An isolated strain of enterogenic Escherichia coli, Escherichiacoli W5

Abstract

The present application relates to a strain of enteric Escherichia coli isolated bacteria E.coli W5, which is preserved in Guangdong Microbial Culture Collection Center, the preservation number is GDMCC 66116, and the preservation date is April 8, 2025.The isolated strain of the present application is Escherichia coli, and there is a difference in the sequence compared with the commonly used Escherichia coli, which is a natural isolated strain of nematode intestine.The strain has higher plasmid transformation capacity compared with the commonly used Escherichia coli E.coli Nissle 1917, the transformation efficiency is increased by 1.5 times, and can maintain a higher colonization level and a longer colonization time during the colonization process in the nematode intestine, the colonization level of W5 after 144h of colonization in the nematode intestine is as high as 2543.2 times compared with E.coli Nissle 1917, and can become a candidate strain for the research of nematode intestine.

Description

Technical Field

[0001] This invention relates to the field of biotechnology engineering, specifically to an Escherichia coli strain with high plasmid transformation capacity and high colonization level, which can colonize the intestine of Caenorhabditis elegans for a long time and be used for nematode intestinal research. Background Technology

[0002] In the field of biology, nematodes are widely used model organisms in the study of many biological processes. Nematodes feed on bacteria, which are digested in the gut and then used as nutrients. This makes nematodes a highly genetically manipulated model organism system for studying the direct and indirect effects of diet, including host-microbe interactions. *C. elegans* is a powerful tool for studying host-microbe interactions. *Escherichia coli*, due to its biological characteristics and ease of genetic manipulation, is helpful in exploring complex issues in nematode and host-microbe biology.

[0003] Currently, the engineered Escherichia coli strains commonly used in gut research are E. coli Nissle 1917 and E. coli MG1655, but their colonization levels and colonization time in nematode intestines are relatively low. Summary of the Invention

[0004] This invention aims to address the problems existing in the prior art by providing a novel *Escherichia coli* strain derived from nematodes, which exhibits superior performance in plasmid transformation efficiency and colonization levels. This new strain will provide a more effective and reliable tool for research related to the gut of *C. elegans*.

[0005] To address the aforementioned technical problems, this invention provides an enterogenic Escherichia coli W5 strain isolated from Caenorhabditis elegans during its L4 life cycle. This strain exhibits good plasmid transformation ability and a high colonization level, and can contribute to research related to the nematode gut.

[0006] To achieve the above-mentioned objectives, the technical solution implemented by this invention is as follows:

[0007] Enterogenic Escherichia coli was isolated from the intestinal lysate of Caenorhabditis elegans, screened by LB and E. coli staining plates, and the 16S rRNA gene sequence was amplified by PCR.

[0008] The 16S rRNA gene sequence obtained by PCR amplification was compared with commonly used E. coli sequences and other closely related species to construct a MEGA phylogenetic tree;

[0009] To compare whether the gene manipulation function of enterogenic Escherichia coli is advantageous, the transformation efficiency of the strain was first tested. The plasmid J23101-sfgfp@pSB3K3, which was already in the laboratory and expressed the green fluorescent protein gene, was transformed into the E. coli to be tested to form the engineered strain. Different concentrations were diluted and spotted on LB and LB+kanamycin solid plates. The transformation efficiency was calculated based on the number of single colonies.

[0010] To compare the colonization level and duration of enterogenic *E. coli* isolated from *C. elegans*, λ-Red homologous recombination gene editing technology was used to insert green fluorescent protein (GFP) and kanamycin resistance genes into the *E. coli* genome. This allowed the strain to be stably expressed in nematodes, facilitating subsequent observation and screening. Nematodes were fed with three knockout bacteria—*E. coli* W5ΔlacZ::sfgfp KmR, *E. coli* Nissle 1917ΔlacZ::sfgfp KmR, and *E. coli* MG1655ΔlacZ::sfgfp KmR—to L4. The nematodes were then transferred to heat-inactivated *E. coli* OP50 NGM solid medium for culture. Nematodes were collected at four time points after feeding: 24h, 48h, 96h, and 144h. The nematode intestinal lysate was serially diluted with different concentrations and spotted onto LB+kanamycin solid plates. The CFU carried by each nematode was calculated based on the number of single colonies to compare the colonization levels of different Escherichia coli.

