Pseudomonas putida PP_3142 gene knockout mutant strain and its application

Abstract

The present invention discloses a knockout mutant strain of Pseudomonas putida PP_3142 and its application. The mutant strain is Pseudomonas putida ΔPP3142, which is obtained by seamlessly knocking out the 72bp to 1190bp region of the sugar transferase gene PP_3142 of Pseudomonas putida KT2440, and the nucleotide sequence of the PP_3142 gene is shown in SEQID NO.1. The mutant strain ΔPP3142 of the present invention has a significantly weakened ability to form a biofilm on the liquid surface, but has no significant difference in adhesion and biofilm formation ability on solid surfaces compared with the wild-type strain KT2440, and can be applied to scenes such as water environment management and restoration.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a liquid surface biofilm-defective Pseudomonas putida PP_3142 gene knockout mutant strain and application thereof. Background Art

[0002] Pseudomonas putida is a Gram-negative bacterium widely found in water and soil environments. It has strong environmental adaptability and can decompose a variety of aromatic compounds and adsorb a variety of heavy metals. It can be used to develop a variety of microbial technologies, including the treatment of environmental pollutants, microbial agents that promote crop growth, and microbial pesticides. It has great application value in environmental management and agricultural production. Pseudomonas putida has a strong aggregation ability and can form biofilms in a variety of environments. Biofilm is a bacterial group structure, mainly composed of bacteria and extracellular matrix secreted by bacteria. It has a certain three-dimensional structure inside, which can help extracellular enzymes to better decompose nutrients. At the same time, it can also help bacterial groups resist external stress factors and improve the environmental adaptability of the population. Studies have shown that after bacteria form biofilms, their resistance to a variety of external stress factors will be significantly improved, such as the host immune system, antibiotics, and oxidative stress factors. The formation of biofilms can enable bacterial groups to stably attach to specific media. Using this characteristic of biofilms, strains with specific functions can be carried on artificial carriers and put into water environments to achieve water management and bacterial recovery processes.

[0003] The main components of the extracellular matrix of Pseudomonas putida are extracellular adhesion proteins and exopolysaccharides. Currently, there are two adhesion proteins identified in the genome of Pseudomonas putida KT2440, namely LapA and LapF. LapA is a key factor in biofilm formation, which directly affects the adhesion of bacteria to the surface of the medium and between bacteria. After knocking out lapA, the strain cannot form a biofilm. LapF is not a necessary matrix component for biofilm development, but it will affect the colonization of the strain in the rhizosphere. There are four extracellular polysaccharide synthesis gene clusters in Pseudomonas putida KT2440, namely PP_1277-1288 (synthesis of alginate), PP_2629-2638 (synthesis of bacterial cellulose), PP_1795-1788 and PP_3132-3142. The main function of extracellular polysaccharides is to improve the stability of biofilms. PP_3142 is the last gene in the PP_3132-3142 gene cluster. The predicted encoding product is a sugar transferase, but its specific function has not been studied in depth.

[0004] Pseudomonas putida can not only form biofilms on the surface of solid media, but also form stable and thick biofilms on the gas-liquid interface. The liquid surface biofilm floats on the surface of the water body, which can interfere with the entry of oxygen into the water body and the growth of microorganisms in the surface water layer, which has a certain impact on the application of Pseudomonas putida in the water environment. Therefore, in order to improve the application potential of Pseudomonas putida, it is necessary to transform it to obtain strains that can form biofilms normally on solid surfaces and will not form stable biofilms on the liquid surface. At present, many strains of Pseudomonas putida have completed genome sequencing and have a complete genetic operation system, making it possible to transform strains using genetic engineering methods, by knocking out or knocking in specific genes, changing the characteristics of the strains to make them have better characteristics. Summary of the invention

[0005] The purpose of the present invention is to provide a Pseudomonas putida PP_3142 gene knockout mutant strain in response to the status of the prior art. The mutant strain has a significantly weakened ability to form a biofilm at the air-liquid interface, but can form a biofilm normally on a solid surface, thereby improving its application potential in aquatic environments. It can be used as a chassis microorganism for further functional transformation and applied to scenarios such as water treatment and restoration.

