Proteomics of Bacillus subtilis: An Overview microbiologystudy

Bacillus subtilis is an aerobic, endospore-forming, gram-positive, rod-shaped soil bacterium widely used for heterologous protein production.

• They are typically 2–6 µm long and less than 1 µm in diameter. The optimum growth temperature of B. subtilis is about 30–35°C.
• B. subtilis is present in unfavorable habitats, such as soil, the gut of terrestrial and aquatic animals consist mammals, industrial installations, and healthcare facilities.
• It has a great protein secretion ability that makes the host secrete extracellular or medicinal proteins and industrial enzymes to degrade various substrates.
• B. subtilis, an ideal multifunctional probiotic, has excellent physiological characteristics and a highly adaptable metabolism.
• Under poor nutrient conditions, it produces a heat-resistant endospore that allows the bacterium to thrive in a continuously changing environment.
• B. subtilis is a model organism for research on gram-positive bacteria or the study of gut and soil microbiomes that helps to understand the sporulation and cell differentiation, biofilm formation, and identify pathogenicity in related pathogens.
• The highly adaptable metabolism properties of an organism make it easy to cultivate on cheap substrates. Cells can easily grow in minimal media with glucose, maltose, cellobiose, or starch.
• Moreover, It helps prevent the growth of pathogenic bacteria and enhances nutrient assimilation.
• B. subtilis is also commonly used as an industrial cell factory for enzymes, vitamins, inositol, acetoin, hyaluronan, and other chemical productions.

Proteomics of Bacillus subtilis

• In 2000, the protein secretion studies in B. subtilis introduced the term ‘secretome’ for the first time.
• The analysis of combined proteomics and molecular biology showed that B. subtilis secreting proteins can be used for different purposes, such as nutrient acquisition and competition with other organisms.
• Proteomics helps to determine the composition of the extracellular proteome (exoproteome), the cell wall proteome, and the membrane proteome. It demonstrates that these sub-proteomes of the cell are highly dynamic entities that alter in response to the growth stage, nutrient availability, and stresses caused by the overproduction of secretory proteins.
• B. subtilis has a single-cell membrane. It makes the proteomic analysis of protein secretion easier, simplifies downstream processing, and reduces the process costs. They are generally recognized as safe (GRAS).
• Bacterial secretory proteins have vital functions, such as cell-to-cell communication, supply of nutrients, environmental detoxification, and killing of potential competitors.
• The secreted proteins of B. subtilis usually contain N-terminal signal peptides essential for export from the cytoplasm. The N-, H-, and C-regions are three characteristic domains of signal peptides. The protein translocation machinery recognizes the signal peptides of secreted proteins. The general secretory (Sec) pathway and the twin-arginine (Tat) translocation pathway are the best pathways for secretory proteins in B. subtilis.
• Three functional stages can be distinguished in B. subtilis protein secretion via the Sec pathway: targeting, translocation, and folding and release.
• According to the B. subtilis genome project, the chromosomal DNA of B. subtilis is 4215 kb in length, and 4100 genes that encode proteins and peptides have been predicted.
• 2D polyacrylamide gel electrophoresis (2D PAGE) separates extracellular proteins and N-terminal sequencing helps to identify extracellular proteins. The visualization of about 200 extracellular proteins can done by two-dimensional (2D) gel electrophoresis and by mass spectrometry using different growth conditions and mutant strains.
• Using classical genetic and biochemical approaches, the protein secretion by B. subtilis includes the export of cytoplasmic proteins, processing of native membrane proteins by type I signal peptidases (SPases), and the release of cell-associated lipoproteins and cell wall proteins into the growth medium.
• During the post-exponential growth phase, the cells of B. subtilis grown in rich media secrete the protein at a high level. At this period, the extracellular proteome of a B. subtilis degU32(hy) mutant exhibits higher extracellular levels of a subset of 13 degradative enzymes.
• Chu et al. identified five extracellular proteins of B. subtilis strain K-1- specifically induced by growth in the xylan-containing medium.
• Proteomics can be used to identify extracellular proteins. Park et al. used a proteomic approach that detected four protein spots with fibrinolytic enzyme activity in the medium of B. subtilis 168. The 2D PAGE gel images should overlaid to detect extracellular protein spots that coincide with clearing zones on a 2D zymogram gel containing bovine fibrinogen.
• One finding by Antelmann et al. on the protein composition of the cell wall defines seven LiCl-extractable proteins present in this compartment of the B. subtilis cell. These proteins comprise the known cell wall-bound proteins LytB and LytC (both involved in cell wall biogenesis), the CWBP23- and CWBP52-processing products of WprA (cell wall-located protease), and processed forms of WapA (structural cell wall-binding protein). The flagellum-related protein Hag and two proteins with unknown functions, YwsB and YqgA, are present in the cell wall proteome.
• Shotgun proteomics provides valuable information from large-scale analysis of protein expression. It is applied to identify the cytosolic proteome and a semi-gel-based approach. This approach helps to identify membrane proteins of vegetative Bacillus subtilis cells. To analyze the heat shock response in B. subtilis, iTRAQ reagents were used.

References

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