What type of bacterial spores exist

In the event of extreme environmental conditions, some gram-positive bacteria, which include the majority of microorganisms pathogenic to humans, form extremely stable bodies of a spherical or elliptical shape — spores.

The nucleation and maturation inside the mother cell gave reason to call them endospores. It is recognized that bacterial spores are the most stable form of life on Earth [1,2].

They fulfill the important function of preserving the bacterial population from extreme environmental influences and are a key factor in the virulence of infectious agents. In some pathogenic species of Bacillus spp. The fight against bacterial spores of pathogenic bacteria in medical institutions is also an acute problem associated with the prevention of nosocomial infections [3,6,7].

In particular, some pathogenic species of Clostridium spp. Among the detected endospores, two categories were identified: those that are enclosed in a spherical outer shell exospore, exosporium , and which do not have this structure. In contrast to the well characterized endospores, knowledge of the role and structure of exosporia is less distinct [5,].

The relevance of studying the listed functions of bacterial spores in the epidemiology and pathogenesis of dangerous infections is associated with the study of their spatial organization, as well as the molecular mechanisms of the formation of dormant resistance. This is important for the development of new sterilization and detection strategies, as well as the use of these unique biological systems as promising models for modern biotechnologies [8,15].

Endospore: A dormant, non-metabolic, and nonreproductive structure. The ability of microorganisms to spore formation was one of the key characteristics of their early classification [16]. Sporulation, on the one hand, is a form of a bacterial reaction to environmental changes, and on the other hand, a strictly regulated sequence of stages of gene expression and the process of cellular morphological differentiation.

As a result of spore formation, metabolically inactive cellular forms dormants are formed, the morphology of which is radically different from the maternal vegetative cell []. The formation of spores is one of the manifestations of the numerous mechanisms of adaptation of microorganisms to carbon and nitrogen starvation, but unlike other adaptive strategies of bacteria, the formation of endospores lasts on average about 8 hours and proceeds in several stages [].

Sporulation, as a rule, begins in the stationary phase of the vegetative cell cycle, is initiated by the depletion of nutrients, and is a morphogenetic process of assembly of supramolecular structures inside the cytoplasm of a vegetative cell sporangia. The formation of spores begins with a signal for the vegetative cell to start a cascade of synthesis of transcription factors that control the sporulation stages.

This transformation is accompanied by successive and significant changes in the morphology, biochemistry, and physiology of bacteria and culminates in the transformation of a vegetative cell into a resting form [11,]. At the beginning of the XXI century, it became apparent that many bacterial reactions to environmental conditions are regulated by the so-called multicomponent molecular genetic systems [16,]. One of these systems, in response to food stress, triggers the activation of sporulation genes, among which the key is spo0A, which encodes the synthesis of Spo0A protein [22,32,33].

As a result, a vegetative cell undergoes a complex, but clearly defined sequence of morphological and biochemical events, which ultimately lead to the formation of mature endospores Figure 1.

After completion of spore formation, the mother cell undergoes programmed autolysis, releasing a mature spore into the environment [36]. The extreme resistance of endospores to extreme environmental conditions is still under study.

However, the mechanism of their heat resistance, apparently, is one of the most complexes. Mature endospores are in a state of reproductive and metabolic rest, reduced enzymatic activity and low content of high-energy compounds ATP and NADH. The presence of several additional membranes compared to a vegetative cell is a barrier against the penetration of water and substances dissolved in it. Dehydration protects the inside proteins from denaturation and irreversible aggregation at extremely high temperatures, which mediates the ability of spores to survive for a long time tens, hundreds or more years without a supply of nutrients under conditions when vegetative cells die [19,].

Heat resistance of endospores is key mechanism of to their survival and preservation of the bacterial population. However, studies conducted by R. Scheldeman and W. Schubert with colleagues showed that endospores can survive after standard thermal sterilization methods, and special heat treatment regimens are necessary for their destruction [40,41]. Figure 1: The sequence of morphological changes occurring at different stages of sporulation in gram-positive bacteria: initiation of sporulation by nutrient deficiencies, replication of chromosomes stage 0 ; the formation of an axial thread of chromatin and condensation of chromosomes at the poles of the cell stage I ; stage II — the formation of an asymmetrically located partition that creates two compartments and separation of nuclear material; stage III — the formation of the primary dispute and sporangia; stage IV — spore bark formation; differentiation of prospore and sporangia; stage V — formation of the outer spore shell; stage VI — spore maturation; stage VII — maternal cell autolysis and spore release.