[0006] The first object of the present invention is to provide a PP_3142 gene knockout mutant of Pseudomonas putida, which is obtained by seamlessly knocking out the 72bp to 1190bp region of the PP_3142 gene of Pseudomonas putida, and the nucleotide sequence of the PP_3142 gene is shown in SEQ ID NO. 1. The Pseudomonas putida is Pseudomonas putida KT2440, and its deposit number is ATCC No. 47054.

[0007] The second object of the present invention is to provide a method for weakening the ability of Pseudomonas putida to form liquid surface biofilm, comprising the following steps: seamlessly knocking out the 72bp to 1190bp region of the sugar transferase gene PP_3142 in the wild strain of Pseudomonas putida, and the nucleotide sequence of the PP_3142 gene is shown in SEQ ID NO.1.

[0008] Preferably, the method for weakening the biofilm formation ability of Pseudomonas putida liquid surface comprises the following steps:

[0009] (1) Using genomic DNA of wild-type Pseudomonas putida as a template, the upstream and downstream fragments of the PP_3142 gene were amplified by PCR;

[0010] (2) pDS3.0 was digested with SacI and the resulting fragments were recovered by gel excision;

[0011] (3) The upstream and downstream fragments of the PP_3142 gene were connected into the linearized plasmid pDS3.0 by a one-step seamless ligation method to construct the knockout vector pDS-3142, which was then transformed into Escherichia coli S17-1;

[0012] (4) Escherichia coli S17-1 carrying the knockout vector pDS-3142 was mixed and incubated with a wild-type strain of Pseudomonas putida for conjugation transfer, so that pDS-3142 entered the wild-type strain of Pseudomonas putida, and then coated on a gentamicin and chloramphenicol resistance screening LB plate to obtain a primary exchange recombinant;

[0013] (5) The single exchange recombinant was cultured in LB liquid medium and spread on an LB plate containing sucrose. A single colony was cultured and picked, and the Pseudomonas putida PP_3142 gene knockout mutant strain in which the PP_3142 gene was knocked out was identified using identification primers.

[0014] Preferably, the wild strain of Pseudomonas putida is Pseudomonas putida KT2440, and its deposit number is ATCC No.47054.

[0015] Preferably, the primer sequences for amplifying the upstream and downstream fragments of the PP_3142 gene are:

[0016] 3142upS: 5'-TACCGCATGCGATATCGAGCTC CCAGCCTGCAAACCTATCTG-3';

[0017] 3142upA: 5'-GCCTGGATGGCGTTACTGA-3';

[0018] 3142dnS: 5'-TCAGTAACGCCATCCAGGCTGACACTTTGTATTCGCAGGAT-3';

[0019] 3142dnA: 5'-TGTGGAATTCCCGGGAGAGCTC CACCTTCAACCGCAACCAC-3';

[0020] The sequence of the identification primer is:

[0021] 3142wS: 5'-CCGGCAACCCTACCTGGA-3';

[0022] 3142wA: 5'-TGCCGAGTGGGAGTTCTACCTG-3'.

[0023] Preferably, the concentration of gentamicin in step (4) in the LB plate is 40 μg / mL, and the concentration of chloramphenicol in the LB plate is 25 μg / mL; the concentration of sucrose in step (5) in the LB plate is 10% m / v.

[0024] The third object of the present invention is to provide the above-mentioned Pseudomonas putida PP_3142 gene knockout mutant, namely, the use of Pseudomonas putida ΔPP3142 as a chassis microorganism in water environment treatment and restoration.

[0025] Preferably, the Pseudomonas putida ΔPP3142 can normally form a biofilm on a solid surface, but its ability to form a biofilm on a liquid surface is reduced.

