A new species of shewanella with antibacterial activity and application thereof

Abstract

The application discloses a new Schumannella species with antibacterial activity and application thereof, and belongs to the technical field of microorganisms. A strain 10F1B-5-1 is identified as a potential new species of Schumannella (Schumannella), and is classified and named as Schumannella sp. The strain 10F1B-5-1 has been preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.1.16698. The Schumannella 10F1B-5-1 provided by the application has effects on gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and gram-negative bacteria (Acinetobacter baumannii), provides a strain resource for research and development of new antibiotics, and has good research value and application potential.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms, and particularly relates to a new species of Schumannella with antibacterial activity and application thereof. BACKGROUND

[0002] The problem of bacterial drug resistance has become one of the most serious challenges in the field of global public health, and the situation in China is particularly severe, so new and efficient antibiotics need to be put into use. Actinobacteria are the main force of antibiotic production, and the species diversity thereof is an important basis for antibiotic research and development. With repeated excavation and screening of ordinary environmental actinobacteria resources, the probability of discovering new compounds therefrom is becoming lower and lower. Actinobacteria inhabiting special environments have unique gene types and metabolic mechanisms, and have become an important breakthrough for new antibiotic research and development.

[0003] Mangrove is a woody plant community growing in the intertidal zone of tropical and subtropical coasts. As an ecological system transitioning from land to sea, mangrove contains unique and rich microbial resources, and has become a hotspot for the development of medicinal actinobacteria. As part of the marine life system, mangrove-derived actinobacteria have the potential to produce compounds with novel structures and unique biological activities. Although the research started late, with the help of the basis and experience of terrestrial natural product research, the secondary metabolites of mangrove actinobacteria have developed rapidly and achieved many important results. These natural products have diverse biological activities, including antibacterial, antiviral, cytotoxic, antioxidant, etc.

[0004] In recent years, the research on secondary metabolites of rare actinobacteria from mangrove has also gradually increased. During 2015-2020, a total of 16 new compounds were isolated from rare actinobacteria from mangrove. Therefore, it is very important to excavate new actinobacteria resources from mangrove ecology, and new species of rare actinobacteria and their secondary metabolites have important research value. SUMMARY

[0005] The purpose of the present application is to provide a new species of Schumannella strain 10F1B-5-1 with antibacterial activity, which is classified and named as Schumannella sp., and has various antibacterial activities, thereby providing a new bacterial source for new antibiotic research and development.

[0006] In a first aspect, the present application claims a potential new species of Schumannella

[0007] The Schumannella new species strain 10F1B-5-1 provided by the present application is isolated from mangrove soil, and is classified and named as Schumannella sp. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) located at No. 1, Beichen West Road, Haidian District, Beijing, and has a preservation number of CGMCC NO. 1.16698 and a preservation date of March 7, 2025.

[0008] The identification characteristics of the new species of Schumannella 10F1B-5-1 are as follows:

[0009] Strain 10F1B-5-1 is a gram-positive, non-motile, rod-shaped bacterium, which forms greenish yellow, smooth, convex, and regular-edged colonies on the culture medium. The length of the 16S rRNA gene sequence of strain 10F1B-5-1 is 1485 bp, and the similarity with Schumannella soli 10F1D-1T is the highest (98.3%), followed by Schumannella luteola DSM 23141T (98.2%). Genome similarity analysis shows that the average nucleotide identity (ANI) of strain 10F1B-5-1 with members of the genus Schumannella is 83.9%-84.3%, and the DNA-DNA hybridization value (DDH) is 27.9%-30.8%. Phylogenetic analysis further shows that strain 10F1B-5-1 clusters with members of the genus Schumannella and forms an independent evolutionary branch. Based on the above results, strain 10F1B-5-1 can represent a new species of the genus Schumannella.