[0011] Beneficial effects:

[0012] To identify *E. coli* bacteria capable of long-term colonization of nematode intestines, this invention isolates *E. coli* from the nematode gut by mimicking the abundant microbial community found in nematodes in the wild. Secondly, to enhance the gene transformation efficiency of *E. coli*, different competent cell preparation methods and transformation conditions are employed to influence the transformation effect. To facilitate the tracking and screening of *E. coli* within the nematode gut, *E. coli* gene knockout bacteria are constructed using λ-Red homologous recombination gene editing technology, enabling their genome to carry the green fluorescent protein gene and the kanamycin resistance gene.

[0013] The above-described method of the present invention comprehensively utilizes the advantages of the green fluorescent protein gene and nematode entero-derived Escherichia coli. Through MEGA phylogenetic tree, transformation efficiency, and colonization level experiments, a nematode entero-derived E. coli strain, E. coli W5, with high efficiency in all aspects was finally screened. Compared with the model Escherichia coli commonly used in nematode gut research in existing articles, this bacterium has higher transformation efficiency and a higher colonization level in the nematode gut, and therefore has greater advantages. Attached Figure Description

[0014] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0015] Figure 1 This is a MEGA phylogenetic tree of the nematode gut-derived isolates of this invention and the previously studied 16S rRNA gene sequences of Escherichia coli.

[0016] Figure 2 This is a comparison of the plasmid transformation effects of the nematode gut-derived isolates of this invention with E. coli Nissle 1917 and E. coli MG1655.

[0017] Figure 3 This is a comparison chart of colony-forming units (CFUs) data of the nematode gut-derived bacteria isolated in this invention with those of E. coli Nissle 1917 and E. coli MG1655 formed in the nematode gut. Detailed Implementation

[0018] Main materials

[0019] Wild-type Caenorhabditis elegans Bristol strain, N2

[0020] 2×Phanta Max Master Mix was purchased from Nanjing Novozymes Biotechnology Co., Ltd.

[0021] 5-Fluorouracil (CAS: 51-21-8) was purchased from Sigma-Aldrich, USA.

[0022] E. coli chromogenic culture medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0023] Sterile PBS buffer was purchased from Seville Biosciences.

[0024] Triton X-100 was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0025] Greiner 96μClear flat bottom microplate was purchased from Guangzhou Dingguo Biotechnology Co., Ltd.

[0026] Other materials not specified were obtained through regular commercial purchases.

[0027] 50 mg / mL kanamycin stock solution: 0.5 g kanamycin, add 10 mL ddH2O, filter through a 0.22 μM filter membrane, aliquot into 1.5 cm centrifuge tubes, and store at -20 °C.

[0028] 5 mg / mL cholesterol: Add 25 mg cholesterol to 50 mL of anhydrous ethanol, filter through a 0.22 μM filter membrane, and store at 4 °C.

[0029] LB solid medium: 10g tryptone, 5g yeast extract, 5g sodium chloride, 20g agar powder, and ultrapure water to 1L;

[0030] LB+Kanamycin solid medium: 10g tryptone, 5g yeast extract, 5g sodium chloride, 20g agar powder, add ultrapure water to 1L, sterilize and cool to 45℃, then add 50mg / mL kanamycin solution at a ratio of 1:1000.

[0031] TB liquid culture medium: 12g tryptone, 24g yeast extract, 17mM KH2PO4, 72mM K2HPO4, 0.4% glycerol, plus ultrapure water to 1L;

[0032] NGM solid culture medium: 3g sodium chloride, 2.5g tryptone, 17g agar powder, 25mL (1M) potassium phosphate solution, 1mL (1M) calcium chloride dihydrate solution, 1mL (1M) magnesium sulfate heptahydrate solution and 1mL (5mg / mL) cholesterol, plus ultrapure water to 1L.

[0033] NGM+5-fluorouracil solid culture medium: 3g sodium chloride, 2.5g tryptone, 17g agar powder, 25mL (1M) potassium phosphate solution, 1mL (1M) calcium chloride dihydrate solution, 1mL (1M) magnesium sulfate heptahydrate solution, and 1mL (5mg / mL) cholesterol are added to ultrapure water to 1L. The mixture is sterilized and cooled to 45℃. Then, 20mM 5-fluorouracil solution is added at a ratio of 1:1000.