[0026] The Pseudomonas putida ΔPP3142 obtained by the present invention is cultured in LB and TSB culture media at 28°C. The ability to form a biofilm on the liquid surface is significantly weakened compared with the wild-type strain (Pseudomonas putida KT2440). It can only form a very small amount of loose and fragile liquid surface biofilm, but the ability to form a biofilm on a polystyrene material is not significantly different from that of the wild-type strain (Pseudomonas putida KT2440). The knockout of the PP_3142 gene does not affect the adhesion properties of the strain on the solid surface, so that the mutant strain can effectively adhere to the carrier medium during the application process, while reducing the formation of biofilms on the liquid surface in the application scenario. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a gel image of PCR identification of the PP_3142 knockout strain ΔPP3142 of Pseudomonas putida. Lane 1: Marker III, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.); Lane 2: PCR amplification product of wild-type KT2440 using primers 3142wS and 3142wA; Lane 3: PCR amplification product of ΔPP3142 using primers 3142wS and 3142wA.

[0028] Figure 2 The liquid surface biofilm phenotypes of the wild-type strain (KT2440) and the PP_3142 gene knockout strain (ΔPP3142) of Pseudomonas putida in LB and TSB media.

[0029] Figure 3 is the amount of biofilm formation of wild-type Pseudomonas putida strain (KT2440) and PP_3142 knockout strain (ΔPP3142) in LB and TSB media in polystyrene 24-well plates. ns: not significant, t-test p>0.05. DETAILED DESCRIPTION

[0030] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0031] The wild-type Pseudomonas putida used in the following examples is Pseudomonas putida KT2440, whose deposit number is ATCC No. 47054, purchased from ATCC.

[0032] Example 1 Construction of Pseudomonas putida engineered strain ΔPP3142

[0033] (1) Construction of recombinant plasmid pDS-3142

[0034] First, based on the PP_3142 gene (nucleotide sequence as shown in SEQ ID NO.1) and its preceding and following sequences in the genome information of Pseudomonas putida KT2440 (NC_002947), two sets of primers 3142upS / 3142upA and 3142dnS / 3142dnA were designed, and a high-fidelity enzyme HD Cloning Kit (TaKaRa) kit recombination ligation, the reaction system is as follows:

[0035]

[0036]

[0037] The recombinant ligation product was introduced into Escherichia coli S17-1 by heat shock transformation, and 10 μL of the reaction solution was added to 100 μL of Escherichia coli S17-1 chemically competent cells, mixed, incubated on ice for 30 min, placed in a 42°C water bath for 90 s, cooled on ice, and then added with 800 μL of LB medium for recovery culture for 1 h. Most of the supernatant was removed by centrifugation, and the cells were suspended and spread on an LB plate containing 20 μg / mL gentamicin. The cells were cultured overnight at 37°C, and multiple colonies were picked and verified by PCR using primers 3142upS and 3142dnA to obtain the vector pDS-3142, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0038] The primer sequences for amplifying the upstream and downstream regions of PP_3142 are as follows:

[0039] 3142upS: 5'-TACCGCATGCGATATCGAGCTC CCAGCCTGCAAACCTATCTG-3';

[0040] 3142upA: 5'-GCCTGGATGGCGTTACTGA-3';

[0041] 3142dnS: 5'-TCAGTAACGCCATCCAGGC TGACACTTTGTATTCGCAGGAT-3';

[0042] 3142dnA: 5'-TGTGGAATTCCCGGGAGAGCTC CACCTTCAACCGCAACCAC-3'.