High heat resistance of endospores is provided by dipicolinic pyridine-2,6-dicarboxylic acid DPA , which forms a chelate complex with calcium and other divalent cations and reduces the water content in spores to a very low level [21,39,42,43]. Despite the fact that DPA was found in bacterial spores quite a long time ago [43], the function of this interesting chemical structure has been not finally determined [19,44,45]. The high concentration of dipicolinic acid and its specificity for bacterial endospores have made this component the main object of scientific research.

At the beginning of the XXI century A. Driks and P. Setlow suggested that this acid, intercalated with DNA and RNA via covalent bonds, forms a gel-like polymer matrix that protects nucleic acids from damage at high temperatures [31,46]. The leading role of DPA in ensuring dehydration [37], creating and maintaining metabolic rest [7], as well as stabilization of basic nuclear proteins undisputed [38,44] and minimizing the likelihood of their denaturation, aggregation, and lysis [7,37,38,44] is indisputable.

During germination, DPA is released from spores, providing the reverse process of hydration of the nucleus and the formation of vegetative cells [19,37]. The unique properties of DPA of endospores have allowed modern biotechnologists to consider it as a promising excipient excipient to increase the stability and highly effectiveness of biopharmaceutical liquid protein preparations and prevent their non-specific aggregation, for example, antibodies [38].

Morphological observations of the changes occurring at the stages of sporulation showed that the formation of the spatial structure of the spore membranes begins with the process of invagination of the cytoplasmic membrane and the final separation of the prospores from the mother cell.

The mature endospore has a concentric multilayer structure, while the number of protein layers varies in different types of bacteria. Its main structural components are the core surrounded by two layers of structurally distinct peptidoglycans in the inner shell, which is characterized by low permeability for small molecules and water [16,25,51], and cortical peptidoglycan [27].

Cortical peptidoglycan structurally corresponds to a similar cell wall polymer of a vegetative cell. These proteins, functioning as a molecular barrier, protect spores from ultraviolet radiation and active oxygen radicals [17,54,55] and serve as a source of amino acids for spores during their germination period [7,55].

Thanks to the use of mass spectrometry in the outer shell, more than 70 different proteins were found that are difficult to separate due to chemical crosslinking that protects endospores from the destructive effects of hydrolytic enzymes and strengthens the structure of the shell. The assembly and arrangement of these protein structural components of endospores ensures their survival and preservation in anticipation of suitable conditions for germination [7,21,56,57].

Scientific and practical interest in disputes is caused not only by their biochemical structures mediating molecular stability strategies, but also by the mechanisms of regulation of their transformation into vegetative forms under favorable conditions. Such conditions occur when spores enter humans or animals, where they germinate and become pathogens of dangerous infections. For example, due to the germination of B.

Like sporulation, spore germination is an equally complex and strictly regulated process that proceeds in several stages [37,45,61]. Spore germination in nature is initiated by nutrients amino acids, carbohydrates, purine nucleosides, or combinations thereof.

Nutrient substrates interact with the receptors of the inner membrane of the spores and start the germination program, which at some point becomes irreversible. The program begins with the release of dipicolinic acid, hydrogen ions and divalent cations and their replacement with water, which causes an increase in the pH of the core from 6.

These changes are important for the subsequent increase in enzymes activity and, ultimately, the metabolism of future cells, accompanied by macromolecular synthesis. In this case, the spore swells and grows in size not only as a result of water absorption, but also as a result of cell growth due to reserve material [5,14,45,62]. Farther, the membranes break under the influence of pressure caused by growth, and a new vegetative cell emerges from the destroyed spore membrane [57].

Partially based on this germination mechanism, a fractional sterilization method is based. Even if the spore has not grown, but hydration has occurred, the process is irreversible; she must first become a vegetative cell [45,56].

For the germination of endospores, the external glycoprotein membranethe exosporium exospore , which provides their connection with the environment, is of particular importance.

The Spatial Organization of Exospores: As already mentioned, in some endospores of spore-forming gram-positive bacteria, an additional outer protein layer, the exosporium, forms a barrier to the environment during sporulation [3,18,63,64,65].

In recent years, studies of exosporium have been carried out mainly on isolates of three main closely related species of spore-forming bacteria Firmicutes type included in the Bacillus cereus sensu lato group: B. In addition to them, exospores form some species of Clostridium spp. The exospore architectures of bacteria in these groups have a similar morphology [13].

Usually they are a flexible, but strong shell — a thin continuous protein basal layer, which has an outer, hair, and inner, crystalline layers. The subtle features of exosporia can vary under different growth conditions and in different types of bacteria [68,]. The use of modern methods of molecular biology has significantly expanded the understanding of the biochemical structure and spatial organization of the exosporial structures of the Bacillus spp family and Clostridium spp.

