Cyclodepsipeptides antibiotic broad-spectrum antagonistic action laterally, the strain of bacteria brevibacillus laterosporus, with a wide range of antagonistic action - producer of antibiotic lateralline

 

(57) Abstract:

The invention relates to the field of biotechnology, in particular to the production of a new antibiotic lateralline. Laterally has an antagonistic effect against bacteria, cyanobacteria, phytopathogenic fungi and protozoa. Laterally is cyclodepsipeptides antibiotic and represent a white amorphous solid, having the formula [cyclo-(L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Leu)], and the molecular weight 1224,4 Da. Producer of lateralline is a strain of bacteria Brevibacillus laterasporus, which is obtained from natural strains of the same species in a multistage breeding and deposited under number VKPM IN-8287. Strain active against a number of G+ and G - bacteria, phytopathogenic fungi and protozoa microorganisms. The use of strain and lateralline allows you to extend the range of antagonists of broad-spectrum. 2 C. p. F.-ly, 4 PL.

The invention relates to Microbiology, biotechnology and, in particular, to the production of bacterial origin for diseases of plants and animals.

Currently, as a means of combating plant pathogens are mainly used Khimich Innovene resistance in pathogens no selective effect toxicity to warm-blooded) dictate the need for new connections. As an alternative plant protection products can be used bacterial antifungal drugs, which include strains of microorganisms with antagonistic activity against different biological objects.

Widespread use of the so-called “classic” of antibiotics has led to the emergence of resistant pathogens, causing serious social and medical problems. One way of solving this problem is the search of new generation antibiotics, which include peptide antibiotics. Interest in them is due to their mechanism of action that causes physical damage to the cell membranes of microorganisms. It is assumed that this mechanism of action to a certain extent reduces the likelihood of resistance of pathogenic bacteria.

Currently known a number of bacillary strains are able to produce peptide antibiotics with antibacterial and/or fungicidal activity. Characteristics bacillary other) and D-amino acids. Thus, the described strains of Bacillus licheniformis, synthesizing low molecular weight (mol. weight 1400-3200) polypeptides and lipopetides, with a broad spectrum of antagonistic activity (1, 2). Strains of Bacillus subtilis produce several antimicrobial factors. Miroballi - anionic 13-membered cyclic polypeptide with mol. mass of ~1800, has a broad spectrum of activity against bacteria, protozoa and fungi (3). A new class lipopeptide fungicides combines active peptides families iturin, surfactin and Fungizone (4). Furini (4) and bucillamine (5) - neutral or anionic Cycloheptane mol. mass of ~1000, combined with a fatty acid or ester bond with the formation of the lactone ring cycle (furini), or a peptide bond in the cycle (ballotini) (6). Iturin and bucillamine have limited antimicrobial activity and a broad spectrum of activity against fungi and yeast. Surfactin - circular lipopeptide, contains seven hydrophobic amino acid residues, connected with-hydroxyzine acid (7). This is a powerful surface-active compound, also known as antibacterial and antifungal agent. Perizin (8) and lipstatin (9) - lipopetides, containing ten amino acids and lipid part, raspologat ornithine and allo-threonine. Known group cyclodepsipeptides (gramicidin S and tyrocidine) produced by strains of Bacillus brevis with weak antibacterial and antifungal activity (10). There is information about the strains of Bacillus laterosporus, inhibiting the growth of bacteria and fungi (11, 19, 20). From a strain of Bacillus laterosporus MK-PNG-276A selected alatini a, b, C, D group cyclic Decapeptide Teroldego family, showing antagonistic activity against gram-positive bacteria Staphylococcus aureus, Staphylococcus ssp., Streptococcus ssp., Enterococcus faecicum and yeast-like fungi Candida albicans, but it is not active against gramotritsatelnymi bacteria Pseudomonas mendocina and Pseudomonas marginata (12, 13). Information about the antagonistic activity of these substances against protozoa, cyanobacteria, yeasts Saccharomyces cerevisiae and pathogenic fungi are absent. Producing strains Molotilov Bacillus laterosporus MK-PNG-276A selected as the closest analogue of the claimed strain with a broad spectrum of antagonistic action.

The task is to expand the Arsenal of tools based on the cyclic Decapeptide used to combat pathogens of plants and animals.

