Burkholderia BK-1 and uses thereof

Abstract

The present disclosure relates to the field of microbial technology, and provides a Burkholderia sp. BK-1, which was preserved in the China Center for Type Culture Collection on September 30, 2024, and has a preservation number of CCTCC NO: M 20242135. The present disclosure also provides an application of the Burkholderia sp. BK-1 in reducing the accumulation of heavy metals in crops. The Burkholderia sp. BK-1 provided by the present disclosure not only has the ability of mercury reduction, but also has the abilities of phosphorus dissolution and siderophore production, and thus can reduce the accumulation of heavy metals in crops, for example, can reduce the accumulation of mercury in rice.

Description

Technical Field

[0001] This disclosure relates to the field of microbial technology, for example to Burkholderia BK-1 and its applications. Background Technology

[0002] Mercury is one of the most toxic heavy metal pollutants. It can travel long distances through the atmosphere and settle into terrestrial and aquatic ecosystems, posing a serious threat to the ecological environment and human health. With the rapid development of industrialization in my country, mercury pollution has become increasingly serious. Every year, 500-600 tons of mercury are released into the atmosphere through human activities, accounting for approximately 30% of global anthropogenic emissions. Surveys of heavy metal pollution in Chinese arable land over the past thirty years show that mercury is the second largest heavy metal pollutant, second only to cadmium. Mercury pollution in farmland soil leads to a decline in soil quality, productivity, and food safety. Due to its toxicity, once absorbed by plants, mercury accumulates permanently in their bodies and eventually enters the human body through the food chain, causing harm to human health.

[0003] Rice is one of my country's main food crops. In 2022, the rice planting area was 29.4501 million hectares, with a total output of 208.195 million tons. The planting area of ​​glutinous rice has remained stable at around 789,300 hectares, with a total output of about 6 million tons. Due to topography, soil parent material, and the development and utilization of mineral resources, the rate of heavy metal exceedance in farmland soil remains high. A large amount of mercury enters the paddy soil through inorganic mercury, causing mercury pollution in paddy soil. Consuming rice from mercury-contaminated areas is one of the main ways for humans to be exposed to methylmercury.

[0004] Soil contains a diverse and abundant community of metabolically active microorganisms, many of which are tolerant to heavy metals and play crucial roles in their absorption, translocation, and degradation. Recent studies have shown that microorganisms can mitigate the toxic effects of heavy metals on crops and regulate their absorption and translocation, playing a significant role in reducing heavy metal accumulation in edible parts of crops. Heavy metal-resistant growth-promoting bacteria are a type of bacteria that can stably grow under heavy metal stress and promote plant growth. Research has found that these bacteria possess multiple heavy metal resistance mechanisms, including redox reactions, intracellular transformation, and extracellular precipitation, which can reduce the availability of heavy metals in the environment and mitigate their toxic effects on plants. Furthermore, they can directly or indirectly promote plant growth and enhance plant tolerance to heavy metals through mechanisms such as the production of indoleacetic acid, siderophores, and phosphorus solubilization and nitrogen fixation. Existing studies have shown that in heavy metal-contaminated environments, inoculation with heavy metal-resistant growth-promoting bacteria can effectively promote the growth of crops such as wheat, rice, and soybeans, improve the crops' tolerance to cadmium, reduce the availability of heavy metals in the rhizosphere, and reduce the absorption of cadmium by crops.

[0005] However, current research on microbial reduction of heavy metals in crops is mostly focused on cadmium, while similar research on mercury is relatively limited. Summary of the Invention

[0006] The purpose of this disclosure is to overcome the shortcomings of the prior art and provide Burkholderia BK-1 and its application. Burkholderia BK-1 not only has the ability to reduce mercury, but also has the ability to solubilize phosphorus and produce siderophores, thus reducing the accumulation of heavy metals in crops, such as reducing the accumulation of mercury in rice.

[0007] The above objective is achieved through the following technical solution:

[0008] On the one hand, a Burkholderia sp. BK-1 is provided. The Burkholderia sp. was deposited at the China Center for Type Culture Collection on September 30, 2024, with accession number CCTCC NO:M 20242135.

