Saccharobacterium tumefaciens and application thereof

Abstract

The invention discloses a strain of Saccharobacterium tumefaciens and application thereof, and belongs to the field of microorganisms. According to the invention, a strain is separated and purified from leaves of Chuxiong saiai plant in Yunnan, and cell morphology observation, physiological and biochemical characteristic detection, cell chemical composition detection, and sequencing and comparison of genome DNA prove that the strain is a new species of the genus beach bacillus, and is named as beach bacillus saiai, and the strain is a new species of the genus beach bacillus and is named as beach bacillus saiai. Experiments and genome analysis prove that the strain has phosphorus solubilizing potential and can degrade PO4 < 3 + > generated by organophosphorus, so that the strain obtained by screening can be used for preparing reagents for degrading organophosphorus.

Description

Technical Field

[0001] This invention relates to a strain of *Bacillus spp.* and its applications, belonging to the field of microbiology. Background Technology

[0002] genus *Bacillus* ( Arenivirga It belongs to the domain Bacteria, phylum Actinobacteria. Actinomycetota Actinomycetes ( Actinomycetes )-Micrococcales ( Micrococcales )- Microbacteriaceae ( Microbacteriaceae In 2017, Moriyuki Hamada et al. isolated a Gram-positive actinomycete from sand samples of mangrove tidal flats in Okinawa, Japan. Through polyphasic taxonomy experiments, they determined its taxonomic position and established a new genus within the Microbacteriaceae family—*Bacillus*. Arenivirga flava As the type species (Hamada, M., Saitou, S., & Tamura, T. (2017). Arenivirga flava gen. nov., sp. nov., a new member of the family Microbacteriaceae isolated from a mangrove tidal flat. International journal of systematic and evolutionary microbiology, 67(9), 3318–3322. https: / / doi.org / 10.1099 / ijsem.0.002113), this genus currently contains only one valid published species.

[0003] This genus of strains consists of aerobic, heterotrophic, Gram-positive short rod-shaped bacteria that are non-motile. The main respiratory quinone component is MK-11, and the main fatty acid component is anteiso-C. 15:0 anteiso-C 17:0 and iso-C 16:0 The main polar lipid components are PG (phosphatidylglycerol), DPG (diphosphatidylglycerol), and GLs (glycolipids). The G+C content of the genomic DNA is 72.2%. Summary of the Invention

[0004] One objective of this invention is to provide a strain of *Bacillus basilata*, which was deposited on June 27, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31114 and taxonomic name [not specified]. Arenivirgasida .

[0005] The Arenivirgasida (Bacillus spp.) is a chemoheterotrophic aerobic bacterium that is Gram-positive. The cells are short rod-shaped, non-motile, and do not produce endospores. On PYG medium, it forms light orange circular colonies with a dry surface and neat edges. The maximum colony diameter is 0.8 mm. The optimal growth conditions are pH 7.0, NaCl concentration of 0-4%, and temperature of 28℃.

[0006] The second objective of this invention is to provide a bacterial agent prepared using Bacillus thuringiensis.

[0007] The third objective of this invention is to provide an application of *Bacillus spp.* or a bacterial agent prepared from *Bacillus spp.* in the preparation of organophosphate degradation reagents.

[0008] The active ingredient of the degrading organophosphorus reagent is *Bacillus basilica* or a bacterial agent prepared from *Bacillus basilica*.

[0009] The beneficial effects of this invention are: (1) Based on the comprehensive phenotypic and genotypic data and phylogenetic analysis results, the *Bacillus basilica* discovered in this invention represents a new species of the genus *Bacillus*. *Bacillus basilica* is a new species of bacteria discovered from the leaves of the plant *Bacillus basilica*.

[0010] (2) It was also found that *Bacillus basilica* has the ability to degrade organophosphates, therefore the strain described in this invention can be used to prepare reagents for degrading organophosphates.

[0011] Deposition description Category Naming: Arenivirgasida ; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures; The abbreviation for the depository institution is CGMCC. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: June 27, 2024; Registered with the China National Collection Center (CGMCC) No. 31114. Attached Figure Description

[0012] Figure 1 A colony photograph of strain YZ-1 grown on PYG agar solid medium at 28°C for 48 hours.

[0013] Figure 2 Specific implementation method for detecting the polar lipid components of strain YZ-1.

[0014] Figure 3A phylogenetic tree constructed based on the 16S rRNA gene sequence of strain YZ-1 shows the phylogenetic position of strain YZ-1.

[0015] Figure 4 Images showing the phosphate-solubilizing ability of strain YZ-1. Detailed Implementation

[0016] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0017] Example 1 Isolation, screening and identification of strains The strain of this invention was collected from Badusan (a traditional Chinese medicine formula) in Chuxiong, Yunnan. Sida szechuensis It was isolated and purified from plant leaves.

