Method for promoting growth of dopamine by using bacteria and application of dopamine in regulation and control of physiological properties of microorganisms

Abstract

The invention discloses application of bacterial dopamine and a synthesis method for improving the yield of the bacterial dopamine, and particularly relates to application of dopamine in regulating and controlling physiological properties of microorganisms, including application in promoting growth of bacterial strains, promoting formation of bacterial biofilms, improving the motility of bacteria and enhancing chemotaxis of the bacterial strains. The invention also discloses a method for improving the synthesis of dopamine in bacteria. The method comprises the following steps: screening bacteria, preparing a solution, preparing a culture medium, inoculating bacteria, detecting and the like. 10 mmol / L of a tyrosine solution is added into an LB and / or MM culture medium, the pH value is adjusted to 7.2-7.4, the synthesis amount of bacterial dopamine is increased, Cy3-labeled DA (Cy3-DA) is successfully synthesized through synthesized DA, the compound and E.coli ATCC 11303 are incubated, a visual basis is provided for studying the action mechanism of DA in bacteria, and the application prospect is broad. And a foundation is laid for deeply exploring a molecular mechanism and a biological effect of interaction between DA and bacteria.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for promoting the production of dopamine using bacteria and the application of dopamine in regulating the physiological characteristics of microorganisms. Background Art

[0002] Thirty years of research in the field of microbial endocrinology has shown that there are complex interactions between bacteria and their hosts, in which Cas (catecholamines) play a key messenger role, promoting information transfer between cells. Studies have shown that CAs have significant effects on bacterial growth, motility, adhesion, and virulence. However, these effects mainly depend on the composition of the culture medium and the density of the inoculum, especially in the minimum salt medium (i.e., serum SAPI) that simulates malnutrition and iron restriction conditions. In the human microbiome, the response of microorganisms to a series of neuroendocrine hormones is increasingly considered to play a key role in disease pathogenesis and homeostasis maintenance, but there have been few reports in this area in recent years.

[0003] CAs are mainly found in humans and animals, and they act as neurotransmitters and hormones to regulate the physiological activities of the body. When they were discovered in plants, the relationship between microorganisms and CAs was also established. CAs can promote induction and communication between organisms, and play the role of messengers between and within different species. Among microorganisms, they were first discovered in intestinal microorganisms. The intestine contains many nerve cells, and their number is comparable to that of neurons in the spinal cord. This neural network is called the enteric nervous system (ENS), which plays a key role in regulating the intestinal environment. More than half of the dopamine (DA) in the human body is produced by the ENS, which affects the bacteria in the intestine and is crucial to maintaining the balance of the intestinal flora.

[0004] Current studies have shown that microorganisms can also synthesize CAs such as DA. For example, some enterococci can synthesize DA. The synthesized DA is mainly transported to the host's brain through the blood for use by the host to regulate the central nervous system. This connection between the brain and the intestine is also called the brain-gut axis. The intestinal microbiota is an important variable in controlling peripheral DA levels. From an evolutionary perspective, microorganisms preceded vertebrates and have long evolved mechanisms for synthesizing and recognizing hormones. At present, there is still a lack of research on how microorganisms synthesize DA and what effect the produced DA has on the microorganisms themselves. It is undoubtedly of great significance to use this as a starting point to explore the effects of DA on the growth and reproduction of microorganisms and the mode of action of DA. Summary of the invention

[0005] The present invention studies the application of dopamine in regulating the physiological characteristics of microorganisms. First, the DA production of common bacteria in the natural environment and the laboratory was detected, and the intracellular DA levels of five strains were quantified by ELISA. E. coli ATCC11303 was selected as a further research object. Through genome analysis, the relevant DA synthesis genes were revealed, and the synthesis pathway of DA in E. coli ATCC11303 was preliminarily clarified. In addition, a series of experiments were conducted to explore the effect of DA on the physiological characteristics of E. coli ATCC11303, and Cy3 fluorescent labeling and laser confocal microscopy were used to study the transport pathway of DA in bacteria. Using 4D-DIA proteomics technology, it was further clarified how DA affects the physiological and biochemical characteristics of the E. coli ATCC11303 strain. The specific operations are as follows:

[0006] First, the DA production of common bacteria in the natural environment and laboratory was tested. The dopamine (DA) detection kit provided by Shanghai Jingkang Biotechnology was used to determine the DA content by double antibody sandwich enzyme-linked immunosorbent assay (ELISA).

[0007] Table 1 Intracellular dopamine concentrations of dopamine-producing bacteria and their strains

[0008]

[0009] Furthermore, based on the above research, by adding tyrosine to the culture medium, the addition of L-tyrosine / D-tyrosine to the culture medium of Escherichia coli ATCC11303 was studied respectively, the changes in intracellular and extracellular dopamine concentrations of the strain were detected, and the effect of tyrosine on the intracellular and extracellular dopamine concentrations of the E. coli ATCC11303 strain was explored.