[0009] On the other hand, a bacterial agent comprising Burkholderia BK-1 is provided.

[0010] On the other hand, we provide an application of Burkholderia BK-1 or the above-mentioned inoculant in reducing the accumulation of heavy metals in crops.

[0011] In some embodiments, the crop includes rice.

[0012] In some embodiments, the heavy metal includes mercury.

[0013] The beneficial effects of this disclosure are:

[0014] The Burkholderia BK-1 disclosed herein not only has the ability to reduce mercury, but also the ability to solubilize phosphorus and produce siderophores, thus reducing the accumulation of heavy metals in crops, such as reducing the accumulation of mercury in rice.

[0015] Biological Preservation

[0016] A Burkholderia sp. BK-1 disclosed herein was deposited on September 30, 2024, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO:M 20242135. The depositary address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, 430072, China. Attached Figure Description

[0017] Figure 1This is a graph illustrating the mercury resistance verification of strain BK-1 in Example 2; where, Figure 1 Figure A shows the results of mercury resistance verification of strain BK-1 at mercury concentrations of 0-20 mg / L. Figure 1 B shows the growth effect of strain BK-1 on LB solid medium at mercury concentrations of 0-20 mg / L.

[0018] Figure 2 This is the phylogenetic tree of strain BK-1 from Example 2;

[0019] Figure 3 This is a graph showing the growth capacity of strain BK-1 in Example 3 under different mercury concentrations, pH levels, and inoculum sizes; where, Figure 3 Figure A shows the growth ability of strain BK-1 at different mercury concentrations. Figure 3 Figure B shows the growth ability of strain BK-1 at different pH values. Figure 3 C shows the growth capacity of strain BK-1 at different inoculum amounts;

[0020] Figure 4 This is a diagram showing the results of the strain characteristic study of strain BK-1 in Example 4, that is, a diagram comparing the characteristics of strain BK-1 on CAS medium, nitrogen-fixing bacteria medium, inorganic phosphorus medium, organic phosphorus medium and cellulose red medium.

[0021] Figure 5 The figures show the mercury removal rate and scanning electron microscopy results of strain BK-1 in Example 5; where, Figure 5 Figure A shows the mercury removal rate results for strain BK-1. Figure 5 B is a graph showing the cell morphology of strain BK-1 at a mercury concentration of 0 mg / L (scale bar = 500 nm). Figure 5 C is a graph showing the cell morphology of strain BK-1 at a mercury concentration of 10 mg / L (scale bar = 500 nm);

[0022] Figure 6 This is a graph showing the electrophoresis results of the merA gene of strain BK-1 in Example 6;

[0023] Figure 7 This is a graph showing the results of real-time fluorescence quantitative analysis of each gene in the mer operon of strain BK-1 in Example 6.

[0024] Figure 8 The effects of strain BK-1 from Example 7 on mercury accumulation in various organs of indica rice variety N1311r and japonica rice variety WN; among which, Figure 8 A represents the effect of strain BK-1 on mercury accumulation in various organs of the indica rice variety N1311r. Figure 8 B represents the effect of strain BK-1 on mercury accumulation in various organs of the japonica rice variety WN. Detailed Implementation

[0025] The technical solutions in some embodiments of this disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments provided in this disclosure, all other embodiments obtained by those skilled in the art are within the scope of protection of this disclosure.

[0026] Example 1: Culture medium formulation

[0027] 1. LB liquid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, add water to 1L.

[0028] 2. LB solid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 15g agar, add water to 1L.

[0029] 3. Ashube medium for nitrogen-fixing bacteria: 0.2g potassium dihydrogen sulfate, 0.2g magnesium sulfate, 0.2g calcium chloride, 5.0g calcium carbonate, 10.0g mannitol, 0.1g calcium sulfate, 15.0g agar, adjust pH to 7.0±0.1, add water to 1L, autoclave at 121℃ for 15min, and set aside.