[0034] M9 buffer: Add 5g sodium chloride, 3g KH2PO4, and 6g Na2HPO4 to ultrapure water to 1L, sterilize and cool to 45℃, then add 1mL (1M) magnesium sulfate heptahydrate solution.

[0035] Example 1: Screening of Escherichia coli from Nematode and Establishment of MEGA Phylogenetic Tree

[0036] (1) Culture of Caenorhabditis elegans

[0037] First, L4-stage nematodes were transferred to NGM plates inoculated with E. coli OP50 and incubated for 24 hours, with E. coli OP50 serving as their food source [Girard LR, Fiedler TJ, Harris TW, et al. WormBook: the online review of Caenorhabditis elegans biology[J]. Nucleic acids research, 2007, 35(Database issue): D472-475.]. This allowed the nematodes to become fully infested with eggs. M9 buffer was added to the NGM plates containing a large number of adult nematodes and eggs for collection. The late-pregnancy adult nematodes were dissolved in bleach (1 mL 4M sodium hydroxide, 1.25 mL sodium hypochlorite, 7.75 mL ddH2O). This process released the eggs, which were then collected to obtain synchronized L1-stage larvae. The simulated rich microbial community in the wild was fed to L1 nematodes until L4 stage, and the nematodes were collected. [The method for constructing the simulated rich microbial community in the wild is referenced He YJ, Hao FR, Fu HR, et al. N-glycosylated intestinal protein BCF-1 shapes microbial colonization by bindingbacteria via its fimbrial protein[J]. Cell Reports, 2023, 42(1)].

[0038] (2) Isolation and identification of entero-derived Escherichia coli

[0039] After washing L4 nematodes 3-5 times with M9 buffer, wash twice with M9 buffer (LM buffer) containing 25 mM levamisole (levamisole inhibits the pharyngeal pump and defecation), then wash twice with LM buffer containing 100 μg / mL gentamicin (to clean bacteria outside the nematodes), and then wash three times separately with M9 buffer. Place the washed nematodes into a 1.5 mL centrifuge tube containing 100 μL of PBS containing 1% Triton X-100, add sterile steel balls, and grind on a homogenizer (60 Hz, 1 min, repeated 9 times) to prepare a nematode intestinal lysis stock solution. Add 10 μL of the stock solution to 1 mL of sterile PBS buffer to obtain 10 μL of lysate. -1 Diluent, and so on, to obtain 10 -2 -10 -4Diluent. Spread 100 μL onto LB solid medium and incubate overnight at 37°C. Pick a single colony that is round with neat edges, a smooth surface, is translucent, and has small bumps resembling E. coli. After streaking twice on LB solid medium, pick another single colony and streak it on E. coli chromogenic solid medium to determine the genus. If the single colony turns blue, it is identified as E. coli.

[0040] (3) Construction of the MEGA evolutionary tree

[0041] Single colonies of Escherichia coli were selected and amplified using the primers in Table 1 and the PCR reaction system in Table 2. The PCR amplification yielded a 16S rRNA gene fragment of approximately 1500 bp.

[0042] Table 1. PCR amplification primers for identifying enteric Escherichia coli isolated from Nematodes

[0043]

[0044] Table 2 PCR reaction system (50 μL)

[0045]

[0046] Table 3 PCR amplification process

[0047]

[0048] After the reaction was terminated, the sample was purified and recovered by 1% agarose gel electrophoresis for later use and then subjected to Sanger sequencing (Sangon Biotech Shanghai Co., Ltd.).

[0049] The study used the Neighbor-Joining (NJ) method of MEGA software to compare the sequences of isolated nematode-derived *E. coli* with those commonly used to study the gut. Sequence differences were found, such as... Figure 1 .

[0050] After the above culture and identification, one of the isolated bacteria was classified and named E. coli W5, which is of nematode origin.

[0051] The nematode-derived enterotrophic Escherichia coli W5 was deposited on April 8, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located in Guangzhou, Guangdong Province, with accession number GDMCC 66116.

