Cellulomicrobe phage DS1 and application thereof

Abstract

The invention discloses a cellulomicrobe bacteriophage DS1 and application thereof, and belongs to the technical field of microorganisms. The cellulomicrobe bacteriophage DS1 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.45680. According to the bacteriophage DS1 obtained through self screening, the bacteriophage DS1 is determined to be the cellulomicrobe bacteriophage through separation identification and whole genome sequencing, and the efficient and continuous bactericidal effect of the bacteriophage DS1 on cellulomicrobe is further verified through experiments. The bacteriophage DS1 provided by the invention can be used for controlling the population number of cellulomicrobe in microbial communities such as soil, water and the like. A selectable basic material is provided for prevention and control of cellulomicrobe, and a bacteriophage seed bank is enriched.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a cellulomicrobacterial phage DS1 and application thereof. Background Art

[0002] Cellulosimicrobium is a very important microbial resource. It is widely used in biotechnology fields such as bioflocculant preparation, microbial fermentation, heavy metal removal from water bodies, and antibiotic removal. The enzyme system contained in its genome plays an important role in the degradation of aromatic compounds in the natural environment. However, its application as a microbial agent has interfered with the stability of the original ecological microbial community in nature to a certain extent, and excessive use has destroyed the structure of the original ecological microbial community. Although it has achieved the effect required by people to a certain extent as a microbial agent, it has inhibited the environmental function of the original microbial community and caused certain damage to the stability of the ecological environment. At the same time, due to the gene transferability of bacteria, once the cellulosimicrobium obtains toxic genes through transformation and other methods, it will cause great harm to human health. Therefore, it is necessary to develop an effective means that is harmless to the human body and the environment and can effectively control the number of cellulosimicrobium organisms.

[0003] Phages have gradually attracted attention due to their high specificity, safety to the human body, and no pollution to the environment. Compared with antibiotics, phage therapy has fewer side effects, high specificity, less destructiveness to the structure of microbial communities, and phages can co-evolve with bacteria and are not easily resisted by bacteria. Although phages have achieved good results in removing bacteria in livestock and poultry farming, aquaculture, and food industry, the type and number of phages are an important factor limiting the further development of phage therapy, and further separation and identification of phages are needed to enrich the phage species library.

[0004] Therefore, the isolation of Cellulosimicrobium phage and its application research are issues that need to be urgently addressed by those skilled in the art. Summary of the invention

[0005] The purpose of the present invention is to provide a fiber microbacteria phage DS1 and its application to solve the problems existing in the above-mentioned prior art. The phage DS1 provided by the present invention can be used to control the population of fiber microbacteria in microbial communities such as soil and water. The present invention provides an optional basic material for the prevention and control of fiber microbacteria and enriches the phage species library.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a strain of Cellulosimicrobium sp. phage DS1, which is deposited in the General Microbiological Center of China National Committee for Microbiological Culture Collection, with a deposit number of CGMCC No.45680.

[0008] The present invention also provides the use of the Cellulosimicrobium bacteriophage DS1 in preparing products that inhibit Cellulosimicrobium.

[0009] The present invention also provides a product for inhibiting fiber microbes, wherein the effective component comprises the fiber microbe phage DS1.

[0010] The present invention also provides the use of the fiber microbacteria phage DS1 or the product in inhibiting fiber microbacteria.

[0011] The present invention also provides the use of the fiber microbacteria phage DS1 or the product in controlling the number of fiber microbacteria populations in an environment.

[0012] Furthermore, the environment includes a soil environment and a water environment.

[0013] The present invention also provides a method for controlling the population of fiber microbes in an environment, comprising applying the fiber microbe bacteriophage DS1 or the product to an environment where a bacterial agent containing fiber microbes has been used, and using the fiber microbe bacteriophage DS1 to lyse the fiber microbes, thereby controlling the population of the fiber microbes.

[0014] Furthermore, the environment includes a soil environment and a water environment.

[0015] The present invention discloses the following technical effects:

[0016] The present invention screened and obtained a phage DS1, and through its isolation, identification and whole genome sequencing, it was determined that the phage DS1 was a cellulomicrobium phage, and further verified through experiments that the phage DS1 had a highly efficient and sustained bactericidal effect on cellulomicrobium.

