rus
Issue #5/2022
M.V.Zubasheva, A.I.Akhmetova, Т.А.Smirnova, N.V.Shevlyagina, Yu.A.Smirnov, V.G.Zhukhovitsky, I.V.Yaminsky
CRYSTAL FORMATION IN BACTERIAL CELLS OF BREVIBACILLUS LATEROSPORUS
CRYSTAL FORMATION IN BACTERIAL CELLS OF BREVIBACILLUS LATEROSPORUS
DOI: https://doi.org/10.22184/1993-8578.2022.15.5.254.261
Bacillus Brevibacillus laterosporus (Bl) is a promising source of bacterial insecticides. The crystal-forming ability of Bl bacteria has not been established until recently. Bacterial cells of Brevibacillus laterosporus were characterized with the aid of scanning and transmission electron microscopy. The formation of protein crystals in bacteria from the first stages of nucleation in the cells up to the stage of free crystals was studied. Molecular resolution images of the crystals have been obtained and the crystal lattice parameters have been determined. In the case of entomocidal bacilli, crystalline protein toxins are formed in the cells, which lead to death of insects after absorption by the bacteria. This is the defense mechanism of these bacterial cells. In such case, the crystals perform the protective function of living organisms.
Bacillus Brevibacillus laterosporus (Bl) is a promising source of bacterial insecticides. The crystal-forming ability of Bl bacteria has not been established until recently. Bacterial cells of Brevibacillus laterosporus were characterized with the aid of scanning and transmission electron microscopy. The formation of protein crystals in bacteria from the first stages of nucleation in the cells up to the stage of free crystals was studied. Molecular resolution images of the crystals have been obtained and the crystal lattice parameters have been determined. In the case of entomocidal bacilli, crystalline protein toxins are formed in the cells, which lead to death of insects after absorption by the bacteria. This is the defense mechanism of these bacterial cells. In such case, the crystals perform the protective function of living organisms.
Теги: brevibacillus laterosporus crystal formation protective function spores toxins transmission electron microscopy защитная функция образование кристаллов просвечивающая электронная микроскопия споры токсины
INTRODUCTION
The formation of crystals or crystalline inclusions is a rare phenomenon in living nature. Mostly crystals are formed in non-living nature. They are represented by crystalline rocks, gemstones, etc. Carbon has several crystalline forms - diamond, graphite and also the fine crystalline powder carbine. Formation of crystals occurs when the total energy of the building units - atoms or molecules – in the crystal is reduced in comparison to their energy in solution. The reverse process, dissolution of crystals, can also occur if there is a clear lack of dissolved matter in the solution. Crystals are present in living organisms, for example, magnetic crystals of iron oxide or iron sulfide are synthesised in magnetotactic bacteria. The size of the crystals ranges within 25–130 nm [1, 2]. These crystals provide protective functions in bacteria due to presence of a toxic divalent ion. The habitat of these bacteria contains excess iron. These bacteria move along magnetic field lines. Magnetic crystals act like a magnetic arrow.
Bacillus thuringiensis (Bt) crystals are the best studied due to their entomocidal activity against Lepidoptera, Diptera and Coleoptera insects. A study of entomocidal Bt crystals revealed several basic morphological types: bipyramidal, spherical-amorphous, flat square, cubic, stick-shaped. Most of the crystals active against Lepidoptera were bipyramidal or cubic in shape. Irregular, spherical and rod-shaped crystals could be mosquito-cidal. Flat, square crystals were active against Coleoptera [3]. Bt spores and crystals are used to control insect pests. However, preparations based on them do not fully meet the necessary requirements. Other environmentally friendly bacterial preparations are currently being sought and studied.
Bacilla Brevibacillus laterosporus (Bl), in contrast to Bt, are poorly studied entomopathogenic bacteria. Their distinctive feature is the presence of a canoeid inclusion attached to the spore. We have previously studied and characterized strains of Bl capable of forming paraspore crystals [4]. The studied Bl strains exhibited crystalline inclusions of different shapes and sizes, similar to those described for the entomopathogenic Bt bacteria. The highest activity of Bl was detected against insects of the Díptera order. This includes mosquitoes of the genera Aedes, Anopheles, Culex and black flies (Simulium vittatum). Many mosquito species are vectors of malaria, yellow fever, dengue fever, haemorrhagic fever and lymphatic filariasis. Synthetic insecticides have adverse effects on humans and nature. In addition, resistance to such insecticides has been found in pathogen-transmitting mosquitoes.
