Saudi Cultural Missions Theses & Dissertations

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    Piptedoglycan Dynamics in Bacillus subtilis
    (Newcastle University, 2024-09) Aljohani, Alaa; Richard, Daniel
    Gram-positive bacterial cell morphology, growth, and division rely on a delicate balance of peptidoglycan synthesis and controlled degradation to maintain the structural integrity of the cell wall. The process of growth involves synthesising new wall material underneath the existing cell wall on the surface of the cell membrane. This growth requires the cell wall degrading enzymes to distinguish between the newly generated wall and the old wall, selectively degrading the outer layers of the wall to facilitate cell enlargement. As the B. subtilis genome encodes 42 genes that are potentially involved in peptidoglycan degradation, systematic deletion of the known autolytic enzymes was used combined with phenotypic analysis for both cell morphological changes and the ability to become motile. From this work, it was found that only 1 of 2 specific autolytic enzymes is functionally required for growth (CwlO and LytE). Although these two autolytic enzymes exhibit a significant degree of functional redundancy, they are required for slightly different aspects of cell morphology. Only CwlO, in concert with CwlQ or CwlS, was also found to be required for the efficient insertion of flagella through the cell wall. Further analysis showed that CwlO activity with respect to cell growth required the activity of a peptidoglycan carboxypeptidase (DacA), but this was not required for flagellar insertion, suggesting that CwlO has two distinct modes of action. In summary, the results of the work presented in this thesis show that the majority of the predicted cell wall degrading enzymes are dispensable, and only 2 enzymes, CwlO and LytE, have critical roles in maintaining normal cell morphology. Interestingly, this study also reveals that the two key autolytic enzymes seem to have distinct modes of action and potentially differ in their substrate specificity. In this respect, a model for cell growth is presented that tries to amalgamate the results of this work with previously published ideas to explain how cell growth is coordinated with respect to peptidoglycan synthesis and degradation without compromising cell integrity and maintaining cell morphology. This model potentially outlines the basic mechanism of cell wall metabolism in Gram-positive rod-shaped bacteria. It also seems that aspects of the mechanism are also conserved in other bacterial species with a different cell morphology as well as in Gram-negative bacteria. These results clearly indicate that the autolytic enzymes and their regulatory mechanisms are potentially interesting novel targets for the development of small molecule antibacterial compounds.
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    High Resolution Imaging and Physical Properties of Gram-Positive Bacterial Cell Walls Using Atomic Force Microscopy
    (The University of Sheffield, 2023-03) Alomari, Anaam; Hobbs, Jamie
    In this work atomic force microscopy (AFM) was used to study the cell wall of Gram positive bacteria at high resolution with particular focus on Staphylococcus aureus and Bacillus subtilis. The bacterial cell wall is a critical component of bacteria, protecting them from cell lysis due to their internal turgor pressure and giving them their shape, as well as acting as the interface with the outside world. As such it is an important target for multiple antibiotics, as well as being of fundamental interest in its own right. This thesis considers three interrelated projects. In the first set of experiments, a comparison between methicillin resistant (MRSA) and methicillin sensitive (MSSA) S. aureus cell walls has been carried out. The average thickness of MRSA and MSSA sacculi in air and liquid conditions were measured. The cell wall of MRSA is thicker than that of MSSA in both conditions. We have observed that the different parts of the sacculi (rings & mesh) have different thicknesses even on the same fragment of sacculus. There is a significant difference between the thickness of the rings & mesh in MSSA and MRSA in both conditions. There is significant difference when comparing mesh between MSSA and MRSA in liquid, while there is no significant difference between the rings in different type of bacteria or when measuring the whole thickness of MSSA and MRSA in liquid. It seems likely that the architectural difference is that the MRSA wall is denser than WT. This is arguably in line with the fact that MRSA has an extra active cell wall synthesis enzyme (penicillin binding protein, PBP), PBP2A which may add additional PG cross-links during initial synthesis. In the second aspect of the work, the very early stage of cell division is studied (known as the “piecrust”) that are the first sign of the division process in S. aureus cells. Under normal growth in WT, we can see the initial septum in the inner side of the sacculi; the septum walls start to grow as a top layer of mesh in the centre of the cell, then, the height of the septum wall becomes higher and at the same time the gap between the two daughters cell walls becomes obvious. The initial septum of MRSA sacculi are largely the same as MSSA sacculi in term of their architecture except that the gap between two septal plates is bigger and the thickness of the whole piecrust is thicker than for MSSA. The impact of pbp1* and rpoB* mutation has also been explored. We noted, in the absence of PBP1 activity, even when PBP2A is acting, the cell is not able to develop the septum beyond the stage of a wide ridge in the cell wall. However, in the presence of rpoB* mutation, a septum can be formed but the separation between the two septal plates is much less clear than is normal. In last section of this work, the architecture of the division process in live cells is investigated. This work was carried out on B. subtilis as immobilization of live S. aureus is problematic. We have seen the growth of the division site at high resolution for the first time, clearly imaging fibres spanning the division plane and their stretching and eventual failure. On the division area of the live cell images, the two daughter cells connect to each other by thick fibres on the top of the gap. In some live cells, we can see division in which a piece of cell wall breaks off between the two dividing cells, apparently because the division is not perfectly aligned around the circumference. The nanomechanical properties of Bacillus subtilis live cells were explored using force spectroscopy. The average of the Young’s modulus for division area is lower than for the cell body and the cell pole. It seems likely that there is some softening of the material in the division area during the splitting process. The poles have a higher modulus than that measured in the division region, so the value in the division region is probably dominated by the spanning fibres.
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