[0043]

[0044]

[0045] (2) Knockout of the PP_3142 gene of Pseudomonas putida using pDS-3142

[0046] The knockout vector pDS-3142 was transformed into Escherichia coli S17-1 to obtain Escherichia coli S17-1 containing pDS-3142. The Escherichia coli S17-1 containing pDS-3142 and wild-type Pseudomonas putida KT2440 were mixed in equal volumes for conjugation transfer. Specifically, Escherichia coli S17-1 containing pDS-3142 was activated in LB medium containing 20 μg / mL gentamicin, and wild-type Pseudomonas putida KT2440 was activated in LB medium containing 25 μg / mL chloramphenicol. 100 μL of each strain was mixed in a 2 mL centrifuge tube, and 1 mL LB medium was added. The mixture was centrifuged at 7000 r / min for 3 min, the supernatant was discarded, and 2 mL LB was added for rinsing and centrifugation to remove the supernatant, the antibiotics were removed, the precipitated bacteria were suspended with 300 μL of culture medium, and the plates were placed at 28°C for 12 h for conjugation transfer. 100 μL was spread on an LB plate containing 40 μg / mL gentamicin and 25 μg / mL chloramphenicol, and the plates were placed at 28°C for 24 h. After pDS-3142 was inserted into the genome of Pseudomonas putida KT2440 through homologous recombination, the strain acquired gentamicin resistance. Pick a single colony and use primers 3142wS and 3142wA for PCR amplification verification. If there is no PCR product, insert plasmid pDS-3142 into the upstream or downstream region of PP_3142 through recombination exchange. Inoculate the correct strain that has undergone the first recombination exchange into LB medium without antibiotics for expansion culture, then streak on multiple LB plates containing 10% (m / v) sucrose, place at 28°C for 24 hours, pick a single colony with good growth, and use primers 3142wS and 3142wA for PCR amplification verification. If there is no amplification product, it means that the second exchange has not occurred. The amplification product fragment size of 2377bp is restored to the wild type strain, and the amplification product fragment size of 1258bp is the target gene knockout mutant. The amplification product of the correct mutant is as follows: Figure 1 As shown, the Pseudomonas putida PP_3142 gene knockout mutant was named Pseudomonas putida ΔPP3142.

[0047] The identification primer sequences are as follows:

[0048] 3142wS: 5'-CCGGCAACCCTACCTGGA-3';

[0049] 3142wA: 5'-TGCCGAGTGGGAGTTCTACCTG-3'.

[0050] Example 2 Detection of the biofilm forming ability of ΔPP3142

[0051] Observation of liquid surface biofilm phenotype: LB medium and tryptone soy broth medium (TSB) were used to detect the biofilm formation ability of the mutant strain of Pseudomonas putida ΔPP3142. The specific steps were as follows: wild-type Pseudomonas putida KT2440 and mutant strain of Pseudomonas putida ΔPP3142 were cultured overnight in LB and TSB liquid medium, respectively, and the OD of the bacterial solution was measured. 600 , dilute the bacterial solution with the corresponding fresh medium to make the final OD 600 =0.1; 2.4 mL of diluted bacterial solution was transferred to a 12-well plate, with 3 replicates per group, and placed in a 28°C incubator for 24 hours to observe the phenotype of the liquid surface biofilm. After the culture, all liquid culture was removed with a pipette, so that the liquid surface biofilm was attached to the bottom of the well plate for easy observation. The results showed that the wild-type Pseudomonas putida KT2440 could form a complete and thick liquid surface biofilm in LB and TSB culture media, while the liquid surface biofilm of the mutant strain of Pseudomonas putida ΔPP3142 was significantly reduced and fragile and loose, such as Figure 2 shown.

