Ultrastructural analysis of Brevibacillus laterosporus bacterial cells using electron and atomic force microscopy
In contrast to some other bacilli, including entomopathogenic, spores of B. laterosporus don’t have exosporium. Using SEM it was shown that such spores have folded uneven surface. At the same time, the structure of spores of B. laterosporus is strain-specific. For example, spore of B. laterosporus 16-92 is surrounded by a fibrillar capsule, which is absent in other strains. Canoe-shaped inclusion has different size and structure at different strains. The crystals produced by B. Laterosporus differ in a shape, size and biological activity .
Despite intensive electron microscopic studies of the morphology of this type of bacilli, details of the ultrastructure of spores and crystals are poorly studied due to the impossibility of working with the native material. As is known, the technique of sample preparation in electron microscopy includes fixation, coloration, dehydration, filling with resin. These methods violate the structural integrity of biological objects and do not allow to accurately determine their sizes. Alternative high-resolution method of studying the native ultrastructure of samples, including bacterial spores, is atomic force microscopy that enables to visualize the architecture of spores at the molecular level per se, without chemical and physical influences.
In this regard, the comparative study of the spores and crystals by electron and atomic force microscopy are of interest.
MATERIALS AND METHODS
The strains of B. laterosporus were obtained from the laboratory collection of the Institute of Genetics (16-92) and collection of the Institut Pasteur (Paris) from Dr. Lecadet (Lat 006). Strains were grown on agar NBY during 72 h at 30 ⁰C. Spores and crystals for studies were washed from the agar, suspended and washed in distilled water.
Electron microscopy was carried out by the methods of negative contrasting and ultrathin sections as described in . The suspension of spores and crystals was prepared for negative contrast in two ways. In the first case, the suspension was applied on a copper formvar-coated grids, reinforced with carbon and stained with 1% aqueous uranyl acetate. In the second case, the suspension was applied to a grids covered with carbon film and stained with 2% aqueous solution of NANOW. Electron microscopes JEM-100 B and LEO912AB were used.
FemtoScan multifunctional scanning probe microscope  and FemtoScan Online software  were used for atomic force microscopy.
Fine structure of the spores and crystals was previously studied by the method of ultrathin sections [1, 2]. Fig.1a and Fig.1b show spores in the sporangia and free spores. In addition to spores, there is a rhombic crystal in sporangia. Spores are bonded with layered canoe and surrounded by a spore sheath and cortex. Canoe is joined to a spore at one side. On the opposite side of spore only folded spore shell is visible. It is evident in the presented images that canoe of strain 16-92 is narrow with tightly packed layers.
Ultrastructure of a strain of B. laterosporus Lat 006 has its own features. A cubic crystal is observed in sporangia near the spore (Fig.1c). The mature spores of the strain Lat 006 have a large friable canoe (Fig.1d). Layers of canoe, probably due to the lack of rigidity, are arranged in waves. A lamellar, square crystals with rounded corners are discovered at Lat 006. During lysis the sporangium with canoe-shaped inclusion and crystals are released separately.
Fig.2 presents images of electron-dense oval spores of B. laterosporus 16-92 with canoe-shaped inclusion.
When using contrasting by means of NANOW, on a surface of spores of a strain 16-92 it is possible to observe numerous long thin outgrowths (Fig.3) which are absent at a strain Lat 006 (Fig.4). Fig.4 shows a large canoe with submerged spore Lat 006. The square electron-dense crystals are also visible in this image.
Spores and crystals of B. laterosporus are studied less than other entomopathogenic bacteria. In particular, it concerns a canoe which function isn't defined, and crystals, whose activity towards different biological objects is poorly studied.
Three-dimensional image of spore of B. laterosporus on the surface of cleaved mica was obtained using a FemtoScan scanning probe microscope (Advanced Technologies Center, Russia). Spores of B. laterosporus were studied using the atomic force microscopy (Fig.5). The observed size of the spore was about 1.5 µm. The construction of the image is performed in the FemtoScan Online software .
Atomic force microscopy allows to obtain information about three-dimensional topography and to estimate the volume of spore at her drying in the air.
The strains of B. laterosporus are specifically active against the larvae of various dipteran insects, in particular against mosquitoes An. stephensi и Ae. Аegypti. High biocidal effect (antibacterial, fungicidal, cyanolytic) allows the use of these strains as a producers of biological plant protection from disease and for fight against toxic microscopic algae (blue-green bacteria). ■
The study was performed with financial support of RFBR in the framework of a research project No. 15-04-07678.