Peptidoglycan architecture and dynamics in Gram-positive bacteria

The major structural determinant of most bacterial cells is the peptidoglycan layer, a network (the sacculus) of glycan strands cross-linked by short peptide stems which encompasses the entire bacterium. However the architecture of peptidoglycan, which must accommodate both the structural requirements of the cell and the dynamic processes of growth and division, is poorly understood. Most architectural studies have addressed the peptidoglycan of rod-shaped bacteria. The architecture in Gram-positive cocci and ovococci is largely uncharacterised, In this study, atomic force microscopy (AFM) of purified sacculi of three species of ovococcus was combined with biochemical analysis and super resolution fluorescence microscopy. A model was developed in which incorporation of long glycan strands from a single mid-cell focus results in preferential orientation, parallel to the short axis of the cell. AFM and fluorescence microscopy of the coccoid bacterium Staphylococcus aureus revealed a dynamic peptidoglycan architecture of rings and knobbles, growth by peptidoglycan maturation, and a system of heritable peptidoglycan ribs. Ribs may provide a structural mechanism for coordinating orthogonal division on three planes. We hypothesised cell wall growth occurs via the activity of N-acetyl-β-D-glucosaminidases. Four putative glucosaminidases were identified, with SagB found to have a major role in glycan strand length determination. Hydrolysis of the septal cross-wall by glucosaminidase activity was required for spherical shape, suggesting a novel mechanism of growth by hydrolysis. My work has highlighted diverse and elegant peptidoglycan architectures, adapted to meet the unique mechanical requirements of bacteria with different morphologies and strategies for growth and division.