The problem is solved by means of:

- selection cyclodepsipeptides of broad-spectrum antibiotic is doziruuschih antibiotic laterally and having a wide spectrum of antagonistic action, due to including the antibiotic latrocinium.

Declare cyclodepsipeptides antibiotic, called by us laterally, not known from the information sources. Laterally has an antagonistic effect against bacteria, cyanobacteria, phytopathogenic fungi and protozoa (table 1). Laterally is a white, amorphous solid, having the formula [cyclo-(L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Leu)], and the molecular weight 1224,4. Laterally soluble in methanol, ethanol and other lower alcohols, insoluble in water.

The inventive strain selected in a multistage selection from natural strain of Brevibacillus laterosporus. He deposited in Russian national collection of industrial microorganisms under the number VKPM B-8287.

The strain of Brevibacillus laterosporus VKPM B-8287 claimed

as a producer of antibiotic lateralline and

as the strain with a broad spectrum of antagonistic action.

The inventive strain of Brevibacillus laterosporus VKPM B-8287 has the following features:

1. Cultural and morphological characteristics.

Aerobic gram-negative motile Bacillus 0.5-0,72,5-5.0 µm capsule form. Vegetate the Noah side of the debate. The size of the dispute 0.9-1,21,5 to 1.7 μm. After 24 hours of growth on rich agar media [NBY g/l: nutrient broth (nutrient broth)-8; yeast extract (yeast extract), 3 - agar-agar - 20; DPS g/l: yeast kaprinovye - 30, corn flour - 15, agar-agar - 20; DG g/l: hydrolyzed yeast - 45, agar-agar 20] strain forms a rounded colonies 3-4 mm in diameter with a smooth surface is cream-coloured and rough edges.

2. Cultivation of the strain.

When grown in liquid nutrient medium [NBY (g/l): nutrient broth (nutrient broth) - 8; yeast extract (yeast extract) - 3] at 28-30C with shaking (250 rpm) uniform growth of the culture is observed in the whole volume. To 96 hours of cultivation occurs mainly 90-95% of spores and 5-10% of the vegetative cells. The titer of the culture after 96 hours of cultivation is ~3,0109Jr.

3. Physiological and biochemical characteristics.

Strain sprayway glucose, maltose, fructose. Not sprayway sucrose and lactose. Forms lecithinase; not hydrolyzes starch; hydrolyzes casein and gelatine. Urea does not break. Forms a twin-esterase.

On the basis of morphology (presence konomini structure attached to a dispute) and physiological and biochemical characteristics of the selected shtam the th activity.

Strain synthesizes the antibiotic laterally and showing antagonistic activity against gram-positive bacteria: Bacillus thuringiensis, Bacillus subtilis, B. megaterium, Bacillus, Bacillus cereus, Bacillus sphaericus, Staphylococcus ssp., Staphylococcus aureus, Streptococcus ssp., Enterococcus ssp., Enterococcus faecium, Sarcina flava (table 2);

gramotritsatelnymi bacteria: Acinetobacter calcoaceticus, Flavobacterium aquatile, Pseudomonas marginata, Pseudomonas mendocina, Xanthomonas spp., Erwinia herbicola, Erwinia carotovora, Erwinia atroseptica. Vibrio nereis (table 2);

cyanobacteria: Nostoc spp. 29411 and Anabaena spp. 5781 (table 3);

phytopathogenic fungi: Fusarium frost, Fusarium nivale, Fusarium solani, Rhizoctonia solani, Sclerotinia sclerotiorum, Phomopsis helianthi, Phoma solanicola, Alternaria tenuis, Aspergillus niger (table 4);

yeast: Saccharomyces cerevisiae (table 4);

easiest: Tetrahymena pyriformis u Entamoeba moshkovskii (example 8).

The invention is illustrated by the following examples

Example 1.

Microbiological synthesis of lateralline strain of Brevibacillus laterosporus VKPM B-8287, isolation and purification of lateralline.