An alternative to address these problems and limitations is biological control using entomopathogenic bacteria. Bacillus thuringiensis subsp. israelensis (Bti) was the first subspecies of Bt to be toxic to twoflies [5]. A study on the nature of the insecticidal factors showed that insects of the Diptera order, mosquitoes and black flies, are sensitive to the paraspore inclusion of Bti, which consists of four proteins Cry4Aa, Cry4Ba, Cry11Aa, Cyt1Aa [6]. Ben-Dov [7] reported that the larvicidal activity consists of four major (134, 128, 72 and 27 kDa) and at least two minor (78 and 29 kDa) polypeptides encoded by the cry4Aa, cry4Ba, cry11Aa, cyt1Aa, cry10Aa and cyt2Ba genes mapped to a 128000 bp plasmid known as pBtoxis. These δ-endotoxins form a complex paraspore crystalline body with extremely high specificity. Electron microscopy has shown that Bti cells form inclusions that consist of different segments, an osmiophilic or lightly stained, crystallized lattice segment with a period of 4.3 nm and an osmophilic, stained lattice segment with a period of 7.8 nm. The complex of several crystals is enclosed in a common shell [8].
The crystalline inclusions of Bl can have a cubic shape. We wrote about cubic shape of crystals earlier [9]. In the present paper we show the results of study of rhombic crystals in Bl cells. This work is devoted to a study of the process of crystal formation in Bl during sporulation and to the analysis of the structure of crystals.
METHODS AND MATERIALS
In this work we made and used the following:
Strain B. laterosporus (Bl) 16–92 was isolated from a dead insect.
The strain was grown on NBY agarized medium for 48–96 h at 30 °C.
Electron microscopy.
Scanning electron microscopy (SEM).
The native spore-crystalline suspension was examined using a Quanta 2003D electron ion scanning microscope (FEI Compani, USA). Samples of the native suspension were placed on a silicon substrate, which was fixed to an aluminum table using carbon tape. The preparations were examined in high and low vacuum. The surface of the sample was scanned at an accelerating voltage of 5–30 kV.
Transmission electron microscopy (TEM)
Bacilli of strain 16–92 were washed off the agar and fixed first by the Ito-Karnowsky method [10]. The material was then fixed in 1% OsO4 solution on 0.2 M cacodylate buffer and in 1% uranyl acetate solution on 0.2 M maleate buffer. The material was dehydrated in alcohol concentrations of 50°, 70°, 96°, 100°. The material was then placed in a mixture of 100° alcohol and LR White resin and then in pure LR White resin. The material was transferred into gelatin capsules, which were placed in a thermostat at 56 °C. Sections were obtained on an LKB III ultra-tome (LKB, Sweden) and contrasted with a 1% solution of uranyl acetate in 70° alcohol and citric acid lead. Spore and crystal suspensions for negative contrast studies were applied to carbon-film-coated grids and stained with 2% aqueous NANOWtm. The preparations were examined on electron microscopes JEM -100 B and LEO912AB.
The images were processed using FemtoScan Online software [11]. A two-dimensional Fourier transform was used to determine the lattice parameters of the observed crystals.
RESULTS
Crystal formation in Bl was studied: from the first stages up to the stage of free crystals. To detect crystals, a spore-crystalline suspension of strain 16–92 was examined using SEM. Figure 1 shows mature spores with a canohedral inclusion and rhombic crystals. The crystals were either bound to the spores or were in a free state.
Electron microscopic examination of ultrathin sections of strain Bl 16–92 by TEM showed that the growing, dividing cells did not contain crystals. Crystals appeared in vegetative cells before entering the sporulation stage. Thereafter, the crystal-forming cells went through all the stages of spore formation. In Fig.2 a prospore can be seen, at some distance from which a rhomboidal crystal is located. In Bl crystals are formed without connection to the spore structures. Near the crystal there is a rarefaction of the cytoplasm.
Lysis of the sporangium cytoplasm around the spore and the crystal increases as the spore matures. In addition to the crystalline inclusion, an overgrowth of a lamellar canoeid inclusion is observed. Fig.3 shows that the cytoplasm of the sporangium is completely lysed and the cell membrane is preserved in most spores, but partially destroyed in some. Figure 4 shows the spore with canoeid inclusion and a crystal after leaving the sporangium. As in Bt, spores and crystals of Bl are released separately during sporangium lysis.
Two or more crystals form in some Bl cells. This can be seen in Fig.5: a crystal of complex structure is distinguishable.
The crystals are enclosed in a common shell (Fig.5, 6, 7, 8).
The crystals have the same electron density, which distinguishes them from the Bti crystal complex. Bti has several crystals in a common shell with different electron densities.
The complex structure of Bl crystals was confirmed by further studies.
The area of crystalline inclusions visible in negative contrast has a diamond shape, as shown in Fig.9.