[0010] Table 2 Effect of tyrosine on intracellular and extracellular dopamine concentrations in E. coli ATCC11303 strain

[0011]

[0012]

[0013] The above studies show that adding +10mmol / L -tyrosine or D-tyrosine to LB / MM medium can significantly improve the intracellular and extracellular DA synthesis capacity of the strain.

[0014] Then, by studying the effect of dopamine on the growth of E. coli ATCC 11303, biofilm quantitative analysis, scanning electron microscopy preparation, strain motility experiment and chemotaxis experiment were carried out based on four indicators: biofilm formation amount, biofilm morphology, motility and chemotaxis, to deeply explore the effect of DA on the physiological characteristics of E. coli ATCC 11303 strain.

[0015] Furthermore, by amide labeling method, DA with amine group was combined with Cy3 containing carboxyl group to synthesize Cy3-DA labeled with Cy3, and its excitation light and emission light wavelengths were determined to be 546nm and 564nm respectively, and its molecular weight was 627. This compound was incubated with E.coli ATCC 11303.

[0016] Furthermore, the effect of DA on the protein expression level of E. coli ATCC 11303 was deeply analyzed with the help of 4D-DIA proteomics technology, and 22,091 peptides and 3,048 proteins were identified, of which 138 proteins showed significant changes between the DA treatment group and the control group, including 60 upregulated and 78 downregulated proteins.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The first systematic study of the interaction between DA and bacteria, preliminarily clarifying the transport pathway of DA in bacteria;

[0019] (2) With DA as the research target, the DA production of common bacteria in the natural environment and laboratory (derived from the environment and human body) was tested. It was found that the ability of bacteria to biosynthesize DA varied among species. Enzyme-linked immunosorbent assay (ELISA) was used to screen DA-producing strains. The levels of DA synthesis in different strains were as follows: E. faecalis YN771>E. coli ATCC11303>E. coli BW25113>B. cereus MYB41-22>S. marcescens KMR-3. In addition, DA concentration was affected by the culture environment. In particular, in the nutrient-poor MM culture medium, DA production was generally higher than that in the nutrient-rich LB culture medium. It was also related to the level of L-tyrosine in the culture medium, further confirming that E. coli ATCC11303 had a DA synthesis pathway similar to that of animals and plants, and it was observed that bacteria could release endogenously synthesized DA into the external environment.

[0020] (3) The effect of DA on the physiological characteristics of E. coli ATCC 11303 strain was investigated, and it was shown that the growth-promoting effect of DA was most obvious in MM medium supplemented with 30% serum, and 50 μmol / L DA had the best effect, and the maximum turbidity value was 1.55 times higher than that of the control group. However, the growth-promoting effect of DA could be offset by 10 μmol / L chlorpromazine, a receptor antagonist, indicating that the promoting effect of DA may be mediated by specific receptors. 200 μmol / L DA treatment significantly increased the biofilm thickness to 1.5 times the original thickness. This effect was mainly achieved by promoting bacterial proliferation and the increase of extracellular secretions. 50 μmol / L DA significantly enhanced the motility of the strain, and the diameter of the motility zone increased by about 2 mm compared with the control group, which could be neutralized by 100 μmol / L chlorpromazine. 50 μmol / L DA significantly promoted the chemotaxis of bacteria, increasing the number of bacteria to about 2.75 times that of the control group, while chlorpromazine did not significantly affect the chemotaxis induced by DA.

[0021] (4) Cy3-labeled DA can penetrate the bacterial cell membrane and enter the cell, which can be used to explore the action site of exogenous DA, determine its transport pathway, and ultimately determine whether it acts inside or outside the cell.

[0022] (5) With the help of 4D-DIA proteomics, we explored the main proteins that affect the physiological characteristics of E. coli ATCC 11303 by DA, and performed subcellular localization of the differentially expressed proteins. Among them, 91.3% of the proteins were located in the cytoplasm, 7.25% in the plasma membrane and 1.45% outside the cell, revealing that DA mainly acts inside the cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Intracellular dopamine concentration of mixed bacteria in natural environment (mean±SD, n=3).

[0024] Figure 2 Dopamine concentration in the screened dopamine-producing strains (mean±SD, n=3)

[0025] Among them, A is the intracellular dopamine concentration of E. faecalis YN771 strain; B is the intracellular dopamine concentration of E. coli ATCC11303 strain; C is the intracellular dopamine concentration of E. coli BW25113 strain; D is the intracellular dopamine concentration of S. marcescens KMR-3 strain; E is the intracellular dopamine concentration of B. cereus MYB41-22 strain.

[0026] Figure 3 Intracellular and extracellular dopamine concentrations in E. coli ATCC11303 strain after addition of L-tyrosine (mean±SD, n=3).

[0027] Figure 4 Intracellular and extracellular dopamine concentrations in E. coli ATCC11303 strain after addition of D-tyrosine (mean±SD, n=3).