[0052] Example 2: Plasmid transformation efficiency of nematode-derived Escherichia coli and commonly used Escherichia coli self-made competent cells

[0053] The laboratory already had the plasmid J23101-sfgfp@pSB3K3, which was constructed based on the iGEM common component library (https: / / parts.igem.org). This plasmid was transformed into each self-made competent E. coli cell. The main components of this plasmid, such as J23101, pSB3K3, and sfgfp, can be found at https: / / parts.igem.org, and were then assembled manually. It should be noted that other plasmids can also be used to evaluate the plasmid transformation efficiency of E. coli, as long as the plasmid used can continuously express fluorescent proteins and resistance genes. The plasmid J23101-sfgfp@pSB3K3 was used only because it is readily available; therefore, the specific construction method of this plasmid is not described in detail. Those skilled in the art, based on the description of this invention, can use other plasmids capable of continuously expressing fluorescent proteins and resistance genes to conduct E. coli transformation efficiency evaluation experiments.

[0054] The competent cells were prepared using the CaCl2 method. A small amount of the preserved bacterial strain was streaked onto LB agar plates and incubated at 37°C. Single colonies were then transferred to 5 mL of LB agar and incubated at 37°C with shaking at 180 rpm for 17 h. The overnight culture was then diluted 400-fold to 200 mL of fresh LB agar and incubated at 37°C with shaking at 180 rpm until the OD value was reached. 600 =0.3-0.4, transfer the culture to 50mL centrifuge tubes and immediately place on ice for 20min. Place the centrifuge tubes in a pre-chilled centrifuge at 4℃ and centrifuge at 3000g for 8min. Discard the supernatant, add 10mL of pre-chilled 50mM CaCl2, gently resuspend on ice, repeat the previous centrifugation step, collect the cells, discard the supernatant again, add 10mL of pre-chilled 50mM CaCl2, gently resuspend on ice, place on ice for 30min, centrifuge again at 3000g for 6min, discard the supernatant again, add 2mL of 50mM CaCl2 and 15% glycerol to each 50mL centrifuge tube, gently resuspend, place on ice for 2h, then aliquot into 1.5mL centrifuge tubes, flash freeze in liquid nitrogen, and store at -80℃.

[0055] The plasmid transformation was performed using a heat shock method. Plasmid J23101-sfgfp@pSB3K3 was transformed into 100 μL of competent *E. coli* cells, resulting in a final concentration of 1 μg / mL. Following standard transformation procedures, the plasmid was added to the competent cells and placed on ice for 30 min, followed by a heat shock at 42°C for 90 s, immediately placed on ice for 3 min, and then 900 μL of fresh LB agar was added for recovery and incubation at 37°C for 1 h. The transformation product was serially diluted, and 3 μL of each product was aliquoted onto LB and LB+kanamycin agar plates for triple replicates, incubated upside down at 37°C for 17 h. The transformation efficiency (%) was calculated based on the number of colonies at the same concentration: (number of single colonies on the LB+kanamycin agar plate) / (number of single colonies on the LB agar plate × dilution factor) × 100%.

[0056] The transformation efficiency of Escherichia coli was calculated and is shown in Table 4. The transformation efficiency of E. coli W5, derived from nematode E. coli, was 1.5 times higher than that of the commonly used E. coli Nissle 1917.

[0057] Table 4. Transformation efficiency of *E. coli* isolated from nematode gut-derived bacteria and commonly used *E. coli* models.

[0058]

[0059] Experiment Example 3: Comparison of colonization levels of nematode gut-derived Escherichia coli and commonly used model Escherichia coli in nematode intestines

[0060] Gene knockout bacteria were constructed using λ-Red recombination technology with the isolated E. coli W5, E. coli Nissle 1917, and E. coli MG1655, and named E. coli W5ΔlacZ::sfgfp KmR, E. coli Nissle 1917ΔlacZ::sfgfp KmR, and E. coli MG1655ΔlacZ::sfgfp KmR, respectively.