It should be noted that the observed diamond-shaped region of 320 × 540 nm (Fig.9) is not a single crystal but rather a cluster of individual smaller crystals. The size of single crystals ranges from 25 to 120 nm. If we consider the surface of a single crystal, the Fourier image rather shows hexagonal packing of proteins in the crystal (Fig.10). Accordingly, 6 reflexes are observed in the Fourier image. The reflexes are not located on a circle, but on an ellipse. There could be two reasons for this:
the protein molecules that make up the crystal have an elongated shape;
it is a hardware effect (artefact).
The presence of several possible arrangements of protein crystals is observed, for instance, in the case of lysozyme. Under different crystallization conditions, a tetragonal or orthorhombic arrangement is obtained [12].
In the case of crystals Bl, the presence of two packings shows that the biochemical composition of the inner compartment of the cell, where crystallization takes place, should differ more or less considerably. This observation shows that the inclusion of Bl is similar to that of Bti.
ACKNOWLEDGMENTS
The study was completed with the financial support of the RFBR and the London Royal Society No. 21-58-10005, RFBR, Project No. 20-32-90036, RSF, project No. 20-12-00389 and from the Foundation for the Promotion of Innovation, Project No. 71108.
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
The formation of crystals or crystalline inclusions is a rare phenomenon in living nature. Mostly crystals are formed in non-living nature. They are represented by crystalline rocks, gemstones, etc. Carbon has several crystalline forms - diamond, graphite and also the fine crystalline powder carbine. Formation of crystals occurs when the total energy of the building units - atoms or molecules – in the crystal is reduced in comparison to their energy in solution. The reverse process, dissolution of crystals, can also occur if there is a clear lack of dissolved matter in the solution. Crystals are present in living organisms, for example, magnetic crystals of iron oxide or iron sulfide are synthesised in magnetotactic bacteria. The size of the crystals ranges within 25–130 nm [1, 2]. These crystals provide protective functions in bacteria due to presence of a toxic divalent ion. The habitat of these bacteria contains excess iron. These bacteria move along magnetic field lines. Magnetic crystals act like a magnetic arrow.
Bacillus thuringiensis (Bt) crystals are the best studied due to their entomocidal activity against Lepidoptera, Diptera and Coleoptera insects. A study of entomocidal Bt crystals revealed several basic morphological types: bipyramidal, spherical-amorphous, flat square, cubic, stick-shaped. Most of the crystals active against Lepidoptera were bipyramidal or cubic in shape. Irregular, spherical and rod-shaped crystals could be mosquito-cidal. Flat, square crystals were active against Coleoptera [3]. Bt spores and crystals are used to control insect pests. However, preparations based on them do not fully meet the necessary requirements. Other environmentally friendly bacterial preparations are currently being sought and studied.
Bacilla Brevibacillus laterosporus (Bl), in contrast to Bt, are poorly studied entomopathogenic bacteria. Their distinctive feature is the presence of a canoeid inclusion attached to the spore. We have previously studied and characterized strains of Bl capable of forming paraspore crystals [4]. The studied Bl strains exhibited crystalline inclusions of different shapes and sizes, similar to those described for the entomopathogenic Bt bacteria. The highest activity of Bl was detected against insects of the Díptera order. This includes mosquitoes of the genera Aedes, Anopheles, Culex and black flies (Simulium vittatum). Many mosquito species are vectors of malaria, yellow fever, dengue fever, haemorrhagic fever and lymphatic filariasis. Synthetic insecticides have adverse effects on humans and nature. In addition, resistance to such insecticides has been found in pathogen-transmitting mosquitoes.
An alternative to address these problems and limitations is biological control using entomopathogenic bacteria. Bacillus thuringiensis subsp. israelensis (Bti) was the first subspecies of Bt to be toxic to twoflies [5]. A study on the nature of the insecticidal factors showed that insects of the Diptera order, mosquitoes and black flies, are sensitive to the paraspore inclusion of Bti, which consists of four proteins Cry4Aa, Cry4Ba, Cry11Aa, Cyt1Aa [6]. Ben-Dov [7] reported that the larvicidal activity consists of four major (134, 128, 72 and 27 kDa) and at least two minor (78 and 29 kDa) polypeptides encoded by the cry4Aa, cry4Ba, cry11Aa, cyt1Aa, cry10Aa and cyt2Ba genes mapped to a 128000 bp plasmid known as pBtoxis. These δ-endotoxins form a complex paraspore crystalline body with extremely high specificity. Electron microscopy has shown that Bti cells form inclusions that consist of different segments, an osmiophilic or lightly stained, crystallized lattice segment with a period of 4.3 nm and an osmophilic, stained lattice segment with a period of 7.8 nm. The complex of several crystals is enclosed in a common shell [8].
The crystalline inclusions of Bl can have a cubic shape. We wrote about cubic shape of crystals earlier [9]. In the present paper we show the results of study of rhombic crystals in Bl cells. This work is devoted to a study of the process of crystal formation in Bl during sporulation and to the analysis of the structure of crystals.