[0030] 4. Organophosphate bacteria culture medium: 10.0g glucose, 0.5g ammonium sulfate, 0.5g yeast extract, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate, 0.03g ferrous sulfate, 0.03g manganese sulfate, 1.0g lecithin, 15g agar. Adjust the pH to 7.0-7.5, add water to 1L, autoclave at 121℃ for 15min, and set aside.

[0031] 5. Inorganic phosphorus bacteria culture medium: 10g glucose, 0.5g ammonium sulfate, 0.5g yeast extract, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate, 0.03g ferrous sulfate, 0.03g manganese sulfate, 5.0g calcium sulfate, 15.0g agar. Adjust the pH to 7.0-7.5, add water to 1L, autoclave at 121℃ for 15min, and set aside.

[0032] 6. CAS detection medium: 0.0605g chromaine (CAS), 0.0729g hexadecyltrimethylammonium bromide (HDTMA), 0.002645g ferric chloride hexahydrate, 0.29525g sodium dihydrogen phosphate dihydrate, 1.2135g disodium hydrogen phosphate dodecahydrate, 0.125g ammonium chloride, 0.0375g potassium dihydrogen phosphate, 0.0625g sodium chloride, 9.0g agar. Adjust pH to 6.8±0.1, add water to 1L, autoclave at 116℃ for 30min, and set aside.

[0033] 7. Cellulose Congo Red Medium: Sodium nitrate 1.0g, disodium hydrogen phosphate 1.2g, potassium dihydrogen phosphate 0.9g, magnesium sulfate 0.5g, potassium chloride 0.5g, yeast extract powder 0.5g, acid-hydrolyzed casein 0.5g, Congo red 0.2g, cellulose powder 5.0g, agar 15.0g. Adjust pH to 7.0±0.1, add water to 1L, autoclave at 121℃ for 15min, and set aside.

[0034] Example 2: Screening and species identification of mercury-resistant strains

[0035] 1. Screening of mercury-resistant strains

[0036] Using four paddy soil samples from the vicinity of a mercury mine in Xiushan Tujia and Miao Autonomous County, Chongqing, the four soil samples were mixed thoroughly under sterile conditions. 5g of each soil sample was placed in a conical flask containing 50mL of sterile water to prepare a bacterial suspension. After shaking for 20 minutes and allowing to stand, 1mL of the supernatant was inoculated using a pipette into a solution containing 5mg / L Hg. 2+ The culture was incubated in LB liquid medium at 28°C and 150 rpm on a shaker. After the medium became turbid, 1 mL of the culture was inoculated into a solution containing 10 mg / L Hg. 2+ In LB liquid medium, repeat this process multiple times until the medium no longer becomes turbid. Take 1 mL of the turbid bacterial culture and dilute it with sterile water for 10 minutes. -1 -10 -6 Dilute and take 10 -4 -10 -6 0.1 mL of each dilution solution was inoculated onto LB solid medium and incubated at 28°C for 24 h. Single colonies with good growth were streaked 2-3 times to obtain single colonies, which were then stored at 4°C.

[0037] 2. Species identification of mercury-resistant strains

[0038] The test strain was cultured in LB liquid medium (28℃, 150 rpm) for 24 h. 2 mL of the bacterial culture was centrifuged at 4℃, 8000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected. 100 μL of TE buffer was added and mixed well, followed by 10 μL of lysozyme. The mixture was then incubated overnight at 37℃ in a constant temperature water bath. Single-cell DNA was extracted according to the Shanghai Sangon Biotech bacterial DNA extraction kit. The extracted DNA samples were stored at -20℃ for later use. PCR amplification of the conserved nucleic acid sequence of the mercury-resistant strain was performed using universal primers 27F and 1492R for the 16S rRNA gene sequence. The PCR products were sequenced by Beijing Qingke Biotechnology Co., Ltd., and the amplified sequences were compared with those obtained on the NCBI website. A phylogenetic tree was constructed using MEGA software.

[0039] 3. Experimental Results

[0040] Thirty-eight strains potentially resistant to mercury were screened using a selective enrichment and screening method. These 38 strains were then inoculated with a medium containing mercury. 2+ A mercury-resistant strain, BK-1, was screened from LB liquid medium at concentrations of 0 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, and 20 mg / L based on the growth of the strains.