[0010] In a second aspect, the present application claims a culture

[0011] The culture claimed by the present application is the material obtained by culturing strain 10F1B-5-1 of the new species of the genus Schumannella described in the first aspect above in an actinomycete culture medium. The culture medium can be in solid or liquid form.

[0012] The term "culture" refers to the collective term for a liquid or solid culture medium inoculated and cultured by humans, in which a microbial population grows. That is, the product obtained by growing or amplifying microorganisms, which can be a biologically pure culture of microorganisms, or can contain a certain amount of culture medium, metabolites, or other components produced during the culture process. The term "culture" also includes a subculture obtained by subculturing microorganisms, which can be a culture of a certain generation, or a mixture of several generations.

[0013] In the specific embodiments of the present application, the actinomycete culture medium is specifically PG medium and ISP 2 medium.

[0014] In a third aspect, the present application claims a metabolite.

[0015] The metabolite claimed by the present application is the metabolite of strain 10F1B-5-1 of the new species of the genus Schumannella described in the first aspect above, which is classified and named as Schumannella sp.

[0016] The term "metabolite" refers to primary metabolites and or secondary metabolites produced in the process of microbial metabolism. Primary metabolites refer to the substances and energy that microorganisms maintain life activities, such as monosaccharides and their derivatives, nucleotides, vitamins, amino acids, fatty acids, and various macromolecular polymers composed of monomers, such as proteins, nucleic acids, polysaccharides, and lipids. Secondary metabolites are complex compounds synthesized from primary metabolites by microorganisms at a specific growth stage, such as antibiotics, hormones, alkaloids, toxins, etc., and their physiological functions to the microorganisms themselves are usually not clear.

[0017] In a fourth aspect, the present application claims protection for the strain 10F1B-5-1 of the new species of Schumannella as claimed in the first aspect above, which is classified and named as Schumannella sp., the culture as claimed in the second aspect above, or the metabolite as claimed in the third aspect above, for use in inhibiting Bacillus subtilis, Staphylococcus aureus, and Acinetobacter baumannii.

[0018] The present application at least includes the following beneficial effects:

[0019] 1. Experiments have proved that the strain 10F1B-5-1 provided by the present application represents a new species of Schumannella, enriching the species diversity of actinomycetes.

[0020] 2. Schumannella 10F1B-5-1 has antibacterial activity against Staphylococcus aureus, Bacillus subtilis, and Acinetobacter baumannii, showing potential application value of the bacterium in the research and development of antibacterial drugs. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Figure 1 is a transmission electron micrograph of the strain 10F1B-5-1;

[0022] Figure 2 Figure 3 is a phylogenetic tree of the strain 10F1B-5-1 constructed based on 16S rRNA gene sequences;

[0023] Figure 3 Figure 4 is a phylogenetic tree of the strain 10F1B-5-1 constructed based on core proteome. DETAILED DESCRIPTION

[0024] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art.

[0025] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0026] All materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0027] Isolation of strain 10F1B-5-1

[0028] The strain 10F1B-5-1 of the present application was isolated from the soil of mangrove in Futian, Shenzhen, and the isolation was performed as follows: bury in-situ culture device containing 0.3% agar in the soil of mangrove, and take back after one month, pick up the intact in-situ culture device for cleaning. In the clean bench, transfer the agar in the in-situ culture device to a sterile 15 mL centrifuge tube, cut into pieces with a sterile scalpel, then add 10 mL sterile water, shake thoroughly to mix. Take 200 μl of the suspension and spread on ISP 5 medium (medium formula: L-aspartic acid 1.0 g / L, glycerol 10.0 g / L, K2HPO4 1.0 g / L, FeSO4•7H2O 0.001 g / L, MnCl2•4H2O 0.001 g / L, ZnSO4•7H2O 0.001 g / L, agar 20.0 g / L, pH value 7.2±0.2), and incubate at 30°C for 2-12 weeks. During the incubation, observe and record the outgrowth and contamination in time. Use YIM 38 medium (medium formula: glucose 4.0 g / L, yeast extract 4.0 g / L, malt extract 5.0 g / L, agar 20.0 g, pH value 7.2±0.2) for strain purification, repeat for many times until single colony is obtained, and use 20% (v / v) glycerol as protective agent for -80°C freezing preservation. Obtain single colony numbered as 10F1B-5-1.