METHODS AND MATERIALS
In this work we made and used the following:
Strain B. laterosporus (Bl) 16–92 was isolated from a dead insect.
The strain was grown on NBY agarized medium for 48–96 h at 30 °C.
Electron microscopy.
Scanning electron microscopy (SEM).
The native spore-crystalline suspension was examined using a Quanta 2003D electron ion scanning microscope (FEI Compani, USA). Samples of the native suspension were placed on a silicon substrate, which was fixed to an aluminum table using carbon tape. The preparations were examined in high and low vacuum. The surface of the sample was scanned at an accelerating voltage of 5–30 kV.
Transmission electron microscopy (TEM)
Bacilli of strain 16–92 were washed off the agar and fixed first by the Ito-Karnowsky method [10]. The material was then fixed in 1% OsO4 solution on 0.2 M cacodylate buffer and in 1% uranyl acetate solution on 0.2 M maleate buffer. The material was dehydrated in alcohol concentrations of 50°, 70°, 96°, 100°. The material was then placed in a mixture of 100° alcohol and LR White resin and then in pure LR White resin. The material was transferred into gelatin capsules, which were placed in a thermostat at 56 °C. Sections were obtained on an LKB III ultra-tome (LKB, Sweden) and contrasted with a 1% solution of uranyl acetate in 70° alcohol and citric acid lead. Spore and crystal suspensions for negative contrast studies were applied to carbon-film-coated grids and stained with 2% aqueous NANOWtm. The preparations were examined on electron microscopes JEM -100 B and LEO912AB.
The images were processed using FemtoScan Online software [11]. A two-dimensional Fourier transform was used to determine the lattice parameters of the observed crystals.
RESULTS
Crystal formation in Bl was studied: from the first stages up to the stage of free crystals. To detect crystals, a spore-crystalline suspension of strain 16–92 was examined using SEM. Figure 1 shows mature spores with a canohedral inclusion and rhombic crystals. The crystals were either bound to the spores or were in a free state.
Electron microscopic examination of ultrathin sections of strain Bl 16–92 by TEM showed that the growing, dividing cells did not contain crystals. Crystals appeared in vegetative cells before entering the sporulation stage. Thereafter, the crystal-forming cells went through all the stages of spore formation. In Fig.2 a prospore can be seen, at some distance from which a rhomboidal crystal is located. In Bl crystals are formed without connection to the spore structures. Near the crystal there is a rarefaction of the cytoplasm.
Lysis of the sporangium cytoplasm around the spore and the crystal increases as the spore matures. In addition to the crystalline inclusion, an overgrowth of a lamellar canoeid inclusion is observed. Fig.3 shows that the cytoplasm of the sporangium is completely lysed and the cell membrane is preserved in most spores, but partially destroyed in some. Figure 4 shows the spore with canoeid inclusion and a crystal after leaving the sporangium. As in Bt, spores and crystals of Bl are released separately during sporangium lysis.
Two or more crystals form in some Bl cells. This can be seen in Fig.5: a crystal of complex structure is distinguishable.
The crystals are enclosed in a common shell (Fig.5, 6, 7, 8).
The crystals have the same electron density, which distinguishes them from the Bti crystal complex. Bti has several crystals in a common shell with different electron densities.
The complex structure of Bl crystals was confirmed by further studies.
The area of crystalline inclusions visible in negative contrast has a diamond shape, as shown in Fig.9.
It should be noted that the observed diamond-shaped region of 320 × 540 nm (Fig.9) is not a single crystal but rather a cluster of individual smaller crystals. The size of single crystals ranges from 25 to 120 nm. If we consider the surface of a single crystal, the Fourier image rather shows hexagonal packing of proteins in the crystal (Fig.10). Accordingly, 6 reflexes are observed in the Fourier image. The reflexes are not located on a circle, but on an ellipse. There could be two reasons for this:
the protein molecules that make up the crystal have an elongated shape;
it is a hardware effect (artefact).
The presence of several possible arrangements of protein crystals is observed, for instance, in the case of lysozyme. Under different crystallization conditions, a tetragonal or orthorhombic arrangement is obtained [12].
In the case of crystals Bl, the presence of two packings shows that the biochemical composition of the inner compartment of the cell, where crystallization takes place, should differ more or less considerably. This observation shows that the inclusion of Bl is similar to that of Bti.
ACKNOWLEDGMENTS
The study was completed with the financial support of the RFBR and the London Royal Society No. 21-58-10005, RFBR, Project No. 20-32-90036, RSF, project No. 20-12-00389 and from the Foundation for the Promotion of Innovation, Project No. 71108.
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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