[0028] Figure 5 Growth curve of E.coli ATCC 11303 strain in LB medium, Note: A: 1nmol / L, B: 1μmol / L.

[0029] Figure 6 Growth curve of E. coli ATCC 11303 strain in MM medium;

[0030] Among them: A: 1nmol / L, B: 1μmol / L, C: 1mmol / L, D: μmol / L after supplementing Fe2+ in MM culture medium.

[0031] Figure 7 Growth curve of E.coli ATCC 11303 strain affected by different concentrations of DA. Note: MM medium.

[0032] Figure 8 Growth curve of E. coli ATCC 11303 strain under different conditions;

[0033] A is the growth curve of E. coli ATCC 11303 strain affected by different concentrations of DA in the presence of serum, and B is the effect of dopamine and dopamine receptor antagonists on the growth of E. coli ATCC 11303 strain.

[0034] Fig. 9 The results of the study on the effect of dopamine on the biofilm formation of E. coli ATCC 11303 strain;

[0035] A represents the effect of dopamine on the biofilm formation of E. coli ATCC 11303 strain; B represents the effect of dopamine and dopamine receptor antagonists on the biofilm formation of E. coli ATCC 11303 strain.

[0036] Fig.10 This is a scanning electron micrograph of E. coli ATCC 11303 strain.

[0037] Among them: Pictures A and B are control groups, C and D are 200μmol / L DA treatment groups, E and F are 200μmol / L DA + 100μmol / L chlorpromazine treatment groups, G and H are 100μmol / L chlorpromazine treatment groups; A, C, E, G are 5000×, B, D, F, H are 20000×

[0038] Fig.11 Soft agar motility plate for E. coli ATCC 11303 strain;

[0039] Among them: A is the control group, B is the 50 μmol / L DA treatment group, C is the 50 μmol / L DA + 100 μmol / L chlorpromazine treatment group, and D is the 100 μmol / L chlorpromazine treatment group.

[0040] Fig.12 The effects of dopamine and dopamine receptor antagonists on the motility of E.coli ATCC 11303 strain.

[0041] Fig.13 The effect of dopamine and dopamine receptor antagonists on the chemotaxis of E. coli ATCC 11303 strain.

[0042] Fig.14 This is a laser confocal image of 5h E.coli ATCC 11303 bacterial solution after incubation with Cy3-dopamine;

[0043] Fig.15 This is a laser confocal image of 12h E.coli ATCC 11303 bacterial solution incubated with Cy3-dopamine;

[0044] Fig.16 This is the polyacrylamide gel electrophoresis of total protein of E.coli ATCC11303;

[0045] Fig.17 Protein infographic.

[0046] Fig.18 The molecular weight distribution of proteins.

[0047] Fig.19 It is the Venn diagram between samples.

[0048] Fig. 20 This is the correlation heat map between samples.

[0049] Fig.21 Cluster analysis of proteins among different samples.

[0050] Fig. 22 The figure is a statistical diagram of differentially expressed proteins.

[0051] Fig.23 GO functional annotations for differentially expressed proteins.

[0052] Fig.24 KEGG functional annotations for differentially expressed proteins.

[0053] Fig.25 EggNOG functional annotation of differentially expressed proteins.

[0054] Fig.26 Pfam functional annotation for differentially expressed proteins. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following invention, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0056] Example 1: Screening and synthesis of dopamine-producing bacteria

[0057] 1. Strain screening: The dopamine (DA) detection kit provided by Shanghai Jingkang Biotechnology was used to determine the DA content by double antibody sandwich enzyme-linked immunosorbent assay (ELISA), and five dopamine-producing bacteria were screened out, including Enterococcus faecalis YN77 (isolated from tooth root canal), Escherichia coli ATCC11303 (purchased from American Type Culture Collection), Escherichiacoli BW25113 (from the Institute of Microbiology, Yunnan University), Serratia marcescens KMR-3 (isolated from sewage), and Bacillus cereus MYB41-22 (isolated from Mingyong Glacier). Figure 2 ).

[0058] 2. Prepare the culture medium. First, prepare the tyrosine solution. Weigh L-tyrosine or D-tyrosine, dissolve it in ultrapure water to prepare the tyrosine solution. Filter it through a filter membrane to sterilize it, and seal it in a sterile screw-capped bottle for later use. Then, add 0.1% tyrosine solution to LB and / or MM culture medium to prepare it.

[0059] 3. Inoculate bacteria to produce dopamine. The selected strain is prepared as a strain suspension and inoculated into the culture medium in step 2 for cultivation. The dopamine content is detected after 6-24 hours of cultivation. The specific operation is as follows (taking the Escherichiacoli ATCC11303 strain with high dopamine production as an example):

[0060] 1. Activation of strains

[0061] (1) Activation of E. faecalis YN771: Take a tube of E. faecalis YN771 culture stored at -80°C, and gently scrape a small amount of colonies with a burned inoculation loop in a clean bench that has been sterilized by ultraviolet for 15 minutes. Use the "three-line method" to inoculate bacteria on a BHI plate. After labeling, place the plate in a 37°C incubator and culture it for 3-5 consecutive times to complete the activation of the strain.