[0018] The specific separation operation is as follows: (1) Rinse the collected plant samples with running water to remove impurities attached to the surface of the plant leaves.

[0019] (2) Next, use deionized water for ultrasonic cleaning (40kHz, 3min / time), clean 3 times, and change the deionized water each time to further remove impurity particles that are tightly bound to the plant. Place the cleaned plant sample in a ventilated and cool place to allow the surface moisture to evaporate and dry.

[0020] (3) The dried plants were treated with the surface disinfection method used for the isolation of endophytic bacteria in mangrove plants (Wei Yuzhen, Zhang Yuqin, Zhao Lili, et al. Isolation, screening and preliminary identification of endophytic actinomycetes in mangrove forests in Shankou, Guangxi [J]. Bulletin of Microbiology, 2010(6):6.). They were soaked in sodium hypochlorite (containing 5% available chlorine) for 5 min, washed 3 times with sterile water, soaked in 75% alcohol for 3 min, washed 3 times with sterile water, and dried. The disinfected plant samples were placed in sterile petri dishes lined with filter paper and dried in a sterile and ventilated environment. The plant leaves were then crushed with a pulverizer for later use.

[0021] (4) Separation method: The treated leaf fragments of the detoxifying agent plant were evenly dispersed and fixed onto PYG isolation medium (PYG isolation medium formula: peptone 3g·L). -1 5g / L of yeast extract -1 10 g / L of glycerin -1 betaine 1.25 g·L -1 Sodium pyruvate 1.25 g·L -1 15g / L agar -1 Incubate on pH 7 at 28°C for 3 weeks. Select single colonies with good growth and different morphological characteristics and culture them on PYG agar solid medium (formula: 3g / L peptone).-1 5g / L of yeast extract -1 10 g / L of glycerin -1 betaine 1.25 g·L -1 Sodium pyruvate 1.25 g·L -1 15g / L agar -1 The culture was repeatedly purified and cultured on pH 7 plates to obtain pure cultures for subsequent research. The obtained pure strains were preserved in liquid nitrogen at ultra-low temperature and frozen at -80°C using 20% ​​(v / v) glycerol as a protectant.

[0022] Obtain a strain that is the closest control bacterium Arenivirga flava A potential new species closely related to Hls16-32T, strain numbered YZ-1.

[0023] Example 2 Cell morphology observation and physiological and biochemical characteristics detection of strain YZ-1 Morphological and physiological / biochemical characteristics were tested using PYG medium. The main formulation of the PYG agar solid medium was the same as in Example 1. Different growth salt concentrations of PYG agar solid medium were prepared by adjusting the mass of NaCl in the medium. Twelve different growth salt concentrations of PYG agar solid medium were prepared, with the salt concentrations in the PYG agar solid medium being: 0 g / 100 ml (0%), 1 g / 100 ml (1%), 2 g / 100 ml (2%), 3 g / 100 ml (3%), 4 g / 100 ml (1%), and 2 g / 100 ml (2%). 00ml (4%), 5g / 100ml (5%), 6g / 100ml (6%), 7g / 100ml (7%), 8g / 100ml (8%), 9g / 100ml (9%), 10g / 100ml (10%), 15g / 100ml (15%); Eight different pH media were obtained by adjusting the pH of PYG agar solid medium (the media were prepared at the optimal salt concentration), and the pH of the PYG agar solid medium was 4, 5, 6, 7, 8, 9, 10, 11.

[0024] Physiological and biochemical characteristics of the strains were determined using API 50CH (bioMérieux, France, catalog number 50400), API ZYM (bioMérieux, France, catalog number 25200), and BiOLOG GEN III (BIOLOG, USA, catalog number 1030) carbon source detection kits. Other physiological characteristics of the strains, including Gram staining properties, oxygen requirements, catalase activity, oxidase activity, gelatin hydrolysis activity, starch hydrolysis activity, and cellulose hydrolysis activity, were mainly determined according to the "Handbook of Actinomycete Systematics" (Xu L H. Actinomycete systematics: principles, methods and practices [M]. Beijing: Science Press, 2007, 93-108.).