[0041] The results of mercury resistance verification of strain BK-1 at mercury concentrations of 0-20 mg / L are as follows: Figure 1 As shown in Figure A; the growth effect of strain BK-1 on LB solid medium at mercury concentrations of 0-20 mg / L is as follows. Figure 1 As shown in B; the sequence of the PCR amplification product of strain BK-1 is shown in SEQ ID No. 1; the phylogenetic tree of strain BK-1 is shown in... Figure 2 As shown, the analysis indicates that strain BK-1 is in the same minimal branch as Burkholderia sp.strain (MK583567.1) and Burkholderia sp.strain (MK459497.1), and has the closest evolutionary distance. Therefore, strain BK-1 is identified as Burkholderia sp.

[0042] 4. Preservation of mercury-resistant strains

[0043] The selected mercury-resistant strain was identified as Burkholderia sp. BK-1, and deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20242135. The deposit address is China Center for Type Culture Collection (CCTCC), Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China. The deposit date is September 30, 2024.

[0044] Example 3: Optimization of growth conditions for mercury-resistant strains

[0045] 1. Experimental Methods

[0046] To determine the optimal growth conditions for the strain, a single-factor experimental method was used to analyze the growth of the strain under different mercury concentrations, pH values, and inoculum sizes. Mercury concentration gradients of 0 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, and 20 mg / L were selected; pH values ​​of 5, 6, 7, 8, and 9 were selected; and inoculum sizes of 5%, 10%, 15%, 20%, and 25% were selected. Strain BK-1 was inoculated into 50 mL of LB liquid medium according to different mercury concentrations, pH values, and inoculum sizes, with three replicates per group. The absorbance (OD) was measured after 12 hours. 600 ).

[0047] 2. Experimental Results

[0048] like Figure 3 As shown in A, strain BK-1 at 15 mg / L Hg 2+ The strain exhibited optimal growth at the specified concentration, reaching a stationary phase within 48-60 hours. Strain BK-1 showed the best growth at 20 mg / L Hg. 2+ It still grows normally at the concentration, but growth is slow. The effect of different pH values ​​on the growth of strain BK-1 in a medium containing 10 mg / L was further determined to identify the optimal growth conditions for strain BK-1 in mercury-contaminated soil. Figure 3 As shown in Figure B, strain BK-1 grows best at pH 7; therefore, strain BK-1 is suitable for neutral soil. Figure 3 As shown in Figure C, strain BK-1 exhibits the best growth when the inoculum concentration is 10%.

[0049] Example 4: Characteristic Study of Mercury-Resistant Strains

[0050] 1. Experimental Methods

[0051] Mercury-resistant strain BK-1 was streaked multiple times on LB solid medium to obtain single colonies. A small amount of bacterial cells was inoculated into Assab medium for nitrogen-fixing bacteria, organic phosphorus bacteria medium, inorganic phosphorus bacteria medium, CAS detection medium, and cellulose Congo red medium using an inoculation loop. After incubation at 28℃ for 72 hours, the colony characteristics were observed according to the characteristics of each medium to determine the functional characteristics of the mercury-resistant strain.

[0052] 2. Experimental Results

[0053] We performed qualitative analysis on other characteristics of strain BK-1, and found that strain BK-1 exhibited different characteristics under different culture medium conditions. For example... Figure 4 As shown, strain BK-1 exhibits a yellow halo in CAS medium, indicating its ability to produce siderophores; strain BK-1 shows a clear zone around its cells in organic phosphorus medium, indicating its ability to dissolve organic phosphorus; strain BK-1 also shows a clear zone around its cells in inorganic phosphorus medium, indicating its ability to dissolve inorganic phosphorus; strain BK-1 did not show a specific response in cellulose Congo red medium.