[0062] (2) Activation of E. coli ATCC11303, E. coli BW25113, S. marcescens KMR-3, and B. cereus MYB41-22: The operation was the same as above, but LB solid culture medium was used for the activation plates of these four bacterial strains. The culture temperature of E. coli ATCC11303 and E. coli BW25113 was 37°C, and the culture temperature of S. marcescens KMR-3 and B. cereus MYB41-22 was 28°C. The culture temperature of all strains was selected according to their optimal growth temperature.

[0063] 2. Strain culture

[0064] (1) Inoculation: After the strain is activated, store it at 4°C for later use. Use a sterile pipette tip to pick up the bacteria and mix them evenly in a test tube containing 5 ml of LB / MM / BHI medium.

[0065] (2) Cultivation: Place the test tube in a 28°C / 37°C constant temperature shaker with shaking at 180 rpm until the OD 600 ≈0.8~1.0, for standby use.

[0066] Table 1 Culture conditions of each strain

[0067]

[0068] 3. Inoculate the activated strain into the culture medium containing the tyrosine solution prepared in step 2 and culture for 6-24 hours, and detect the concentration of dopamine in the bacterial culture.

[0069] The dopamine (DA) detection kit provided by Shanghai Jingkang Biotechnology was used to determine the DA content by double antibody sandwich enzyme-linked immunosorbent assay (ELISA). First, the ELISA microplate was coated with a specific microbial DA antibody to form an immobilized antibody layer. The DA in the sample will bind to the DA antibody labeled with horseradish peroxidase (HRP) to form a complex. After a series of incubation and washing steps, TMB was used as a substrate for color development, which reacted with HRP to show blue and turned yellow in the presence of acid. The DA concentration is positively correlated with the color depth of the solution. The absorbance value was measured at a wavelength of 450nm to estimate the DA concentration in the sample (the detection range of the kit: 4.78ng / L~200ng / L and 0.4nmol / L~25nmol / L).

[0070] The results are as follows Figure 3As shown in the figure, after adding 0.1% 10mmol / L L-tyrosine to the culture medium, it was found that the intracellular and extracellular DA concentrations increased at different time points in two different culture media (LB, MM), among which the intracellular changes were more obvious than the extracellular changes, and were more significant in the LB culture environment. For details, see Table 2. According to the preliminary experimental results, Escherichia coli may have used exogenously added L-tyrosine to synthesize DA.

[0071] The results are as follows Figure 4 As shown in the figure, after supplementing the culture medium with 0.1% 10mmol / L D-tyrosine, it was found that the intracellular and extracellular DA concentrations increased at different time points in the two different culture media, but its effect was not as obvious as that of L-tyrosine. In general, there was no significant difference in DA concentration between the 0.1% tyrosine treatment group and the control group. For details, see Table 2. It is preliminarily believed that bacteria may have related isomerases that can convert D-tyrosine into L-tyrosine for the synthesis of CAs, which further verifies that there is a pathway related to CAs synthesis in E. coli ATCC11303, and it is related to the tyrosine level in the culture medium.

[0072] Example 2: A method for increasing bacterial dopamine synthesis, comprising the following steps:

[0073] Step 1: Screening dopamine-producing bacteria, using enzyme-linked immunosorbent assay (ELISA) to screen the strain Escherichia coli ATCC11303 with a higher concentration of synthesized DA as a strain that synthesizes dopamine (DA);

[0074] Step 2: prepare an L-tyrosine solution with a concentration of 10 mmol / L, wherein the L-tyrosine is dissolved in ultrapure water, and an appropriate amount of acid can be added during the process to promote the dissolution of the L-tyrosine, and the pH is adjusted to 7.2-7.4, and the solution is filtered and sterilized with a filter membrane (0.22 μm), and sealed in a sterile screw-mouth bottle for later use;

[0075] Step 3: Prepare the culture medium, add 0.1% tyrosine solution (concentration of 10 μmol / L) to LB and / or MM culture medium, and store at 4°C for later use;

[0076] Step 4: Strain culture:

[0077] (1) Transfer: Inoculate the Escherichia coli ATCC11303 plate into 5 mL of LB and MM liquid medium and culture overnight at 37°C and 180 rpm.

[0078] (2) Cultivation: Transfer the inoculum with a 1% inoculum to 100 mL of the above-prepared LB and MM medium containing 0.1% L-tyrosine, culture at 37°C, 180 rpm, and collect the bacterial liquid after 6 h, 12 h, and 24 h respectively;

[0079] (3) Separation of supernatant and precipitate: Centrifuge the bacterial solution collected at different stages at 3000 rpm for 20 min, take the supernatant into a new sterile EP tube, label it and store it at 4°C in the dark for later use;

[0080] (4) Washing the precipitate: Pipette PBS buffer and gently blow the bacterial cells to rinse, repeat three times, mark and weigh, and store at 4°C in the dark for later use;

[0081] (5) Cell disruption: Add a certain amount of lysozyme based on the dry weight of the cells and react at 37°C for 25 min;

[0082] (6) Collecting the supernatant: After disruption, centrifuge at 3000 rpm for 20 min and collect the supernatant as the intracellular sample for detection.