[0025] The identification results showed that strain YZ-1 is a chemoheterotrophic aerobic bacterium, Gram-positive, with short rod-shaped cells, non-motile, and does not produce endospores. It forms light orange, round colonies on PYG agar solid medium, with a dry colony surface, neat edges, and a maximum colony diameter of 0.8 mm. Figure 1 It can grow in a pH range of 6.0-10.0, a NaCl concentration range of 0%-7.0%, and a temperature range of 10-40℃; the optimal growth conditions are pH 7.0, NaCl concentration of 0-4%, and 28℃. It can grow on ISP (International Streptomyces Research Program) media. It can ferment the following carbon sources to produce acids: glycerol, D-arabinose, D-xylose, L-xylose, β-methyl-D-xylose, glucose, sorbitol, rhamnose, inositol, α-methyl-D-mannose, α-methyl-D-glucoside, arbutin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, xylitol, gentiobiose, D-minobiose, D-lythose, D-rockose, L-rockose, D-arabinitol, L- It contains arabinitol, gluconate, and 2-keto-gluconate; it possesses alkaline phosphatase, lipoesterase, leucine aromatic aminoaminase, valine aromatic aminoaminase, cystine aromatic aminoaminase, trypsin, acid phosphatase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-glucosaminease, β-fucoside enzyme, esterase, and lipase activities, but lacks β-uronic acid glycoside and naphthol-phosphohydrolase activities. It is negative for aescin hydrolysis, hydrogen sulfide production, starch hydrolysis, gelatin liquefaction, oxidase (API), VP test, and methyl red test, but positive for nitrate reduction, catalase, and urea hydrolysis.

[0026] Example 3 Detection of cytochemical components of strain YZ-1 The cytochemical components of strain YZ-1, including fatty acids, quinone types, and polar lipids, were detected by gas chromatography (GC), high-performance liquid chromatography (HPLC), and thin-layer chromatography (TLC). (Sasser M. Identification of bacteria by gasghromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE:MIDI inc; 1990. Minnikin DE, O'Donnell AG, Goodfellow M, Alderson G, Athalye Met al. An integrated procedure for the extraction of bacterial isoprenoidquinones and polar lipids.) J Microbiol Methods 1984;2:233–241.). The fatty acid composition of strain YZ-13 is shown in Table 1. The results show that the main fatty acid of strain YZ-1 is antesio -C 15 : 0 , iso -C 16 : 0 and anti so -C 17:0 In strain YZ-1, the dominant methylnaphthoquinone is MK-11; the polar lipid components include phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), some unknown glycolipids (GLs), phosphatidylinositol (PI), and a polar lipid (PL) with an unknown structure containing phospholipid groups, such as... Figure 2 As shown.

[0027] Table 1. Fatty acid composition of strain YZ-1 Example 4 Genomic DNA was extracted from strain YZ-1 of this invention and sequenced. The 16S rRNA gene sequence (as shown in SEQ ID NO: 1) was then compared online in an internationally authoritative bacterial taxonomy database (http: / / www.ezbiocloud.net / ) (Kim OS, Cho YJ, Lee K, et al . 2012,Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol(62:716-721). The results showed that the 16S rRNA gene sequence of the strain YZ-1 of this invention showed high similarity to species of the Microbacteriaceae family, among which it was the only species of the genus *Cladosporium*. Arenivirga flava The highest similarity was 97.79% (see Table 2). This value is significantly lower than the threshold of 98.65% for distinguishing different bacterial species (Stackebrandt E, Ebers J (2006). Taxonomic parameters revisited: tarnished gold standards). ​ (33, 152-155.); Based on this, it is inferred that strain YZ-1 is a new species member of the genus *Bacillus*.

[0028] Table 2 Information on closely related bacteria of strain YZ-1 A phylogenetic tree was constructed by selecting strains within the Microbacteriaceae family that showed high similarity to strain YZ-1's 16S rRNA gene sequence. ​ In the phylogenetic tree based on the 16S rRNA gene sequence, strain YZ-1 is related to the type strain of the only species in the genus *Cladosporium*. ​ Hls16-32T clustered on the same evolutionary branch. This confirmed that strain YZ-1 is a novel species within the genus *Cladosporium*.

[0029] To further clarify the phylogenetic position of strain YZ-1, the whole genome sequence of strain YZ-1 was compared and calculated with closely related control strains on EZbiocloud. ​ The average nucleotide similarity (ANI value) of the whole genome sequence of Hls16-32T, strain YZ-1 and ​ The ANI value of Hls16-32T is 83.1%, which is lower than the ANI value (95~96%) used to classify the two gene species (Yoon SH, Ha SM, Lim J, Kwon S, Chun J. A large-scale evaluation of algorithms to calculate average nucleotide identity). ​ ​ 2017;110:1281-1286.).

