Dokument

Summary:
For many years, efforts for discovering new antibiotics were mainly focused on the inhibitors of the cell wall synthesis machinery. We tried to find other antibiotics that target the cell wall by making use of the cell wall degradation enzymes, which lyse the cell wall and therefore kill bacteria. LytC is an autolysin from B. subtilis, which is capable of degrading the bacterial cell wall and make it a potential candidate antibiotic. In this dissertation, I cloned B. subtilis lytC gene and over-expressed LytC protein in E. coli. In order to characterize the function of different LytC regions, we designed different constructs of lytC. I used the Gram-negative bacterium E. coli and Gram-positive B. subtilis to test if LytC can degrade the cell wall components when used as an external additive in cell medium. Different constructs of purified LytC protein were used, the results showed that LytC constructs can efficiently and greatly inhibit the growth of E. coli and B. subtilis, yet it is only effective for exponentially growing cells. The cell wall of stationary cells was more resistant to LytC and cell lysis was not observed. I proved that the C-terminal fusion of LytC with a strep-tag can be successfully over-expressed and purified while the N-terminal fusion cannot. Overexpression of LytC leads to a fast drop in OD600 after one hour induction with 0.5 mM IPTG. The construct of LytCF6 only contains the functional region and the purified protein can still function like full length protein, which means the four cell wall binding (CWB) and the „low complexity region“ (LCR) domains are not necessary for LytC activity. I used strep elution buffer with BSA (bovine serum albumin) as control to verify that it is the LytC protein that is responsible for cell growth inhibition. I deleted 22 amino acids from the C-terminus of the functional region, and protein activity of this partially deleted LytCF2R2 was compared with LytCF2 (which has the full length of functional region). As a result, LytCF2R2 lost almost half of the protein activity in comparison to LytCF2. This further demonstrates that LytCF2 contains the functional region and the deletion of it can result in loss of activity. We reviewed the localization and the single molecule trajectories of proteins (MreBCD, Pbp1A, RodZ and PDH complex) in exponentially grown B. subtilis cells. Phosphofructokinase (PfkA), which is a fast moving protein, was used as a control for MreB in the single molecule tracking study. In the protein localization study, we found that MreB, MreC, MreD, RodZ and Pbp1A are localized along the cell membrane and have a similar localization pattern. The results indicate that MreB, MreC, MreD, RodZ and Pbp1A are membrane proteins. We confirmed the phenomenon that MreB forms discontinuous filaments structure underneath the cell membrane, both in B. subtilis and in E. coli. Upon reduction in its expression level, MreB forms patchy and short filament structures in B. subtilis. MreC and MreD also form patchy spots structures besides the membrane staining pattern. From our calculation, two thirds cells have PdhA localized at one cell pole, one third of the cells does not contain PdhA spots. The localization studies of PdhB, PdhC and PfkA showed that they are all uniformly distributed within the cytoplasm of the cell, which indicates they are cytoplasmic proteins. PdhD has two localization patterns, one is the same as chromosome DNA staining, and the other one is uniformly distributed within the cytoplasm. We found single molecules of MreB are composed of two populations. One is the immobile population that forms the filamentous structures. The other one is the mobile population that freely diffuses within the cell. The same conclusions apply to the Pbp1A and PDH subunits proteins, as well as for the MreB colocalized proteins (MreC, MreD and RodZ). The single molecules of the PfkA compose two mobile populations. The fraction and movement speed of MreB mobile single molecules are lower than those of PfkA. We also deduced that MreB is a membrane-associated protein and that PdhC (E2 subunit) is the core of PDH complex from our data analysis, because the fraction and diffusion coefficient of mobile PdhC single molecules are the slowest of the four PDH subunits.

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