[0083] Step 5: Dopamine detection: ELISA was used to determine the concentration of dopamine in the bacterial culture. The results are shown in Table 3:

[0084] Table 3 Changes in intracellular and extracellular dopamine concentrations in E. coli ATCC11303 strain after adding L-tyrosine

[0085]

[0086] In summary, the DA production of common bacteria (from the environment and human body) in the natural environment and laboratory was tested, and it was found that the ability of bacteria to biosynthesize DA was different among different genus. The levels of DA synthesis in different strains were as follows: E.faecalis YN771>E.coli ATCC11303>E.coli BW25113>B.cereus MYB41-22>S.marcescens KMR-3. In addition, the DA concentration was affected by the culture environment. In particular, in the nutrient-poor MM medium, the DA production was generally higher than that in the nutrient-rich LB medium. It was also related to the level of L-tyrosine in the culture medium, which further confirmed the existence of a DA synthesis pathway similar to that of animals and plants in E.coli ATCC11303, and observed that bacteria could release endogenously synthesized DA into the external environment.

[0087] Example 3: Effect of dopamine on the growth of E. coli ATCC 11303

[0088] Effects of different culture media on the growth of E. coli ATCC 11303 strains. Select the LB medium that is most suitable for the growth of E. coli ATCC 11303 strains, prepare 1 nmol / L and 1 μmol / L concentration solutions, and detect the OD of the bacterial solution every 20 min within 300 min. 600 , draw the growth curve, the results are shown in Figure 5Compared with the control group, no obvious promoting or inhibiting effect was found in the experimental groups of CAs, L-Tyr and L-dopA5.

[0089] Then we selected the simplest MM medium, which only contained carbon source, nitrogen source and inorganic salts, which could provide basic growth requirements for bacteria. We prepared solutions with concentrations of 1nmol / L, 1μmol / L and 1mmol / L. The experimental results are shown in Figure 6 Because the strain grows slowly in MM medium, the OD was measured every 6 h within 72 h. 600 , Figure A: At 1 nmol / L, there was no significant difference between the experimental group and the control group in CAs, L-Tyr and L-dopA5 group; Figure B: At 1 μmol / L, there was no significant difference between the experimental group and the control group in CAs, L-Tyr and L-dopA5 group, only the DA group and the NE group showed a growth-promoting effect at 12h (growth period), 24h and 72h (stable period) of strain growth; Figure C: At 1 mmol / L, there was no significant difference between the experimental group and the control group in CAs, L-Tyr and L-dopA5 group, only the L- Tyr and L-dopA promote the growth of the strain, but the concentration is high. These two substances can be used as nutrients. DA, NE and E will show a certain degree of oxidative discoloration at high concentrations. Among them, DA has the highest degree of oxidation and is black. The quinone substances generated inhibit bacterial growth, followed by NE and E, which are oxidized to reddish brown, but will not cause bacteria to stop growing completely. However, their color causes inaccurate absorbance, so this group cannot judge the bacterial growth by OD value; Group D: After supplementing Fe2+ in MM medium, 1μmol / L CAs solution was added, and OD value appeared only after 24h. 600 However, the reduced Fe2+ easily forms complex precipitation with oxidized CAs, interfering with OD 600 The value was detected, and there may be late complexes interfering with bacterial growth, resulting in slower growth of the strain in the CAs group after 36 hours. Based on the previous research, DA was selected as the research target, MM medium was used as the culture environment, and the μmol / L concentration of DA solution was used to test the effect on the growth and physiological characteristics of E. coli ATCC 11303 strain.

[0090] Based on the existing research, the MM medium was improved by adding 30% (v / v) serum to the MM medium and then adding the same concentration of DA solution. Figure 7There was no significant difference in the growth of the strains between the DA-treated group and the control group; but at the same DA concentration, when supplemented with 30% serum, the growth of the DA-treated group was significantly better than that of the control group, among which 50μmol / L DA had the most obvious growth-promoting effect, with the maximum turbidity reaching 1.55 times. When there was only serum, the growth was inhibited compared with the control group. It is speculated that some substance in the serum works together with DA to promote the growth of E.coli ATCC 11303 strain.

[0091] In further experiments, we investigated whether mammalian catecholamine receptor antagonists could neutralize the stimulatory effect of DA on growth in serum-supplemented MM medium containing 50 μmol / L DA. Figure 8 B As can be seen, the dopaminergic antagonist chlorpromazine neutralized the DA-induced growth, but the antagonist did not affect the growth of E. coli ATCC 11303 strain.