[0017] The bacteriophage DS1 provided by the present invention can be used to control the population of fiber microbes in microbial communities such as soil and water. The present invention provides an optional basic material for the prevention and control of fiber microbes and enriches the bacteriophage species library. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is the double-layer plate plaque morphology of bacteriophage DS1;

[0020] Figure 2 The transmission electron microscopic morphology of bacteriophage DS1 is shown, and the scale bar is 100 nm;

[0021] Figure 3 This is the circle map of the bacteriophage DS1 genome;

[0022] Figure 4 is the OD after adding bacteriophage DS1 to the suspension of cellulomicrobium 600 . DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] The host bacteria Cellulosimicrobium sp. used in the examples were obtained by the research group of Liu Xinchun from the School of Resources and Environment, University of Chinese Academy of Sciences, and were identified as Cellulosimicrobium sp. by 16S sequencing.

[0029] R2A culture medium and culture medium were purchased from Qingdao Hi-Tech Park Haibo Biotechnology Co., Ltd.; PEG8000 was purchased from Amresco; DNase and RNaseA were purchased from Sigma; proteinase K was purchased from Amresco; 0.22 μm microporous filter membrane was purchased from Shanghai Xinya Purification Device Factory; chloroform was purchased from Beijing Dingguo Biotechnology Co., Ltd.

[0030] Example 1 Isolation and purification of bacteriophage

[0031] The sewage sample used in the experiment of the present invention was collected from a small sewage treatment system in the laboratory in September 2019, and was used as the water sample for isolating bacteriophages.

[0032] The sewage sample was centrifuged for 15 min, and 25 mL of sewage was filtered and sterilized using a 0.22 μm microporous filter membrane; CaCl was added 2 The mother solution was adjusted to a final concentration of 1mmol / L and allowed to stand; 20mL of R2A liquid culture medium was added, and then 2.0mL of logarithmic host bacterial suspension (cellulosic bacteria) was added to a 50mL centrifuge tube and mixed; after standing for 15min, oscillated at 37℃ overnight (10h). The next day, 2mL of chloroform was added, and centrifuged at 4℃, 8000g for 30min; 15mL of supernatant was taken, and 20mL of R2A culture medium and 1.0mL of logarithmic host bacterial suspension were added, and the mixture was shaken and placed at room temperature for 30min; oscillated at 37℃ for 120r·min -1 After 24 hours of culture, take out; centrifuge at 4°C, 8000g for 30 minutes, and the supernatant is the stock solution containing the phage of this host bacteria. Use the spot-test method to identify whether the stock solution contains phages. Take an appropriate amount of the stock solution and drop it on the R2A solid culture medium coated with host bacteria, culture at 37°C for 24 hours, and observe whether plaques grow. If clear and transparent plaques appear on the plate, it means that phages exist in the stock solution. Otherwise, it means that phages have not been separated and re-sampling and separation is required.

[0033] The isolated phage may not be a single phage, and the present invention uses a double-layer plate method for further purification. Pick up the plaques on the plate, immerse them in a sterile tube containing 1mL of SM solution, and after leaving it at room temperature for 1 hour, take 0.1mL of the above solution and dilute it in a gradient manner. Take 0.1mL of the diluted solution and 0.1mL of the host bacterial suspension and add them to a 5mL tube, followed by adding 4mL of R2A semi-solid culture medium, and then quickly pour it into R2A solid culture medium to spread it on the entire double-layer plate. Incubate at 37°C for 24 hours and observe the growth of the plaques. Then pick up a single plaque and repeat this step 5-6 times until the size and morphology of each plaque in the double-layer plate are basically the same. Figure 1 The single phage isolated at this time was named phage DS1.

[0034] Example 2 Morphological observation of bacteriophage

[0035] Take 20 μL of phage DS1 suspension and drop it on the copper mesh. After it is naturally precipitated for 10 minutes, use dry filter paper to absorb it from the side. After leaving it in the air for about 1 minute, add 1 drop of 1% uranyl acetate on the copper mesh and stain it for 2 minutes. Then use dry filter paper to carefully absorb the excess dye from the side. After leaving it in the air for 30 minutes away from light, observe it with a transmission electron microscope (JEM-1400).