[0029] Classification and identification of strain 10F1B-5-1

[0030] 1. 16S rRNA gene sequence analysis

[0031] The DNA of the strain 10F1B-5-1 was extracted by using the chelex-100 method, the 16S rRNA gene of the strain was amplified by using primers 27F and 1492R, the PCR product was cloned and sequenced, and the nearly full-length 16S rRNA gene sequence was obtained, the 16S rRNA gene sequence is shown as SEQ ID NO. 1, and is submitted to the GenBank database of NCBI. The homology search of 16S rRNA gene was performed by using the online comparison of EzBioCloud website. The phylogenetic tree was constructed by using Mega 7.0 software and Kimura 2-parameter model by using the maximum parsimony method (ML). The 16S rRNA gene sequence length of the strain 10F1B-5-1 is 1485 bp (as shown in SEQ ID NO. 1), and the GenBank accession number is MK589790. The sequence comparison result shows that the strain 10F1B-5-1 has the highest 16S rRNA gene sequence similarity with Schumannella soli 10F1D-1T (98.3%), followed by Schumannella luteola DSM 23141T (98.2%), and the 16S rRNA gene sequence similarity with other members of the Microbacteriaceae family is lower than 97.3%. The ML phylogenetic tree constructed based on the 16S rRNA gene sequence shows that the strain 10F1B-5-1 is clustered with the members of the Schumannella genus, and forms an independent evolution branch. Figure 2 ).

[0032] 2. Whole genome sequencing and sequence analysis

[0033] The genomic DNA was extracted by using the CTAB method, and after agarose gel electrophoresis and Qubit quality detection, the whole genome sequencing was performed on the Illumina platform, and the sequence was spliced by using SOAPdenovo2. The DNA G+C content of the strain 10F1B-5-1 was calculated based on the whole genome sequence. The core proteome of the strain 10F1B-5-1 and the similar species was obtained by using the M1CR0B1AL1Z3R website, the phylogenetic tree was constructed by using Mega 7.0 software and Jones-Taylor-Thornton model by using the ML method. At the same time, the average nucleotide homology (ANI) between the strain 10F1B-5-1 and the phylogenetically similar species was calculated by using the online tool EzBioCloud, and the DNA-DNA hybridization (DDH) value was determined by using the online tool Genome-to-Genome Distance Calculator.

[0034] The above analysis shows that the full length of the genome of strain 10F1B-5-1 is 4,259,791 bp, the contigs are 153, and the G+C content is 74.1%. The ML system evolution tree constructed based on the core proteome shows that strain 10F1B-5-1 forms an independent evolution branch in the evolution spectrum of Shewanella Figure 3 The ANI and DDH values of strain 10F1B-5-1 with Shewanella members are less than 85% and 31% respectively (Table 1), while the definition criteria of prokaryotic species are ANI value of 95-96% and DDH value of 70%.

[0035] Table 1, ANI and DDH values of strain 10F1B-5-1 with similar species

[0036]

[0037] 3, culture characteristic analysis

[0038] Strain 10F1B-5-1 was incubated at 30℃ for 2 days on TSA medium, and its colony morphology and cell morphology were observed by optical microscope (model BH 2; Olympus) and scanning electron microscope (JEM-1400; JEOL) respectively. Strain 10F1B-5-1 was inoculated in LB, ISP 2, R2A, TSA and NA medium and incubated at 30℃ for 14 days, and its growth characteristics were observed. ISP 2 medium containing different NaCl concentrations (0-10% (w / v), with an interval of 1%) was used to detect the salt tolerance range of the strain. The pH of R2A medium was adjusted to 4.0-13.0 (with an interval of 1 pH unit), and the growth pH range of the strain was observed. Strain 10F1B-5-1 was inoculated into R2A medium, and its growth at different temperatures (4, 8, 10, 15, 20, 25, 28, 30, 32, 35, 37, 40, 45℃) was observed.