[0092] Example 4: Effect of dopamine on the amount of biofilm formation of E. coli ATCC 11303

[0093] The amount of biofilm formation of E. coli ATCC 11303 strain was quantified by crystal violet staining in a polystyrene 96-well plate, and the effect of DA on the amount of bacterial biofilm formation was evaluated by comparing the changes in OD590 values ​​after crystal violet staining. Fig. 9 As shown in A, after staining with 1% crystal violet, in the presence of 200μmol / L DA, the biofilm formation of E.coli ATCC 11303 strain was the highest. At 400μmol / L, the biofilm generation decreased, because at this concentration, the bacterial solution was found to be black, visible to the naked eye, and there were quinone substances in the flocculent matter, which inhibited bacterial growth. In this case, the black substance adhered to the wall of the 96-well plate and was difficult to clean, resulting in a certain degree of experimental error. When DA was 800μmol / L, the bacterial culture wells turned completely black, and the experimental data were meaningless.

[0094] In the next step, we investigated whether dopaminergic receptor antagonists could neutralize the effect of DA on the amount of biofilm formation. Fig. 9 B, The optimal effective concentration of DA was 200 μmol / L, and the same trend as the growth experiment was shown. 100 μmol / L chlorpromazine could neutralize the contribution of DA to the biofilm formation of E. coli ATCC 11303 strain.

[0095] Example 5: Effect of dopamine on biofilm morphology of E. coli ATCC 11303

[0096] In order to more clearly determine whether the effect of DA on the biofilm of E. coli ATCC 11303 strain is still to promote the increase of bacterial numbers, thereby leading to the thickening of the biofilm, or to cause the bacteria to produce a large amount of extracellular substances, such as proteins, polysaccharides, DNA, RNA, peptidoglycan, lipids and phospholipids, we used scanning electron microscopy (SEM) to observe the morphology of the biofilm of the strain. The results are shown in Figure 2. Fig.10 As shown in the figure, the number of bacteria in the 200 μmol / L DA treatment group was large, and they were in a focused state, mostly stacked together, with increased adhesion and bonding between bacteria, and many irregular substances on the periphery, which were speculated to be secretions, and the individual bacteria had no obvious deformation. The control group had fewer bacteria than the DA experimental treatment group, and the number of bacteria in the DA and chlorpromazine co-treatment group was less than that in the DA single treatment group, but more than the control group and the chlorpromazine treatment group, which was consistent with the biofilm quantitative experimental data. It can be seen that DA may affect the number and morphology of biofilms by increasing the number of bacteria and extracellular substances.

[0097] Example 6: Effect of dopamine on motility of E. coli ATCC 11303

[0098] Effects of DA on the motility of E. coli ATCC 11303 strain by plate motility assay

[0099] 1. Preparation of sports plates: Add 0.4 g of bacterial grade agar to every 100 mL of LB medium, sterilize at high temperature, pour the plates, wait for solidification, and place them in a clean bench for later use.

[0100] 2. Prepare dopamine solution: prepare 50μmol / L, 100μmol / L, 200μmol / L and 400μmol / L DA solutions. Make sure to prepare and use them immediately and sterilize them by filtering.

[0101] 3. Strain culture: Culture E. coli ATCC 11303 strain to OD 600 The value is between 0.8 and 0.9.

[0102] 4. Point board:

[0103] (1) Mix DA solutions of different concentrations with bacterial solution at a ratio of 1:1;

[0104] (2) Place in a 37°C constant temperature incubator away from light for 24 h;

[0105] (3) Use a low-speed centrifuge at 3000 rpm for 10 min to collect the cells;

[0106] (4) Wash the cells three times by slowly blowing them with PBS buffer, and finally resuspend the cells with 200 μL PBS;

[0107] (5) Pipette 2.5 μL of the resuspended bacterial solution and gently drop it into the center of the motility plate;

[0108] (6) After sealing, place the plate in a 37°C constant temperature incubator and observe the movement after 8 hours.

[0109] The results are as follows Fig.11 The motility of E. coli ATCC 11303 strain on soft agar motility plate is shown. After incubation of the bacteria with 50 μmol / L DA, the motility of the bacteria will increase. Similarly, 100 μmol / L chlorpromazine will offset part of the effect of DA. Fig.12 From the above data, the average diameter of the motion halo in the 50 μmol / L DA treatment group was about 2 mm larger than that in the control group, showing the same trend as that of growth and biofilm.

[0110] Example 7: Effect of dopamine on chemotaxis of E. coli ATCC 11303

[0111] Chemotaxis is a way for bacteria to respond to certain compounds. It refers to the bacteria "navigating" in the environmental gradient of various chemicals to locate conditions that are conducive to growth. Experimental exploration was carried out using the optimal effective concentrations of 50μmol / L, 100μmol / L and 200μmol / L obtained in the above experiment. The results are shown in Fig.13 , 50μmol / L, 100μmol / L and 200μmol / L DA were added as chemokines, of which 50μmol / L and 200μmol / L DA had the best attracting effect on E.coli ATCC 11303 strain, but the three repeated experiments showed that the data of 200μmol / L were scattered and the differences within the group were large. In the 50μmol / L DA experimental treatment group, the three experimental effects were relatively stable. Taking this as an example, after the gun tip with chemokine was washed, it was found that the number of colonies in 50μmol / L DA was slightly higher than that in the control group by 2.75 times. When further exploring the effect of chlorpromazine on dopamine receptors, the data showed no significance, and it was speculated that DA may have different modes of action with chemotaxis, growth, and biofilm.