The strain of Brevibacillus laterosporus VKPM B-8287 cultivated in a liquid nutrient medium NBY 72 hours at 30 ° C on a circular shaker (250 rpm). The culture fluid was centrifuged at 10000 g for 15 min, the obtained supernatant was discarded and washed three times with water at tsentralnoi temperature. The resulting alcoholic extract was separated by centrifugation (15 min at 10000g), the resulting solution was evaporated under S to obtain crystals of yellow-brown color. Alcohol solution of the crystals was fractionally by HPLC (HPLC) on a reversed-phase column (Delta Pak C-18, 300 , 20 μm (19300 mm, Waters) at a flow rate of 2 ml/min in a linear gradient (from 0 to 100%) solution B (methanol - acetonitrile - water, 75:22,5:2,5) in solution A (methanol - acetonitrile - water, 60:5:35) for 100 min and then in solution B for 20 min On the column was applied to 10 μl of a solution. The obtained fractions exhibiting antagonistic activity were combined, evaporated to dryness and dissolved in ethanol. Then, the solution (10 μl) was applied to a reversed-phase column Vydac C-18, 300 , 5 μm (4,6250 mm, Vydac). The elution was performed at a flow rate of 0.5 ml/min in a linear gradient (from 10 to 100% of solution B in solution a for 60 minutes and then solution B for 20 min Fractions exhibiting antibacterial activity, were combined and evaporated to dryness. The obtained homogeneous antibiotic was an amorphous substance of white color.

Example 2.

The primary structure of lateralline.

To define the structure of the selected antibiotic used NMR spen spectrometer Varian Unity 600 with an operating frequency of 600 MHz. Based on the analysis of the NMR spectrum COSY, TOCSY and ROESY using standard procedures (17) conducted a full assignment of the proton signals of the investigated sample, which corresponds to the amino acid sequence of [cyclo-(L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Leu)]. After analyzing the SRS database revealed that the peptide with the above sequence has no analogues. This peptide, called by us latrocinium, belongs to the family tyrocidine antibiotics.

Example 3.

Determination of molecular weight of lateralline.

To determine the molecular weight of lateralline used mass spectrometric analysis.

MALDI (matrix-assisted laser desorption ionization) mass spectrometrically analysis of the peptide was performed on a mass spectrometer VISION 2000 (ThermoBioAnalysis, OK). As the matrix used 0.15 M 2,5-dihydroxybenzoic acid (DHB) in a mixture containing 25% methanol and 0.1% triperoxonane acid. The sample was irradiated with a UV laser with a wavelength of 337 nm and a maximum energy of 250 µj in a pulsed mode with a frequency of 3 NS. When the mass spectrometry analysis of peptide (laser power is 57%, the number of laser shock - 20) was obtained peak with m/z 1224,4; the corresponding molecular ion of a new antibiotic laterized">

Quantitative evaluation of the antagonistic action of lateralline was performed by determining the minimum inhibitory concentration (MIC). For this purpose, the received laterality was dissolved in the minimum volume of absolute ethanol and prepared the initial solution containing 200 μg/ml of a 5% ethanol. Minimum inhibitory concentration of lateralline was determined by the standard method of serial twofold dilution of the original solution of the antibiotic (18) against each test organism (table 1).

From the data presented in table 1, it follows that laterally has a broad spectrum of antagonistic action, the highest level of antagonistic activity he manifests towards phytopathogenic fungi of the genus Fusarium, the simplest, cyanobacteria, pathogenic bacteria group streptococci, stafilokokkove and enterococci.

Example 5.

The definition of the spectrum of antibacterial action of the strain VKPM B-8287 and its comparison with the closest analogue.

Antibacterial action of cultures of strain was identified by the method of the hole. In 25 ml of agar medium NBY was added 1 ml of 4-hour culture of the test bacteria, cut holes with a diameter of 8 mm were made on 50 CFU effect of strain recorded after 24 hours by measuring the zones of inhibition of growth of test microorganisms. The results are shown in table 2.

Example 6.

Definition tionalities activity of the strain VKPM B-8287.

Tionalities activity of the strain was identified in mixed cultivation. The culture fluid 72-hour culture VKPM B-8287 were made to the suspension 14-day culture of cyanobacteria Nostoc spp. 29411 or Anabaena spp. 5781 (16). Measured optical density at a wavelength of 590 nm) of the mixture at zero time and after incubation at 25C and round-the-clock coverage. Tionalities activity was assessed by the drop in optical density after 24 hours of incubation. The strain VKPM B-8287 caused the drop in optical density is 3-4 times that testified to its lytic action on cyanobacteria (table 3). Lysis of the cells of cyanobacteria were detected also in optical microscopy.