[0061] First, pick a single colony from a fresh E. coli OP50 streak plate and inoculate it into 400 mL of LB medium. Incubate at 37°C with shaking at 250 rpm for 16–22 hours. Concentrate the cultured E. coli OP50 (200 mL) to 100 mL (half volume) by centrifugation (2000 g, 15 min). Vortex to resuspend the cells. Heat-inactivate the concentrated E. coli OP50 in a 90°C water bath for 120 minutes. Spread 500 μL of the heat-inactivated OP50 (HK-OP50) onto an LB plate and observe colony growth. Incubate the plate at 37°C for 16–22 hours; no bacterial growth confirms successful heat inactivation. 0.3 mg / mL of vitamin B2 (riboflavin) was spread on NGM plates to maintain the normal development of nematodes. Then, 300 μL of HK-OP50 was spotted onto NGM-5 fluorouracil (5-Fu) plates to prepare HK-OP50-NGM-5fu medium.

[0062] A single colony of each of *E. coli* W5ΔlacZ, *E. coli* Nissle 1917ΔlacZ, and *E. coli* MG1655ΔlacZ was picked from streak plates and inoculated into 5 mL of LB broth. The culture was incubated at 37°C with shaking at 250 rpm for 16–22 hours. 0.5 mL of the cultured knockout bacteria was diluted into 50 mL of fresh LB broth. The diluted bacteria were cultured at 37°C until OD600 = 1. The bacterial suspensions were collected, and the three suspensions were concentrated to 25 mL (half volume). The suspensions were vortexed to resuspend the bacteria. 300 μL of the bacterial suspensions were spotted onto 6 cm NGM plates to prepare NGM media for feeding nematodes.

[0063] Centrifuge synchronized L1-stage nematodes in a 15 mL test tube, remove the M9 medium, resuspend the synchronized L1-stage nematodes in 500 μL of M9 medium, and then take 10 μL of L1-stage nematodes and place them on a microscope slide for counting. The ideal number of nematodes is 100-200 per 10 μL. Synchronized L1-stage nematodes were inoculated (40 μL) onto E. coli W5ΔlacZ::sfgfpKmR, E. coli Nissle 1917ΔlacZ::sfgfpKmR, and E. coli MG1655ΔlacZ::sfgfpKmR NGM media and cultured at 20°C for 48 h to reach the L4 stage. The L4-stage nematodes were washed 5-6 times with M9 medium and transferred to HK-OP50-NGM-5fu medium. The nematodes were transferred to fresh HK-OP50-NGM-5fu medium daily. The nematodes were collected at 24 h, 48 h, 96 h, and 144 h after feeding, and the nematode intestines were lysed.

[0064] At selected time points, wash the nematodes three times with M9 buffer, and then wash the washed nematodes three times with phosphate-buffered saline (PBS) to remove excess M9 buffer. Add to 100 μL of 18% iodixanol (CBI).

[10] The nematodes were placed in 1.5 mL centrifuge tubes containing 1% Triton X-100 PBS. The CBI in the PBS helps to keep the nematodes in a uniform suspension, facilitating nematode counting. A 10 μL sample was taken for counting to estimate the number of nematodes in the remaining 90 μL. The remaining 90 μL was then homogenized using a homogenizer to rupture the nematode cells and release the intestinal bacteria. The colony-forming units (CFUs) of three strains of *C. elegans* in the gut were measured, and the CFUs in the gut of each nematode were calculated using a formula.

[0065]

[0066] Each condition was replicated at least six times. The results showed that E. coli W5 colonized the gut of *C. elegans* at a much higher level than E. coli Nissle 1917 and E. coli MG1655. Figure 3 .

[0067] Figure 1 This invention presents a MEGA phylogenetic tree of the 16S gene sequences of the nematode enteric isolate E. coli W5, commonly used Escherichia coli E. coli MG1655, E. coli Nissle1917, and other closely related species. The 16S gene sequences of other species besides E. coli W5 were obtained from the NCBI database, with Enterococcus faecium strain LMG 11423 classified as an outgroup.

[0068] Phylogenetic trees constructed using the neighbor-joining (NJ) method based on the 16S rRNA sequences of 18 bacteria showed that strains E. coli W5 and E. coli R94 had a similarity of 94%.