Figure 2. The exosporium of these bacteria is a thin and flexible shell structure, which is usually much larger than the dense endospore located inside [5,14].

The main structural element of exospores in different types of bacteria is a thin crystalline basal protein layer [12,13,80]. It turned out that this glycoprotein plays a significant role in protecting spores from phagocytosis [21,78,79]. In addition, it was recently shown that it mediates the mechanism of immune inhibition, which contributes to the preservation of spores in the lungs of mice [1,83].

The region between the basal layer of the exosporium and the outer shell of the endospore is called the intermediate space [26,60] and is approximately nm [67]. In some places, the basal layer is located in close proximity to the outer layer of the endospore membrane. The exosporium basal layer has a thickness of approximately 12 to 16 nm and consists of two sublayers approximately 5 nm thick [26,67].

The basal layer has a crystalline structural organization with 6-fold symmetry and a periodic interval of 7 nm. The outer surface of this structure consists of a series of hexagonal concave cups in the form of honeycombs, with open ends oriented outwards [17].

This channel structure provides the barrier properties of exosporia [17,67,84]. Hairy filaments of exospore, usually consisting of BclA protein, have a length of 14 to 70 nm and cover the entire surface of the outer shell of the basal layer [67,81,82,86,87].

Recent studies have shown that, unlike other bacteria of the B. Synthesis of Exosporium and its Biochemical Structure. The biochemical structure of exosporium, which differs significantly from the structure of endospores, has been the subject of many reviews [13,26,88,92].

The initiation of synthesis occurs at the central pole of the spore. First, the embryo of the future exospore appears in the form of a small layered structure in the spore-forming mother cell. There are variations in endospore morphology. Examples of bacteria having terminal endospores include Clostridium tetani, the pathogen that causes the disease tetanus.

Bacteria having a centrally placed endospore include Bacillus cereus, and those having a subterminal endospore include Bacillus subtilis. Sometimes the endospore can be so large that the cell can be distended around the endospore. This is typical of Clostridium tetani. When a bacterium detects environmental conditions are becoming unfavorable it may start the process of endosporulation, which takes about eight hours.

The DNA is replicated and a membrane wall known as a spore septum begins to form between it and the rest of the cell. The plasma membrane of the cell surrounds this wall and pinches off to leave a double membrane around the DNA, and the developing structure is now known as a forespore. Calcium dipicolinate is incorporated into the forespore during this time. Next the peptidoglycan cortex forms between the two layers and the bacterium adds a spore coat to the outside of the forespore.

Sporulation is now complete, and the mature endospore will be released when the surrounding vegetative cell is degraded. While resistant to extreme heat and radiation, endospores can be destroyed by burning or by autoclaving.

An indirect way to destroy them is to place them in an environment that reactivates them to their vegetative state. They will germinate within a day or two with the right environmental conditions, and then the vegetative cells can be straightforwardly destroyed. This indirect method is called Tyndallization.

It was the usual method for a while in the late 19 th century before the advent of inexpensive autoclaves. Prolonged exposure to ionising radiation, such as x-rays and gamma rays, will also kill most endospores. This results in the creation of two compartments, the larger mother cell and the smaller forespore.

These two cells have different developmental fates. Intercellular communication systems coordinate cell-specific gene expression through the sequential activation of specialized sigma factors in each of the cells. Next Stage III , the peptidoglycan in the septum is degraded and the forespore is engulfed by the mother cell, forming a cell within a cell. Finally, the mother cell is destroyed in a programmed cell death, and the endospore is released into the environment.

The endospore will remain dormant until it senses the return of more favorable conditions. Some Epulopiscium -like surgeonfish symbionts form mature endospores at night. These spores possess all of the characteristic protective layers seen in B. These are the largest endospores described thus far, with the largest being over times larger than a Bacillus subtilis endospore.

The formation of endospores may help maintain the symbiotic association between these Epulopiscium -like symbionts and their surgeonfish hosts.

Since endospore formation coincides with periods in which the host surgeonfish is not actively feeding, the cells do not need to compete for the limited nutrients present in the gut at night.

The protective properties of the endospores also allow them to survive passage to new surgeonfish hosts. The fish may also benefit from this relationship because it is able to maintain stable microbial populations that assist in digestion and may receive a nutritional gain from microbial products released during mother cell death and spore germination.

Endospore formation in some Epulopiscium -like symbionts follows a daily cycle: A Polar septa are formed at the poles of the cell.

B Forespores become engulfed.