[0054] Example 5: Determination of mercury removal rate and electron microscopic observation of mercury-resistant strains

[0055] 1. Experimental Methods

[0056] Take 1 mL of bacterial culture of strain BK-1 grown under optimal growth conditions and inoculate it into LB liquid medium containing different mercury concentrations. The control group contains no mercury. 2+LB liquid medium was used. After 48 hours of incubation, the bacterial culture was collected, and the Hg concentration in the medium at different mercury concentrations was detected using an atomic fluorescence spectrometer. 2+ The residual amount of Hg is calculated using the formula. 2+ Removal rate.

[0057]

[0058] Where C represents the mercury removal rate, and C0 represents the initial Hg in the sample at 0h. 2+ Concentration, C1 represents the residual Hg in the sample after 48 hours of treatment with the strain. 2+ concentration.

[0059] Collect bacterial suspension at 10 mg / L and centrifuge at 10,000 rpm for 5 min. Discard the supernatant and wash the sample 2-3 times with 1×PBS (pH 7.0) for 15 min each time. After rinsing, centrifuge at 5,000 rpm for 3 min, discard the supernatant, add 1 mL of 2.5% glutaraldehyde solution for fixation, gently shake to mix thoroughly, resuspend the bacterial suspension, fix at 4℃ for 4 h, and then send the sample for further processing. Wash 3 times with phosphate buffer. Dehydrate stepwise with different concentrations of ethanol solution: 30%-50%-70% (30%-50%-70% dehydration at 4℃), 80%-90% (dehydration at room temperature), each dehydration stage for 15 min. 100% ethanol solution requires two dehydration steps. Then replace with isoamyl acetate solution twice, 20 min each time. Dry the bacterial cells using a critical dryer, then coat the bacterial cells with gold using a gold-plating device. Finally, observe and photograph using a field emission scanning electron microscope.

[0060] 2. Experimental Results

[0061] like Figure 5 As shown in Figure A, a comparison of the mercury removal rates of strain BK-1 on LB solid medium with mercury ion concentrations of 5 mg / L, 10 mg / L, 15 mg / L, and 20 mg / L shows that, in the presence of Hg... 2+ At a concentration of 5 mg / L, strain BK-1 exhibited the highest mercury removal rate; the mercury removal rate of strain BK-1 was within the range of Hg. 2+ At a concentration of 5 mg / L, it can still maintain above 80%, but gradually decreases as the ion concentration increases. For example... Figure 5 As shown in B-5C, in Hg 2+ When the concentration was 10 mg / L, electron microscopy revealed that the bacterial cells shrank and became concave in the presence of mercury ions.

[0062] Example 6: Extraction of bacterial RNA from strain BK-1 and real-time quantitative PCR

[0063] 1. Experimental Methods

[0064] Bacterial RNA was extracted using the column-based bacterial total RNA extraction kit from Sangon Biotech Co., Ltd., catalog number: B518655-0050. Detailed operating procedures are described in the kit's instruction manual. cDNA preparation: The reverse transcription kit from Takara Bio Inc. was used. RTMasterMix (TaKaRa, Japan) is used for reverse transcription of bacterial RNA. The specific method is as follows:

[0065] 1) Removal reaction of gDNA

[0066] The reaction system for the removal reaction is shown in the table below:

[0067]

[0068] After reacting at 37℃ for 20 min, 2.5 μL of 0.5 mmol / L EDTA was added and reacted at 80℃ for 2 min. Then, the volume was adjusted to 100 μL with RNase-free ddH2O.

[0069] 2) RT reaction

[0070] The reaction system for the RT reaction is shown in the table below:

[0071]

[0072] The reaction conditions for the RT reaction are shown in the table below:

[0073]

[0074] Real-time quantitative PCR:

[0075] Real-time quantitative PCR was performed using the SYBR Green dye method. The total reaction volume was 25 μL, consisting of 2 μL cDNA, 1 μL each of Primer F and Primer R, 12.5 μL of 2×SYBR Premix Ex Taq™ II, and 2.5 μL of ddH2O. The reaction program was: 95℃ / 30 sec, 95℃ / 5 sec, 60℃ / 30 sec, for 40 cycles. The 16S rRNA housekeeping gene was used as an internal reference gene, with 3 biological replicates and 3 or more technical replicates.

