Vesicles In Gram Positive Bacillus Subtilis Essay Example

Abstract. The aim of the study was to validate the existence of vesicles in gram positive bacteria, bacillus subtilis. The visualization of the vesicles was carried out by various microscopy techniques. The measurement of vesicle size was performed and the amount of vesicles compared between various strains. The laboratory cultured strains was more effective in the study as they exhibited hyper production of the vesicles. The production of vesicles was also validated in environmental strain, however in much smaller amount. The vesicles were also visualized and isolated from biofilms that act as the natural environment of the bacteria. Proteomic analysis on the vesicle prep demonstrated the existence of twenty four proteins, including five siderophore binding proteins. Radio labeling technique was used to quantitate the level of the vesicles in lab strain vs. the environmental strain. It was also demonstrated that a gene, sfp; was deleted in lab strain and was its non functional form was associated with the hyper production of vesicles in lab strain. This sfp gene is possibly the first vesicle associated gene identified. The vesicles can be utilized as adjuvant agents for vaccine development and identification of therapeutic and vaccine targets.
The visualization of the vesicles was carried out by electron microscopy techniques. The lab strain was used to visualize the vesicles as large amount of vesicle were produced in the lab strain. The author used embedded transmission electron microscopy to visualize the vesicles by slicing through the pellet and looking through the membrane. A greater number of putative vesicles were identified from the lab strain as opposed to the environmental strain which differed in size and density. 3D versions of the images were obtained by using scanning electron microscopy (SEM). Gram staining was performed to see the vesicles.
The size of the vesicles was measured by dynamic light scattering, which presented a distribution of size of vesicles. The range of the vesicle size was 50 and 200nm. The authors also approximated the vesicle size from the 2D EM images with the use of a correction factor. The size from the images was measured to be 150 nm, possibly an underestimation due to various preparation stages. Following the measurement of vesicle sizes in the liquid medium culture, the author utilized biofilms to visualize the vesicles and measure their sizes. The size of vesicles present in biofilms was measured to be 100 nm and less. Dynamic light scattering resulted in somewhat larger size estimation. The vesicles growing on biofilms were visualized by using SEM and embedded TEM. Proteomic analysis was performed on the vesicle preps. Proteomic analysis demonstrated that 24 proteins were present in the vesicle sample including 5 siderophore binding proteins and an immunity protein.
The amount of vesicles was quantitated by radio labeling of vesicle preps. Radio labeled C14 that localized in the vesicle was measured in the two strains. A greater amount of radioactivity was measured in the lab strain vesicles. The membrane lipids were separated based on polarity and visualized by using TLC. The radioactivity from various experiments was lower in the environmental strain and difficult to visualize suggesting the lower amount of vesicles in them. GXM, polysaccharide molecule that could be antibody-tagged was used to demonstrate that the intact cellular vesicles were formed by the intact cells and not formed in the medium itself. As the intact vesicles were formed by the cells they did not uptake any GXM, however sonicated vesicles that were prepared from intact vesicles and allowed to reform took in GXM which was antibody tagged with gold label. This intact and sonicated vesicle prep was also used to measure the vesicular diameter. The intact vesicle had a large range of sizes with the median size of 125 nm. The sonicated and reformed vesicles had a more homogenous range of sizes and were smaller than intact vesicles. Gradient centrifugation method on discontinuous layered column was also used to measure the density of the vesicles.
A gene sfp was identified that affected the amount of vesicles produced. The lab strain had a mutation in the sfp gene and it was not functional, while the environmental strain had a functional sfp. When the sfp was rendered inactive in environmental strain by deletion, the amount of vesicle produced increased and similarly when functional sfp was introduced in lab strain their vesicle amount went down. The sfp gene is associated with synthesis of surfactin (an antibiotic), bacillibactin (siderophore) and other unknown molecules by non ribosomal method that involves addition of phosphor pentathiene. Bacillibactin, which is a siderophore, is an iron chelating compound which binds to siderophore binding protein to supply iron into cells. It was demonstrated that the Bacillibactin and Surfactin were nonfunctional in lab strain, similar to SFP gene.
Conclusion: This study establishes that vesicles are produced by bacillus subtilis which exhibit similar structural features and dimension to the vesicles produced from other bacterial cells. These vesicles were visualized, their size determined and their amount quantitated among lab strain and environmental strain. It was demonstrated in the study that the lab strains produced large amount of vesicles making them easy to work with. The hyperproductivity of vesicles in the lab strain could be utilized for commercial or other uses. It was also proven that the intact vesicles were formed by the cells on intention and not formed in the growth medium randomly. A number of proteins were identified in the vesicles. A gene sfp was identified that was associated with the amount of vesicle production. Functional sfp was associated with low vesicle production while mutant/non functional form (seen in lab strain) had a greater amount of vesicles produced.