[0112] In summary, Examples 1-7 show that the effect of DA on promoting the growth of E. coli ATCC 11303 in different culture media was compared and analyzed. The results showed that DA did not promote the growth of E. coli ATCC 11303 in LB culture medium, and the promoting effect in MM culture medium was limited. It showed a slight promoting effect in MM culture medium containing 1 μmol / L DA, but in MM culture medium supplemented with 30% serum, DA significantly enhanced growth, and 50 μmol / L DA had the best growth promoting effect, with the maximum turbidity point increased by 1.55 times. 100 μmol / L chlorpromazine (dopamine receptor antagonist) can weaken the growth promoting effect of DA. 2. Effect of DA on the amount of biofilm formation: In MM culture medium supplemented with 30% serum, 200 μmol / L DA significantly promoted biofilm formation, increasing by about 1.5 times, while 100 μmol / L chlorpromazine partially neutralized this effect. 3. Effect of DA on biofilm morphology: 200μmol / L DA can significantly increase the thickness of biofilm at 36h, mainly by increasing the number of bacteria and secretions such as extracellular polysaccharides. 4. Effect of DA on strain motility: 50μmol / L DA significantly increased the motility of E.coli ATCC 11303. Compared with the control group, the diameter of the motion halo increased by about 2mm on average. 100μmol / L chlorpromazine can partially neutralize the effect of DA. 5. Effect of DA on strain chemotaxis: 50μmol / L DA significantly enhanced the chemotaxis of the strain, making the number of colonies in the treatment group 2.75 times that of the control group, while chlorpromazine had no significant effect on the chemotaxis enhancement induced by DA.

[0113] Example 8: Study on the transport pathway of dopamine

[0114] Cy3-DA was prepared by attaching the Cy3 fluorescent group to the DA molecule through an amidation reaction. The dynamic distribution and transport pathway of extracellular DA in bacteria were visually observed using laser confocal microscopy.

[0115] E. coli ATCC 11303 culture medium was selected to culture the culture medium. The culture medium at the 5-hour growth period was taken and incubated with the labeled Cy3-dopamine. The direction of DA was observed using a laser confocal microscope. The experimental results are as follows: Fig.14 As shown in the figure, E. coli ATCC 11303 bacterial fragments still remain in the supernatant, and Cy3-dopamine in the supernatant has entered the cell. At the same time, Cy3-dopamine monomers still remain in the supernatant, and their color is brighter than the fluorescence entering the bacteria. The washed precipitate was diluted and 10 -3 By observing the gradient solution, it can be clearly seen that Cy3-dopamine enters the cells of E. coli ATCC 11303 strain, and the bacteria emit orange-red fluorescence.

[0116] For further verification, the bacterial culture was collected during the 12-hour stable period and incubated with Cy3-dopamine to observe the fluorescence. Fig.15 As shown in Figure 2, many secretions appeared in the supernatant of E. coli ATCC 11303 strain in the late growth stage, and the fluorescence was weak. Cy3-dopamine fragments were still present in the supernatant; however, in the precipitate (diluted 10 -3 ) found that E. coli ATCC 11303 cells aggregated and the membrane was broken and the contents flowed out, and the fluorescence was significantly weakened compared with 5h, and there was a difference between the two. In summary, it can be roughly determined that DA enters the cells of E. coli ATCC 11303 strains to exercise its function, and the best period of action is in the growth period, which is consistent with the DA growth promotion experiment.

[0117] The observation results of the above experiment using laser confocal microscopy revealed that Cy3-labeled DA can penetrate the bacterial cell membrane and enter the cell, laying the foundation for further revealing the mechanism of action of DA in bacteria.

[0118] Example 9: The effect of DA at an effective concentration of 50 μmol / L on protein expression of E. coli ATCC11303 strain was deeply analyzed using 4D-DIA proteomics technology, using the ideas and methods of comparative proteomics. The setting included a control group and a DA treatment group, with 3 biological replicates in each group, covering the extraction of total protein from each group of samples, peptide sample preparation, DIA sample mass spectrometry detection and DIA data analysis. The specific results are as follows:

[0119] (1) According to the analysis of polyacrylamide gel electrophoresis, Fig.16 As shown in the figure, the protein spectrum of E. coli ATCC11303 showed obvious changes after DA treatment. Although the control group and the DA treatment group were generally similar in band composition, some bands showed significant differences. Compared with the control group, the band density at approximately 37k Da and 50k Da in the DA treatment group decreased. At the same time, in the three repeated experiments of the two groups, the results showed good consistency.

