B.
subtilis
is a bacteria that a member from genus Bacillus. This bacteria is a
gram-positive, rod-shaped and found in different environment such as
terrestrial, aquatic or animal intestine (Earl et al., 2008). Many of studies used the bacteria as a
model gram-positive bacteria, because the process of chromosome duplicate
rapidly and accurately (Graumann,
2012).
Therefore, the bacteria used to investigate the expressed protein as a host.
The bacteria could grow in nutrient media and salt condition, sugar as a carbon
source, and ammonium salt or other amino acid as nitrogen sources (Harwood and Wipat, 2013),
however, under lack of nutrient and environmental stress conditions, induced
bacteria enhances to use metabolic activity and forms endospore to survive. In
that time, it secretes protein into the medium, however it doesn’t produce
endotoxins (Shahcheraghi et al., 2015),
therefore, the bacteria is not pathogen against mammalian (Dubnau, 2012; Shahcheraghi et al., 2015).
Because of that, the bacteria mostly used in laboratory studies to understand
protein expression from recombination, and applied as vaccines technology.
The life cycle of B.
subtilis is happened of three stages, there are vegetative, sporulation,
and germination stage (Fig. 1)(Sella et
al., 2014).
This stage is affected of environment conditions, especially nutrient
availability. B. subtilis quite often found in vegetative form rather
than in endospore form (Earl et
al., 2008).
When the nutrient is available, the bacteria would be in the vegetative stage
which indicated from binary fission cell growth. In this process, the bacteria
active to do replication lead to cell division. However, if the environment is
not suitable for the B. subtilis, the bacteria will form layers to
protect itself against stress conditions, this process is called sporulation (Sella et al., 2014).
Some environments
like high mineral composition, lack of nutrient, neutral pH, temperature, and
high cell density tend to induce the vegetative cell into endospore (Sella et al., 2014). According to
Sella et al. (2014), there are seven steps of sporulation, after the bacteria
finish the DNA replication and contain two chromosome, it will stretch to
different axis (axial), second, the division begin asymmetrically near one of
the poles, and forming a septum (Hilbert
and Piggot, 2004; Sella et al., 2014). Third, the
chromosome get engulf from the septum which differentiate the mother cell and
small forespore. This mother cell provides the needs of forespore development
into spore. Fourth, the forespore has form three layers, inner, cortex, and
outer layer. Inner and outer layer is
composed of protein, while the cortex consists of a thick layer of peptidoglycan. Thus, the
coat is synthesized on the outer layer. The coat is composed from ~80 of mother
cell protein which can protect the spore against stress condition. Sixth, the
spore doesn’t have any morphological change, but it get maturated (Hilbert and Piggot, 2004).
Finally, the mother cell get lysis of lytic enzyme which releases the mature
spore into the environment. Therefore, the spore which can survive in the
extreme conditions has the suitable advantages to display antigen.
The germination occurs when the environment has
suitable, or the nutrient has available lead to break the dormancy of endospore
(Sella et al., 2014). This process is
started of initiation of spore by germination molecules such as amino acid,
sugar or purine nucleosides are detected by the receptors, known as germination
receptors (GRs). The binding of molecule with GR will induce the ion H+, K+,
Na2+, and Ca2+ pumped out lead to increase the pH of the core to suitable pH.
The lytic enzymes activated causing of cortex degradation. The next step, the
pyridine-2,6-dicarboxylic acid (DPA) compound is degraded and released lead
water to enter the spore core (rehydration), thus initiate the protein and
activate the process, followed spore coat hydrolysis will generate new
vegetative cell (Sella et al., 2014).
 Fig. 1 Bacillus subtilis sporulation (Hilbert and Piggot, 2004) |
The endospore structure consists of coat layer, outer
membrane, cortex, germ cell wall, inner membrane, and core (Fig. 2) (Sella et al., 2014). The coat layer
is the outer of the layer which can protect the spore against stress condition.
This layer majorly composes of protein
and minor carbohydrate components. Approximately 70 spore specific protein has
been studied in the spore coat, and each of them has covalent modifications (Henriques and Moran, 2007; Plomp et al., 2014). Most
of protein in the layer of coat don’t have specific function, but most
importantly, the proteins which are essential for coat morphogenesis such as
CotE, CotH, CotO, SafA, and SpoVID (Plomp
et al., 2014). The protein has been known acted as carrier for
displaying bioactive protein especially antigens (Sella et al., 2014). Therefore, the features may apply to
make as a vaccine. The spore surface may determine the environmental conditions
through protein of the surfaces in resulting adapted spore.
The outer membrane envelops the cortex layer. The
membrane is to protect the inner layer. Between outer and inner membrane there
were cortex and germ cell wall which composed of peptidoglycan, however, the
peptidoglycan in cortex has structural modifications, while in germ cell wall
is mostly identical to vegetative cell (Sella et al., 2014). Inside the inner membrane, there is core
which contains nucleotides, ribosomes, and enzymes, but the enzymes is dormant
because the core is relatively dehydrated. The conditions inside the core is
related to the tolerance of the spore which prevent DNA damage (Sella et al., 2014).
Fig. 2. Scanning electron of Bacillus spores (A), and
vegetative cells (B) (Sella et al., 2014)
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