferase, and the application is for non-disease diagnostic and therapeutic purposes; The restriction modification system comprises a DNA methyltransferase and a restriction endonuclease LYSZa7hsdR, wherein the DNA methyltransferase includes the LYSZa7hsdS subunit and the LTSZa7hsdM subunit. The LYSZa7hsdS subunit has the amino acid sequence shown in SEQ ID NO.1; The LYSZa7hsdM subunit has the amino acid sequence shown in SEQ ID NO.2; The restriction endonuclease LYSZa7hsdR has the amino acid sequence shown in SEQ ID NO. 5; The bacteria in question is Pseudomonas aeruginosa.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the LYSZa7hsdS subunit is shown in SEQ ID NO.3; The nucleotide sequence of the gene encoding the LTSZa7hsdM subunit is shown in SEQ ID NO.4; The nucleotide sequence of the gene encoding the restriction endonuclease LYSZa7hsdR is shown in SEQ ID NO.6.

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