There are numerous reports of vesicle production in Gram-positive bacteria including Staphylococcus aureus, Bacillus anthracis and Mycobacterium tuberculosis, where vesicles may play a role in virulence. We established that Bacillus subtilis produces vesicles using a variety of techniques including light scattering, metabolic labeling, and electron microscopy. Vesicles produced by B.subtilis contain proteins, associated with the cellular membrane, suggesting a possible site of origin. Proteins encased in these vesicles belong to a variety of pathways suggesting an involvement in metabolism, iron transport, and antibiotic resistance. B. subtilis could provide a good model for studying the mechanism of vesicle production in pathogenic and hard to work with Gram-positives. It also shows that vesicle production is an evolutionary conserved event, also observed in Gram-negative bacteria, mammalian cells, and archaea.
Objective
The objective of the experiment is to determine the production of extra cellular vesicles from bacillus subtilis. These extra cellular vesicles are produced in samples in the natural environment and not in samples in the controlled environment. The set-up of the experiment is very simple; we are trying to establish if the cells produce the vesicles. If the cell is responsible for producing the vesicles, the molecules will not get inside the cell. If the cell is not producing the vesicles, the cell will form a cover around the vesicle produced. The experiment aims to establish the necessary conditions that have to be present in order for the B.subtilis to produce these extra cellular vesicles. The bacillus subtilis vesicles range in size from 48mm to 400mm in diameter. In order to get an accurate measurement, the vesicles are sliced to look at the membrane instead of looking from the top. The vesicles measured range from 50mm to 200mm in diameter. When using dynamic light battery to measure the size, the median got is 125mm. this is below the average from other methods, which is 200mm.
This is because in dynamic light battery, we are trying to measure a three dimensional diameter from a two dimensional image. A correction factor is applied to the measurements, which gives an average diameter of 150mm, which is still below the 200mm. Our initial studies on extracellular enzyme synthesis in B. subtilis focused on a characterization of several of the major secretory proteins. In this experiment, we describe a biochemical analysis of the extracellular and membrane-bound forms of the major proteases in the same hyperactive secreting strain. These results emphasize that B. subtilis appears to contain membrane-bound forms of all secreted enzymes. It has been established that the vesicles have many proteins. These proteins are of anti-biotic in nature and we are trying to establish if the same can be found in the natural environment as compared to the controlled environment. The set-up of the experiment is very simple; we are trying to establish if the cells produce the vesicles. If the cell is responsible for producing the vesicles, the molecules will not get inside the cell. If the cell is not producing the vesicles, the cell will form a cover around the vesicle produced. Membrane-bound alkaline and neutral proteases were indistinguishable from the extracellular enzymes by the criteria of molecular weight, immuno-precipitation, and sensitivity to inhibitors.
These experiments were undertaken to characterize the extracellular and membrane-bound. Proteases from a hyper-secreting Marburg strain of B. subtilis. Information on the time of synthesis and structure of proteases is needed to examine the process of secretion. An alkaline serine protease and a neutral metallo-protease account for more than 99% of the protease activity of the culture supernatant from controlled strain. Previous reports have described extracellular alkaline and neutral proteases and an esterase having low protease activity from various strains of B. subtilis M proteases in controlled strain, are approximately 15- to 30-fold greater than the levels in the natural strains. The biochemical bases for these effects are presently obscure. Perhaps because the level of secreted esterase was very low, it appears that the membrane bound form was not detected. The possibility that protease M3 or M4 is a membrane-bound form of the secreted esterase cannot be completely excluded until these enzymes are purified to homogeneity.
Conclusion
In the natural environment, most of the vesicles are outside of the cell. In the natural environment, vesicles have few molecules formed inside the cell. Vesicles in a controlled environment have many molecules of the protein formed inside the cell. It has also been established that natural cells do not have the ability to break up the media. They have a large median size of 125mm. this is unlike the cells in a controlled environment, which have the ability to break up the media and come back together to form smaller, cell sizes. Natural cells have the ability to perform in a large variety of sizes. When cells have been split, they lose the ability to produce vesicles. In the natural environment, the cells are able to produce vesicles. When one gene is removed from the bacillus subtilis cell, it loses the ability to produce these vesicles. If the cell without the gene is re-introduced into the natural environment, they are able to produce the vesicles again. This shows that the bacillus subtilis cell has the ability to acquire the gene responsible from other organisms in the environment.
