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.