[0036] Transmission electron microscopy results are shown in Figure 2 The results showed that bacteriophage DS1 has an icosahedral head and a retractable tail. The head is about 123.5nm long, the head diameter is about 51nm, and the tail is about 220nm long. According to the eighth report on virus classification of the International Taxonomy of Viruses (ICTV), this strain of bacteriophage DS1 can be preliminarily classified as the Myotailed Bacteriophage family.

[0037] Example 3 Phage genome analysis and identification

[0038] Phage whole genome sequencing and analysis: Illumina Next Seq 500 was used to sequence the DNA of the samples by PE 2×150. The optimized sequences were then spliced ​​using spades v.3.11.1 splicing software to obtain the best assembly results. The open reading frames of the genome were predicted and analyzed using the biological software PHASTER, and the preliminary annotation of functional genes was completed using NCBI Blastp. tRNAs were predicted online using tRNAscan-SE (http: / / lowelab.ucsc.edu / / tRNAscan-SE / ), and the whole genome circle map was drawn using CGView Server software (http: / / cgview.ca / ) (see Figure 3 ),

[0039] The whole genome has been uploaded to gene bank, with the accession number MW590223. Whole genome analysis showed that the phage genome size was 46217bp, with a G+C content of 55.32%. Analysis showed that the phage had 54 CDS and 2 tRNA, of which 24 CDS were in the positive strand and 30 CDS were in the negative strand. 13 of the 54 CDS were annotated by two databases, of which 8 CDS were annotated proteins, and 5 CDS were predicted proteins obtained by comparison with the phage database. The CDS related to other phage hosts were Gordonia, Arthrobacter and Microbacterium. The remaining 41 CDS were hypothetical proteins, and most of the hypothetical proteins and whole genome Blast results indicated that this was a new phage.

[0040] Phage DS1 is a strain of Cellulosimicrobium sp.phage (Latin name: Cellulosimicrobium sp.phage), which was deposited on October 26, 2023 in the General Microbiology Center of the China Microorganism Culture Collection Administration, referred to as CGMCC, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.45680.

[0041] Example 4 Study on the application of bacteriophage DS1 in cellulolytic microbes

[0042] Take 0.1 mL of the logarithmic phase suspension of the fiber microorganisms and add it to 100 mL of R2A liquid culture medium and cultivate it in a 37°C constant temperature incubator. At 16 hours, take the cells that have completed proliferation and have a titer of 10 8 pfu / mL of phage DS1 stock solution was added to the culture medium at the optimal MOI of 0.1 as the experimental group (with phage), and the control group (control) was added with an equal amount of PBS buffer. Samples were taken from the experimental group and the control group every 4 hours to measure the OD of the cellulosic bacteria 600 .

[0043] The results showed that adding bacteriophage DS1 to the culture medium of cellulosic bacteria in the logarithmic growth phase at 16 h could reduce the OD of cellulosic bacteria to 1. 600 The OD of the control group of fibroblasts decreased from 0.34 to 0.04. 600 The OD of the cellulosic bacteria with phage DS1 reached 0.55 after 36 h of continuous cultivation. 600 is 0.01, indicating that bacteriophage DS1 has a high efficiency and sustained bactericidal effect on cellulosic bacteria. Figure 4 .

[0044] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Cellulosimicrobium sp. phage DS1, characterized in that: The cellulomicrobium muscle tail phage DS1 is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.45680.

2. Use of the Cellulosimicrobium phage DS1 as claimed in claim 1 in the preparation of products that inhibit Cellulosimicrobium.

3. A product for inhibiting fiber microbes, characterized in that: The active ingredient comprises the cellulomicrobacteriophage DS1 described in claim 1.

4. Use of the cellulomicrobacteria phage DS1 according to claim 1 or the product according to claim 3 in inhibiting cellulomicrobacteria.

5. Use of the fiber microbacteria phage DS1 according to claim 1 or the product according to claim 3 in controlling the number of fiber microbacteria in the environment.

6. The use according to claim 5, characterized in that: The environment includes soil environment and water environment.

7. A method for controlling the number of fiber microbes in an environment, characterized in that: The method comprises applying the fiber microbacteria phage DS1 of claim 1 or the product of claim 3 to an environment where a bacterial agent containing fiber microbacteria has been used, and utilizing the fiber microbacteria phage DS1 to lyse the fiber microbacteria, thereby controlling the population of the fiber microbacteria.

8. The method according to claim 7, characterized in that The environment includes soil environment and water environment.

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