[0030] Based on the above results, the 16S rRNA gene sequence of strain YZ-1 (as shown in SEQ ID NO: 1) and its phylogenetic analysis show that strain YZ-1 is a member of the genus *Cladosporium*. The cytochemical components of strain YZ-1, including its dominant fatty acid components, major respiratory quinones, and polar lipid components, support its classification within the genus *Cladosporium*. Furthermore, strain YZ-1 and... ​ The 16S rRNA gene similarity of Hls16-32T was 97.79%, and the intergenomic ANI value was 83.1%. These values ​​are far below the species threshold, indicating that strain YZ-1 is a species distinct from other strains within the genus *Bacillus*. ​ A new species of Hls16-32T; strain YZ-1 and ​ Hls16-32T strains are distinguishable from each other in terms of phenotypic, physiological, biochemical, and cytochemical characteristics (such as the specific composition of fatty acids and polar lipids), supporting the fact that strain YZ-1 represents a new species of the genus *Bacillus*, named *Bacillus basilata*. ​ ).

[0031] Example 5 Using lecithin as the sole phosphorus source in a solid culture medium, the alkaline phosphatase activity of bacterial strains was assessed through plate screening. The specific steps are as follows: Prepare an organic phosphorus solid screening medium (3 g / L glucose). -1 Ammonium sulfate 0.5 g·L -1 Sodium chloride 0.3 g·L -1 Potassium chloride 0.3 g·L -1 Ferrous sulfate heptahydrate 0.03 g·L -1 0.03 g·L⁻¹ manganese sulfate heptahydrate -1 Calcium carbonate 5g·L -1 Lecithin 1g·L -1 15g / L agar -1 The culture medium (pH 7) was autoclaved at 115°C for 30 min. Subsequently, single colonies of strain YZ-1 in the logarithmic growth phase were picked and inoculated onto organophosphorus solid selection medium. After incubation at 28°C for 4 days, the bacterial colony diameter (d) and phosphate-solubilizing zone diameter (D) were measured, and the solubility index (D / d) was calculated.

[0032] Experimental results show that ( ​ The weakly positive control bacteria in the figure are ​ CPCC203993T, the negative control bacteria is ​ CPCC 204279T. The strain YZ-1 of this invention can produce phosphate-solubilizing zones on organophosphorus solid selection medium, with a solubility index of 3.79.

[0033] Simultaneously, liquid screening of alkaline phosphatase activity was performed on strain YZ-1. Strains in the logarithmic growth phase were inoculated into 20 mL of organic phosphorus liquid medium (3 g / L glucose). -1 Ammonium sulfate 0.5 g·L -1 Sodium chloride 0.3 g·L -1 Potassium chloride 0.3 g·L -1 Ferrous sulfate heptahydrate 0.03 g·L -1 0.03 g·L⁻¹ manganese sulfate heptahydrate -1 Calcium carbonate 5g·L -1 Lecithin 1g·L -1 In this study, a blank culture medium without inoculation was used as a control group. After culturing at 28℃ for 14 days, the supernatant was centrifuged and the concentration of organic phosphorus in the culture medium was measured. The blank group was used as a control, and the PO4 produced by the strain in degrading organic phosphorus was calculated. 3+ Concentration change (mg / L) (using ΔPO4) 3+ (Indicated). Based on the standard curve prepared from the phosphate standard solution, M0 represents the PO4 detected in the blank control group. 3 + Concentration, M1 is the PO4 detected in the experimental group. 3+ concentration.

[0034] △PO4 3+ Concentration = M1 - M0 Calculations showed that strain YZ-1 of this invention possesses alkaline phosphatase activity, which degrades ΔPO4 produced by organophosphates. 3+ The concentration was 0.48 mg / L.

[0035] In addition, the genome data of strain YZ-1 was compared with the eggNOG database (http: / / eggnog.embl.de / ) and the KEGG database (http: / / kobas.cbi.pku.edu.cn / home.do) using Diamond software, with an E value lower than e -5 The alkaline phosphatase A (pho A) and alkaline phosphatase E (pho E) genes were found in the genome of strain YZ-1.

[0036] Genomic analysis and phenotypic experiments fully confirmed that strain YZ-1 has phosphate-solubilizing potential.

Claims

1. A strain of *Bacillus spp.* (a type of bacteria) Arenivirgasida ), characterized by: The accession number is: CGMCCNo.31114.

2. A microbial agent, characterized in that: The bacterial agent contains the *Bacillus spp.* as described in claim 1.

3. The application of the *Bacillus spp.* of claim 1 or the bacterial agent of claim 2 in the preparation of organophosphate degradation reagents.

4. A reagent for degrading organophosphorus compounds, characterized in that: The active ingredient of the reagent is *Bacillus spp.* or the bacterial agent described in claim 2.

5. The use of the *Bacillus sphaeroides* of claim 1 in the preparation of the bacterial agent of claim 2.

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