[0052] Determination of biofilm formation ability on solid medium surface: LB medium and tryptone soy broth medium (TSB) were used to detect the biofilm formation ability of Pseudomonas putida mutant strain ΔPP3142. The specific steps were as follows: wild-type Pseudomonas putida KT2440 and Pseudomonas putida mutant strain ΔPP3142 were cultured overnight in LB and TSB liquid medium, respectively, and the OD of the bacterial solution was measured. 600 , dilute the bacterial solution with the corresponding fresh medium to make the final OD 600 =0.1; 1.2mL of diluted bacterial solution was transferred to a TC-treated 24-well plate made of polystyrene, with 4 replicates per group. The plate was placed in a 28°C shaker and cultured at 160r / min for 16 hours to allow the strain to form a biofilm on the wall of the 24-well plate. The biofilm was quantified using the crystal violet staining method. First, the liquid culture was removed and rinsed with saline to remove free bacteria. 2.4mL of 0.1% crystal violet stain was added to each well for staining. After 10 minutes, the stain was removed and the crystal violet that was not bound to the biofilm was rinsed with saline. Then, 2.4mL of 95% ethanol was added to each well to dissolve the crystal violet from the biofilm. After 20 minutes, 200μL was taken to a 96-well ELISA plate and the absorbance at 590nm was measured with an ELISA instrument to measure the amount of biofilm formation. The results are as follows Figure 3 As shown, in LB or TSB medium, the biofilm formation amount of the Pseudomonas putida mutant strain ΔPP3142 was not significantly different from that of the wild-type Pseudomonas putida KT2440.

[0053] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A Pseudomonas putida strain with impaired ability to form liquid surface biofilm PP_3142 A gene knockout mutant, characterized in that Pseudomonas putida PP_3142 The gene was obtained by seamlessly knocking out the region from 72 bp to 1190 bp. PP_3142 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the Pseudomonas putida is Pseudomonas putida ( Pseudomonas putida )KT2440, whose deposit number is ATCC No.47054.

2. A method for weakening the ability of Pseudomonas putida to form biofilm on liquid surface, characterized in that: Glycotransferase gene in wild strain of Pseudomonas putida PP_3142 The region from 72 bp to 1190 bp was seamlessly deleted. PP_3142 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the Pseudomonas putida is Pseudomonas putida ( Pseudomonas putida )KT2440, whose deposit number is ATCC No.47054.

3. The method according to claim 2, characterized in that The following steps are involved: (1) Using wild-type Pseudomonas putida genomic DNA as a template, amplify the PP_3142 Upstream and downstream segments of the gene; (2) Use SacI to digest pDS3.0 and recover the fragments by gel excision; (3) PP_3142 The upstream and downstream fragments of the gene were connected into the linearized plasmid pDS3.0 by one-step seamless ligation to construct the knockout vector pDS-3142, which was then transformed into Escherichia coli S17-1; (4) Escherichia coli S17-1 carrying the knockout vector pDS-3142 was mixed and incubated with a wild-type strain of Pseudomonas putida for conjugation transfer, so that pDS-3142 entered the wild-type strain of Pseudomonas putida, and then spread on a gentamicin and chloramphenicol resistance screening LB plate to obtain a single exchange recombinant; (5) Culture the exchange recombinants in LB liquid medium and spread them on LB plates containing sucrose. Culture and pick single colonies and use identification primers to identify knockouts. PP_3142 Pseudomonas putida PP_3142 Knockout mutant strains.

4. The method according to claim 3, characterized in that The amplification PP_3142 The primer sequences for the upstream and downstream fragments of the gene are: 3142upS: 5'-TACCGCATGCGATATCGAGCTC CCAGCCTGCAAACCTATCTG-3'; 3142upA: 5'-GCCTGGATGGCGTTACTGA-3'; 3142dnS: 5'-TCAGTAACGCCATCCAGGCTGACACTTTGTATTCGCAGGAT-3'; 3142dnA: 5'-TGTGGAATTCCCGGGAGAGCTC CACCTTCAACCGCAACCAC-3'; The sequence of the identification primer is: 3142wS: 5'-CCGGCAACCCTACCTGGA-3'; 3142wA: 5'-TGCCGAGTGGGAGTTCTACCTG-3'.

5. The method according to claim 3, characterized in that: The concentration of gentamicin in step (4) in the LB plate is 40 μg / mL, and the concentration of chloramphenicol in the LB plate is 25 μg / mL; the concentration of sucrose in step (5) in the LB plate is 10% m / v.

6. The Pseudomonas putida according to claim 1 PP_3142 Application of gene knockout mutants in water environment management and restoration.

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