[0120] (2) The results of the Meiji protein bioinformatics analysis platform showed that the DA treatment group and the control group had a large number of common proteins, with 3043 proteins accounting for 99.84% of the total proteins. There were only two unique proteins, namely phosphoribosylaminoimidazole succinocarboxamide synthase (SAICAR) and aconitate hydratase B, which are encoded by purC and acnB-2, respectively. They are key enzymes in purine synthesis and TCA cycle, and play a key role in multiple life processes such as bacterial genetic material replication, energy metabolism and signal transduction. For details, see Fig.18 .observe Fig. 20 and Fig.21 The difference between the DA-treated group and the control group can be clearly seen, and the three parallels within each group have good consistency.

[0121] (3) Differential protein analysis, the results after DA treatment are as follows Fig. 22 After differential expression analysis, a total of 138 differentially expressed proteins were identified, accounting for 4.52% of all identified proteins, of which 60 were upregulated, accounting for 1.96%; Fig.23 As shown, there are 14 proteins whose expression levels increased by more than two times.

[0122] There were 78 genes whose expression was downregulated, accounting for 2.55%. Fig. 22 Figure B shows the distribution of differentially expressed proteins. The X-axis is the differential expression multiple of the two groups of proteins, and the Y-axis shows the p-value. Each point in the figure corresponds to a specific protein. Red dots represent down-regulated proteins, and blue dots represent up-regulated proteins. The closer the position of the dots is to the edge of the figure, the more significant the difference between the two groups of proteins is. Fig. 22 ).

[0123] According to Figure 23-26 As shown in the figure, 18 proteins were annotated as down-regulated in the GO database, mainly in the regulation of molecular metabolism, cell components, cell engineering, metabolic processes and transporter activity-related proteins. According to the annotation of the KEGG database, the expression of proteins related to microbial metabolism, metabolic pathways, synthesis of secondary metabolites, quorum sensing and lactam resistance in different environments was reduced. According to the annotation of the EggNOG database, the most down-regulated proteins are still some proteins with unknown functions, and the rest are proteins related to energy production and conversion, amino acid metabolism and translation. Among them, the expression of proteins related to coenzyme transport and metabolism is also down-regulated. The decrease in the expression of proteins with down-regulated expression is not large, and none of them exceeds 1 times.

[0124] (4) Effects of dopamine on metabolic pathways of E. coli ATCC11303 strain

[0125] iPath3.0 was used to visualize the metabolic pathway of dopamine on E.coli ATCC11303 strain. The results are as follows Fig.25 As shown in the figure, DA has an effect on the overall metabolic pathway of E. coli ATCC11303 strain. After DA treatment, many bacterial pathways (such as purine metabolism, pyrimidine metabolism, amino acid metabolism and secondary metabolite production) were affected. In general, its promoting effect was greater than the inhibiting effect.

[0126] It should be understood that the above specific embodiments of the present invention are only used for illustrative inventions or to explain the principles of the present invention, and do not constitute limitations on the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims, or the equivalent forms of such scope and boundaries.

Claims

1. Application of dopamine in regulating the physiological characteristics of microorganisms.

2. The use according to claim 1, characterized in that: The use of dopamine in promoting the growth of bacterial strains.

3. The use according to claim 1, characterized in that: The application of dopamine in promoting bacterial biofilm formation.

4. The use according to claim 1, characterized in that: The use of dopamine in improving bacterial motility.

5. The use according to claim 1, characterized in that: The application of dopamine in enhancing the chemotaxis of strains.

6. The use according to claim 1, characterized in that: The use of dopamine in bacterial protein response.

7. A method for promoting dopamine production by using bacteria, characterized in that: The following steps are involved: Step 1: Screening bacteria, screening dopamine-producing bacteria, using enzyme-linked immunosorbent assay (ELISA) to screen dopamine-producing strains; Step 2: Prepare a tyrosine solution by weighing tyrosine and dissolving it in ultrapure water to prepare a tyrosine solution. Filter the solution through a membrane to sterilize the solution and seal it in a sterile screw-capped bottle for later use. Step 3: Prepare the culture medium by adding 0.1% of the tyrosine solution prepared in step 2 to the LB and / or MM culture medium; Step 4: Inoculation test: prepare the strain selected in step 1 into a strain suspension and inoculate it into the culture medium in step 3 for cultivation. After 6-24 hours of cultivation, detect the dopamine content.

8. The method according to claim 7, characterized in that In the step 2, the tyrosine includes L-tyrosine or D-tyrosine.

9. The method according to claim 7, characterized in that: In the step 2, the concentration of the tyrosine solution is 10 mmol / L, acid is added to dissolve the tyrosine, and the pH is adjusted to 7.2-7.

4.

10. The method according to claim 7, characterized in that The filter membrane in step 2 has a pore size of 0.22 μm.

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