Information about tionalities activity of the strain - the closest analogue in the sources of information not found.

Example 7.

Determination of the fungicidal activity of the strain of Brevibacillus laterosporus VKPM B-8287.

To identify the fungicidal activity of the strain against fungi and yeast used holes. Culture of test organism was placed in the centre of Petri dishes with ecost 72-hour culture In-8287. The cups were incubated in the dark at room temperature. After 48 hours was assessed by the presence or absence of zones of inhibition of growth of fungi.

The inventive strain inhibited the growth of mycelium of the following pathogens: Fusarium frost, Fusarium nivale, Fusarium solani, Rhizoctonia solani, Sclerotinia sclerotiorum, Phomopsis helianthi, Phoma solanicola, Alternaria tenuis, Aspergillus niger and Saccharomyces cerevisiae, but was not active against Candida albicans (table 4), in contrast to the nearest equivalent.

Example 8.

Determination of the activity of the strain VKPM B-8287 against protozoa.

The activity of the strain VKPM B-8287 against protozoa was determined in mixed cultivation of strain and protozoa. 72-hour culture fluid of strain contributed to the suspension of cells is simple. Used 4-day culture of the ciliate Tetrahymena pyriformis (15-2010, the number of ciliates were counted microscopically) or 7-day culture of amoebae Entamoeba moshkovskii (5-8103the number of amoebae were counted microscopically). After 24 hours of incubation, the death of the protozoa was 100%.

Information about antagonistic activity closest analogue against protozoa are missing.

Thus, we obtained a strain of bacteria Brevibacillus laterosporus VKPM B-8287 with a wide spectrum of antagonistic the bacteria, cyanobacteria, phytopathogenic fungi, yeast and protozoa. He is a producer first isolated cyclodepsipeptides antibiotic called us latrocinium, which also has a wide, but slightly different from that of the producer strain spectrum of antagonistic action. Both can find independent use as antibacterial, antifungal and Antiprotozoal funds to fight diseases in plants and animals.

Bibliography

1. De Lucca, A. J., Walsh, T. J. 1999. Antifungal peptides: novel therapeutic compouds against emerging pathogens. Antimicrobial Agents and Chemotherapy. 43:1-11.

2. Galvez A. M., Maqueda, M., Martinez-Bueno M, Lebbadi M., Valdivia, E. 1993. Isolation and physico-chemical characterization of an antifungal and antibacterial peptide produced by Bacillus licheniformis A 12. Appl. Environ. Biotechnol. 38:438-442.

3. Egorov N. With. 1994. The basic teaching about the antibiotics. Publishing house of Moscow state University. 422-423.

4. Klich, M. A., Lax, A. R., Bland, J. M. 1991. Inhibition of some mycotoxigenic fungi by iturin A, A peptidolipid produced by Bacillus subtilis. Mycopathology. 116:77-80.

5. Mhammedi A. F., F. Peypoux, F. Besson, Michel G. 1982. Bacillomycin F, a new antibiotic of the iturin group. Isolation and characterization. J. Antibiot. 35: 306-311.

6. Moyne A. L., Cleveland I.e., Tuzun S. Small peptides with antipathogenic activity, treated and methods for treating same. U. S. Patent No. 6183736.

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13. Gerald J. M., Haden, P., Kelly, M. T., Andersen, R. J. 1999. Loloatins A-D, cyclic decapeptide antibiotics produced in culture by a tropical marine bacterium. J. Nat. Prod., 62:80-85.

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15. Shida O., Takagi, H., Kadowaki, K., Komagata K. 1996. Proposal for new genera, Brevibaciillus gen.nov. and Aneurinibacillus gen.nov. Int. J. Sysyt. Bacteriol., 45:939-946.

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1. Cyclodepsipeptides antibiotic broad-spectrum antagonistic action laterally with revibacillus laterosporus VKPM B-8287, with a wide range of antagonistic action - producer of antibiotic lateralline.

 

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