Using STORMForce for the investigation of bacterial peptidoglycan structure

STORMForce is the combination of Stochastic Optical Reconstruction Microscopy (STORM) and Atomic Force Microscopy (AFM) imaging techniques. The integration method of STORMForce and application on biological samples is presented in this work. The STORMForce technique was developed to gain a greater understanding of bacterial samples to aid the combat against antibiotic resistance. Here I have studied the cell wall structure of peptidoglycan in E. coli and B. subtilis. Peptidoglycan is a material that provides the cells with mechanical strength to resist the internal turgor pressure, and is the primary target for many antibiotics. The details of the architecture and the way in which that architecture is formed is still poorly understood.
Firstly a STORMForce image was obtained through imaging on separate equipment, which were then overlayed. A protocol was developed for sample production that is suitable for both AFM and STORM. A STORMForce image was obtained from correlating images on separate instruments. Once this overlay was obtained, STORM components were added to the AFM to allow STORM and AFM (STORMForce) imaging to take place on one instrument. The sample protocol was developed further to allow AFM imaging to take place before or after STORM imaging, depending on the sample. The development of equipment and method used for imaging is presented.
STORMForce was used to identify a difference in the structure of the peptidoglycan of E. coli in different areas of the sacculi during the exponential growth phase. A blank band of fluorescence was identified in the STORM images in the middle of the sacculi; AFM then targeted the same area to identify the architectural difference. Literature suggests the blank stripe was due to the removal of the peptide cross-link by amidase enzyme activity. AFM was used to obtain high resolution images showing a reduced fibre spacing in the area of the blank stripe when compared to the surrounding area. This suggests the spacing is reduced due to the lack of peptide cross-link, although resolution was not achieved to image the molecular difference. An amidase deleted mutant was also imaged; results showed there was no longer a visible blank stripe of fluorescence.
Peptidoglycan insertion within the septum and the main rod body of B. subtilis is also investigated via STORMFORCE in this study. Using fluorescently pulse labelled material, STORMForce results showed that although newly inserted peptidoglycan is mostly added to the leading (internal) edge of the septum as it develops, material is also sparsely added through the entire septum. High-resolution AFM images show large holes present within the septum material, suggesting newly inserted material also targets these areas to fill in the large holes to maintain structural integrity. Structured illumination microscopy (SIM) images where also taken of the same pulse labelled material and similar insertion patterns where observed. A quantitative analysis is given for both SIM and STORMForce data.
Peptidoglycan insertion within the main rod body of B. subtilis during cell elongation showed a banding insertion in the pulse labelled material. As well as STORMForce, SIMForce was also used; this was achieved by adapting the imaging protocol by using correlative glass grids. To truly understand the periodicity of the banding structure, single layers of peptidoglycan were imaged via SIMForce as well as double layered intact sacculi. The data was then quantitatively analysed, showing that the frequency of the banding is approximately doubled in a double layer of material compared to a single layer. As MreB is thought to be responsible for the banding insertion of the peptidoglycan, a deleted MreB mutant was also imaged. The mutant showed a loss of any banding formation within the peptidoglycan and a loss of rod shape. This confirms the role of MreB in governing cell shape and controlled peptidoglycan insertion within the rod of the cell.