[0076] 2. Mercury reductase gene analysis

[0077] The mer operon includes genes such as merA, merB, merC, merD, merP, merR, and merT. Among the proteins encoded by these genes, those that affect Hg... 2+ The transformation is primarily driven by mercury reductase encoded by the merA gene, which transforms Hg... 2+ Restored to Hg 0Other genes encode promoter proteins and transport proteins that play auxiliary roles. Therefore, this study mainly uses the detection of the merA gene to determine whether mercury-resistant strains contain Hg. 2+ The restoration process.

[0078] like Figure 6 As shown, the merA gene was detected in strain BK-1, indicating that it has the ability to transform Hg. 2+ process.

[0079] 3. Response of key genes for mercury ion reduction in mercury-resistant strains

[0080] like Figure 7 As shown, in strain BK-1, the expression level of the merR gene was upregulated at 2h, indicating that the merR gene initiated the expression of the mer operator during this period. The expression level was downregulated at 6-12h, and then upregulated again at 18h, showing a secondary expression phenomenon. The expression level of the merC gene was continuously upregulated from 2-12h, and began to downregulated at 18h, indicating that the merC gene has a long duration of Hg(II) transport and is a key gene for Hg(II) transport. The expression level of the merT gene was the highest at 2h, and the expression level was upregulated again from 6-18h, but the upregulation rate decreased. The expression level of the merA gene began to upregulate at 12h, and then downregulated after 18h.

[0081] Therefore, the presence of the mercury reductase gene merA in strain BK-1 indicates that strain BK-1 possesses mercury reduction capabilities. Furthermore, real-time quantitative PCR results show that the genes of the mer operon are involved in Hg reduction. 2+ The sequential expression of the mer operon during the reduction process indicates that the mer operon is involved in the transformation of Hg by strain BK-1. 2+ It played an important role in the process.

[0082] Example 7: Effects of inoculating strain BK-1 on mercury accumulation in various organs of rice from different mercury-tolerant varieties.

[0083] Indica rice variety N1311r and japonica rice variety WN were selected as materials for a pot experiment. After adding 5 mg / L of exogenous mercury, strain BK-1 was inoculated to study the effect of strain BK-1 on mercury accumulation in various organs of different mercury-tolerant varieties.

[0084] The results are as follows Figure 8As shown in A-8B, in the rice roots, the total mercury contents of N1311R1 and WN were 31.97 mg / L and 28.52 mg / L respectively without inoculation. After inoculation with strain BK-1, the total mercury contents decreased to 27.92 mg / L and 19.80 mg / L respectively. In the first stem node, the total mercury contents of N1311R1 and WN were 7.46 mg / L and 5.52 mg / L respectively without inoculation. After inoculation with strain BK-1, the total mercury contents decreased to 27.92 mg / L and 19.80 mg / L respectively. The total mercury content decreased to 2.26 mg / L and 4.71 mg / L, respectively. In leaves, the total mercury content of WN was 13.03 mg / L without inoculation, but decreased to 12.07 mg / L after inoculation with strain BK-1. In grains, the total mercury content of N1311R1 and WN was 0.04 mg / L and 0.03 mg / L, respectively without inoculation, but decreased to 0.03 mg / L and 0.02 mg / L, respectively, after inoculation with strain BK-1.

[0085] Therefore, it can be seen that inoculating with strain BK-1 can reduce mercury accumulation in various organs of rice varieties with different mercury tolerance.

[0086] In summary, the strain BK-1 provided in this disclosure not only has the ability to reduce mercury, but also the ability to solubilize phosphorus and produce siderophores, thus enabling it to reduce the accumulation of heavy metals (e.g., mercury) in crops (e.g., rice).

Claims

1. A Burkholderia sp. BK-1 bacterium, characterized in that, The Burkholderia species was deposited at the China Center for Type Culture Collection on September 30, 2024, with accession number CCTCC NO:M 20242135.

2. A bacterial agent comprising Burkholderia BK-1 as described in claim 1.

3. The application of Burkholderia BK-1 as described in claim 1 or the bacterial agent as described in claim 2 in reducing mercury accumulation in rice.

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