[0039] The above experimental results show that the colony of strain 10F1B-5-1 is cyan and yellow, round, smooth and convex on the surface, the cell is rod-shaped, non-flagellated and non-spore forming, and the transmission electron microscope image of strain 10F1B-5-1 is shown in Figure 1 The strain grows well on LB, ISP 2, R2A, TSA and NA medium, and the growth conditions are as follows: temperature tolerance range is 10-35℃ (optimum temperature is 28-30℃), NaCl tolerance range is 0-7% (optimum content is 0%), and pH tolerance range is 6.0-9.0 (optimum pH is 7.0).

[0040] In summary, strain 10F1B-5-1 can represent a new species of Shewanella. The strain belongs to Shewanella Schumannella , and is classified and named as SchumannellaThe strain was preserved in China General Microbiological Culture Collection Center on March 7, 2025, and the preservation number was CGMCC NO. 1.16698.

[0041] Example 3, Antimicrobial application of strain 10F1B-5-1

[0042] Using one strain many compounds (OSMAC) strategy, strain 10F1B-5-1 was fermented in small scale using four different media (Table 2). About 1 cm 2 of agar containing bacteria was inoculated into 100 mL of liquid fermentation medium, and cultured at 28°C with 180 rpm shaking for 7 days. After centrifugation, the supernatant and mycelium were obtained, and the supernatant was extracted with an equal volume of ethyl acetate three times. The ethyl acetate phase was concentrated and dried to obtain the fermentation crude extract. The crude extract was dissolved in methanol to obtain a solution with a concentration of 50 mg / ml. HPLC was used to analyze the fermentation crude extract of the four media. In addition, paper disc diffusion method was used for antimicrobial activity determination. 25 μL of fermentation crude extract solution was placed on a 6 mm diameter sterile filter paper disc, and after the methanol was evaporated and dried, it was attached to the medium containing 1.5 x 10 7 CFU / ml of test bacteria, and incubated at 37°C overnight. The size of the inhibition zone was recorded.

[0043] Table 2, Fermentation media used in OSMAC strategy

[0044]

[0045] The results of antimicrobial activity detection showed that the crude extract obtained by fermentation of strain 10F1B-5-1 in PG and ISP 2 medium had antimicrobial activity against Bacillus subtilis CPCC 100029, Staphylococcus aureus ATCC 29213 and Acinetobacter baumannii ATCC 19606 (Table 3).

[0046] Table 3, Antimicrobial activity of fermentation crude extract of strain 10F1B-5-1 in different media

[0047]

[0048] Note: -, no antimicrobial activity.

[0049] Although the present application has been described in detail with general description and specific embodiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.

Claims

1. A Shewanella bacterium, characterized in that, The strain 10F1B-5-1 of the Shewanella belongs to the genus of Shewanella Schumannella , is classified as Schumannella sp., is preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC NO.1.16698 and a preservation date of March 07, 2025.

2. The S. oneidensis of claim 1, wherein, The Schumannella The 16S rRNA gene sequence of Bifidobacterium sp. is shown as SEQ ID NO.

1.

3. The use of the crude extract of Schumannella fermentum fermented in PG and ISP 2 medium according to claim 1 in the preparation of a drug for resisting Bacillus subtilis CPCC 100029.

4. The use of the crude extract of Schumannella fermentum fermented in PG and ISP 2 medium according to claim 1 in the preparation of a drug for resisting Staphylococcus aureus ATCC 29213.

5. The use of the crude extract of Schumannella fermentum fermented in PG and ISP 2 medium according to claim 1 in the preparation of a drug for resisting Acinetobacter baumannii ATCC 19606.

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