[0069] Figure 2 : Comparison of plasmid transformation effects of the nematode enteric isolate E. coli W5 and commonly used model Escherichia coli E. coli MG1655 and E. coli Nissle1917. (a) Plate area of ​​bacterial solution after different dilutions after bacterial transformation, where 0 represents the mother liquor, -1 to -12, etc. represent 10 -1 ~10 -12 (a) Iso-dilution factor; (b) Colony distribution of E. coli MG1655, E. coli Nissle 1917, and E. coli W5 on LB+kanamycin solid medium; (c) Colony distribution of E. coli MG1655, E. coli Nissle 1917, and E. coli W5 on LB solid medium; (d) Dilution factor, single colony count, and transformation efficiency of E. coli MG1655, E. coli Nissle 1917, and E. coli W5 on LB+kanamycin solid medium and LB solid medium.

[0070] The transformation efficiency was calculated based on the number of colonies at the same concentration. Transformation efficiency % = (number of single colonies on LB + kanamycin solid plate) / (number of single colonies on LB solid plate × dilution factor) × 100%. This calculation yielded the transformation efficiency of *E. coli*, showing that the transformation efficiency of *E. coli* W5, derived from nematodes, was 1.5 times higher than that of the commonly used *E. coli* Nissle 1917.

[0071] Figure 3 The following is a comparison of the colonization levels of the nematode gut-derived bacterium E. coli W5 and commonly used gut research bacteria Escherichia coli, E. coli Nissle 1917, and E. coli MG1655 in the gut of *C. elegans*. (a) Schematic diagram of bacterial suspensions after different concentrations of dilution following nematode gut lysis, where 0 represents the mother liquor, and -1 to -4 represent 10... -1 ~10 -4 (a) Iso-dilution factor; (b) Colony distribution of E. coli W5, E. coli Nissle 1917, and E. coli MG1655 on LB+kanamycin solid medium; (c) CFU statistics of E. coli W5, E. coli Nissle 1917, and E. coli MG1655 on LB+kanamycin solid medium; (d) Comparison of CFU data of E. coli W5, E. coli Nissle 1917, and E. coli MG1655 in the intestine of Caenorhabditis elegans.

[0072] The collected intestinal lysate was diluted to an appropriate dilution gradient and then subjected to bacterial spotting. The number of colonies of each E. coli was counted to calculate CFU, and the results were obtained by data comparison. After 144 h of colonization in the nematode intestine, the colonization level of E. coli W1 was 2543.2 times that of E. coli Nissle 1917.

[0073] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. An enterogenic Escherichia coli E. coli W5 isolated from the intestine of Caenorhabditis elegans, characterized in that, Its accession number is GDMCC 66116, the depository is Guangdong Provincial Center for Microbial Culture Collection, and the deposit date is April 8, 2025.

2. The *C. elegans* enterogenic *Escherichia coli* according to claim 1, characterized in that, The strain exhibits high plasmid transformation capacity and high colonization efficiency; its plasmid transformation efficiency is 1.5 times higher than that of Escherichia coli Nissle 1917; and the colonization level of W5 after 144 hours of colonization in the nematode gut is 2543.2 times higher than that of E. coli Nissle 1917.

3. The method for culturing enterogenic Escherichia coli W5 according to claim 1, characterized in that, Includes the following steps: A small amount of the preserved Escherichia coli W5 strain was streaked onto LB agar plates and incubated at 37°C. White, translucent, round, and smooth colonies were formed after incubation at 37°C on LB agar plates.

4. The application of the enterogenic Escherichia coli W5 according to any one of claims 1 to 3 in animal intestinal research.

5. The application as described in claim 4, characterized in that, The animal gut studies include plasmid conversion studies.

6. The application as described in claim 4, characterized in that, The animal gut studies include studies on microbial colonization.

7. The application as described in claim 4, characterized in that, The animal described is *Cryptocele*, a nematode.

8. The application as described in claim 4, characterized in that, The application described is to study the host-microbe interaction mechanism under different environmental conditions.

9. The application as described in claim 8, characterized in that, The host-microbe interaction mechanism includes analyzing multiple metabolic pathways to reveal the biological regulatory functions in host-microbe metabolic processes.

10. The application as described in claim 8, characterized in that, The host-microbe interaction mechanism includes utilizing the long-term colonization advantage of the bacteria as a chassis cell for microbial sensors to monitor biomarkers in the organism.

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